Note: Descriptions are shown in the official language in which they were submitted.
WO 95/03831 PCT/US94/08511
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MONOGENOUS PREPARATIONS OF CYTOTOXIC CONJUGATES
FIELC) OF THE INVENTION
This invention is related to the preparation and use of cytotoxic
conjugates. In particular, substantially monogenous preparations of
cytotoxic conjugates, homogeneous compositions of cytotoxic conjugates
and methods for preparing such cytotoxic conjugates are provided.
BACKGROUND OF THE INVENTION
One goal in pharmacology is to design specific agents that act with
high specific activity only on targeted cells or tissues. This aim is of
particular importance, for example, in the design of agents for treatments
of diseases, such as neoplastic disease and diseases of viral origin, in
which the ratio of toxic dose to therapeutic dose is very low and the
dosage must be minimized. Numerous approaches to achieving this goal
have been developed. Among these are the use of agents, such as growth
factors, that act only on specific cells, and the use of toxins that are
relatively non-toxic unless delivered intracellularly.
riLrobl&:,L growth factors and riLrol~lasl growth factor receptors
During the last twenty-five years, a great deal of attention has been
directed towards the identification and characterization of factors that
stimulate the growth, proliferation and differentiation of specific cell types.
Numerous growth factors and families of growth factors that share
structural and functional features have been identified. Many of these
factors have multifunctional activities and affect a wide spectrum of cell
types.
One family of growth factors that has a broad spectrum of activities
is the fibroblast growth factor (FGF) family. This family of proteins includes
FGFs designated FGF-1 through FGF-9 (or acidic FGF (aFGF), basic FGF
(bFGF), int-2, hst-1/K-FGF, FGF-5, FGF-6/Hst-2, keratinocyte growth factor
(KGF), FGF-8 and FGF-9, respectively). These proteins share the ability to
bind to heparin, induce intracellular receptor-mediated tyrosine
WO 95/03831 PCT/US94/08511
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phosphorylation and the expression of the c-fos mRNA transcript, and
stimulate DNA synthesis and cell proliferation.
Acidic and basic FGF, which were the first members of the FGF
family that were characterized, are about 55% identical at the amino acid
5 level and are highly conserved among species. Basic FGF has a molecular
weight of approximately 16 kD, is acidic and temperature sensitive and has
a high isoelectric point. Acidic FGF has an acidic isoelectric point. The
other members of the FGF family have subsequently been identified on the
basis of amino acid sequence homologies with aFGF and bFGF and
10 common physical and biological properties, including the ability to bind to
one or more FGF receptors. Basic FGF, int-2, hst-1/K-FGF, FGF-5, hst-
2/FGF-6 and FGF-8 are oncogenes. For example, bFGF is expressed in
melanomas, int-2 is expressed in mammary tumor virus and hst-1 /K-FGF is
expressed in angiogenic tumors. Acidic FGF, bFGF, KGF and FGF-9 are
15 expressed in normal cells and tissues.
FGFs exhibit a mitogenic effect on a wide variety of mesenchymal,
endocrine and neural cells. They are also important in differentiation and
development. Of particular interest is their stimulatory effect on collateral
vascularization and angiogenesis. Such effects have stimulated
20 considerable interest in FGFs as therapeutic agents, for example, as
pharmaceuticals for wound healing, neovascularization, nerve regeneration
and cartilage repair. In addition to potentially useful proliferative effects,
FGF-induced mitogenic stimulation may, in some instances, be detrimental.
For example, cell proliferation and angiogenesis are an integral aspect of
25 tumor growth. Members of the FGF family, including bFGF, are thought to
play a pathophysiological role, for example, in tumor development,
rheumatoid arthritis, proliferative diabetic retinopathies and other
complications of diabetes.
The effects of FGFs are mediated by high affinity receptor tyrosine
30 kinases on the cell surface membranes or FGF-responsive cells (see, e.~.,
Imamura et al. (1988) Biochem. BioPhvs. Res. Comm. 155:583-590;
WO 95/03831 PCT/US94/08511
~ ~t ~641
Huang et al. (1986) J. Biol. Chem. 261:9568-9571, which are incorporated
herein by reference). Lower affinity receptors also play a role in mediating
FGF activities. The high affinity receptor proteins, which are single chain
polypeptides with molecular weights ranging from 110 to 150 kD,
5 depending on cell type, constitute a family of structurally related FGF
receptors. Four FGF receptor genes have been identified, and, at least two
of these genes generate multiple mRNA transcripts via alternative splicing
of the primary transcript.
Ribosome-inactivating ,urot~i.,s
Ribosome-inactivating-proteins (RlPs), which include ricin, abrin and
saporin, are plant proteins that catalytically inactivate eukaryotic
ribosomes. Some RlPs, such as the toxins abrin and ricin, contain two
constituent chains: a cell-binding chain that mediates binding to cell
surface receptors and internalizing the molecule; and a chain responsible for
15 toxicity. Such RlPs are type ll RlPs. Single chain RlPs, such as the
saporins, do not have a cell-binding chain. As a result, unless internalized,
they are substantially less toxic to whole cells than the RlPs that have two
chains.
RIPS inactivate ribosomes by interfering with the protein elongation
20 step of protein synthesis. For example, the RIP saporin (hereinafter also
referred to as SAP) has been shown to inactivate 60S ribosomes by
cleavage of the n-glycosidic bond of the adenine at position 4324 in the rat
28S ribosomal RNA (rRNA). The particular region in which A4324 is located
in the rRNA is highly conserved among prokaryotes and eukaryotes. A4324
25 in 28S rRNA corresponds to A2660 in Escherichia coli (E. coli) 23S rRNA.
Several RlP's also appear to interfere with protein synthesis in prokaryotes,
such as E. coli.
Several structurally related RlP's have been isolated from seeds and
leaves of the plant SaPonaria officinalis (soapwort). Among these, SAP-6
30 is the most active and abundant, representing 7% of total seed proteins.
Saporin is very stable, has a high isoelectric point, does not contain
,
WO 95/03831 PCT/US94108511
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4-
carbohydrates, and is resistant to denaturing agents, such as sodium
dodecyl sulfate (SDS), and a variety of proteases. The amino acid
sequences of several saporin-6 isoforms from seeds are known and there
appear to be families of saporin RlPs differing in few amino acid residues.
5 Because saporin is a type I RIP, it does not possess a cell-binding chain.
Consequently, its toxicity to whole cells is much lower than other toxins,
such as ricin and abrin. When internalized by eukaryotic cells, however, its
cytotoxicity is 100- to 1000-fold more potent than ricin A chain.
Cytotoxic conjugates
Cytotoxins, such as saporin and ricin A chain, have been covalently
linked to cell surface binding proteins to produce cytotoxic chemical
conjugates or have been linked to antibodies to produce immunotoxins that
are targeted to, and internalized by, specific cells. For example, basic
fibroblast growth factor (bFGF) has been chemically conjugated to saporin-
15 6 to produce the mitotoxin bFGF-SAP (see, e.q., U.S. Patent No.
5,191,067 to Lappi et ak; and Lappi et al. (1989) Biochem. and BioPhvs.
Res. Comm. 160:917-923). The resulting FGF-SAP conjugates have been
used to treat restenosis (see, e.q., International Patent Application No. W0
92/11872, which is based on U.S. Application Serial No. 07/637,074; see,
20 also U.S. Patent No. 5,308,622) and other FGF-mediated disorders.
Treatment is effected by local or intravenous administration of a
therapeutically effective amount of the FGF conjugate following, for
example, balloon angioplasty. Basic FGF-SAP conjugates also have shown
promise as agents for the treatment of certain tumors. The growth of
25 melanomas and other tumors that express receptors to which FGFs bind
can be inhibited by FGF-SAP (see, e.~., published International Application
W0 92/04918, which is based on U.S. Application Serial No. 07/585,319,
filed 9/19/90; published International Application No. W0 92/04918, which
is based on U.S. Patent Application Serial No. 07/585,319; and Beitz et al.
30 (1992) Cancer Research 52:227-230) .
wo 95/03831 ~1 6~64-1 PCT/USg4/08511
Conjugates are often synthesized by the use of reactive sulfhydryls
either found naturally, as in the case of ricin A chain, in the cytotoxic
moiety and the targeting moiety. If not present, sulfhydryls are introduced
into the cytotoxic agent using a chemical coupling agent so that
5 conjugation is possible for antibodies and for RlPs, such as SAP, that are
devoid of native or available sulfhydryls. The chemistry of conjugation,
however, gives rise to various structures, resulting in a heterogeneous
population of products that are difficult to separate from each other. These
structures can include conjugates containing more than one RIP attached to
10 the targeting moiety, more than one targeting moiety attached to the RIP,
or more than one RIP attached to more than one targeting moiety. The
resulting structures also form aggregates because of interactions among
the conjugates, particularly among free sulfhydryls in the conjugates.
Because of the difficulties encountered in separating the resulting
15 conjugates with different structures, heterogeneous mixtures are often
used in experiments and even therapeutic applications.
For example, bFGF is conjugated via a cysteine residue to saporin,
which is first derivatized with N-succinimdyl-3(2-pyridyldithio)propionate
(SPDP). Basic FGF has at least two cysteines available for reaction with
20 SPDP-derivatized saporin. Consequently, reaction of the bFGF with the
SPDP-derivatized SAP results in an array of molecules, which probably
differ with respect to biologically relevant properties and may not be ideal
for in vivo applications. Gel electrophoresis and western blotting verify that
a number of higher molecular weight species are formed. The species
25 contain SAP to FGF ratios of 0.5, 1, 2 and other oligomeric combinations.
There is very little information on the relative activities of the various
constituents of the heterogeneous population, though it has been reported
that polymeric RlPs have increased non-specific toxicities.
To develop FGF-SAP and other cytotoxic agents into acceptable
30 pharmaceutical agents for treating deleterious disease states, it would be
desirable to have a monogenous molecule that is well-characterized
WO 95/03831 PCT/US94/08511
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physically, chemically and biologically. Therefore, it is an object herein to
provide methods for the production of monogenous preparations of
cytotoxic FGF conjugates and of homogeneous compositions containing
conjugates of FGF and cytotoxins. It is also an object herein to provide the
5 FGF cytotoxic conjugates that are produced by these methods. It is also an
object herein to provide compositions that contain homogeneous
populations of FGF cytotoxic conjugates or mixtures of monogenous
cytotoxic conjugates.
SUMMARY OF THE INVENTION
Monogenous preparations of cytotoxic conjugates and compositions
containing homogeneous (non-aggregated) populations of cytotoxic
conjugates are provided. The cytotoxic conjugates contain a polypeptide
that is reactive with an FGF receptor (also referred to herein as an FGF
protein), such as bFGF, linked to a cytotoxic agent. In a given preparation
substantially all of the cytotoxic conjugates have the same ratio of the
polypeptide that is reactive with an FGF receptor to cytotoxic agent. In
preferred embodiments, the cytotoxic conjugates contain one molecule of
FGF protein per molecule of cytotoxic agent.
Polypeptides that are reactive with an FGF receptor (FGF proteins)
include any molecule that reacts with FGF receptors on cells that bear FGF
receptors and results in internalization of the linked cytotoxic agent.
Particularly preferred polypeptides that are reactive with an FGF receptor
include members of the FGF family of polypeptides, muteins of these
polypetides, and chimeric or hybrid molecules that contain portions of any
of these family members, as long as the resulting polypeptide binds to FGF
receptors and internalizes a linked cytotoxic agent and the resulting
preparation of cytotoxic conjugates that contain the FGF protein is
monogenous (i.e. each conjugate in a preparation of such conjugates has
the same molar ratio of FGF protein to cytotoxic agent).
The cytotoxic agents include any molecule that, when internalized, is
cytotoxic to eukaryotic cells. Such cytotoxic agents include, but are not
WO 95/03831 PCT/US94108511
~tb~647
limited to, ribosome inactivating proteins, inhibitors of DNA,RNA and/or
protein synthesis and other metabolic inhibitors. In certain embodiments,
the cytotoxic agent is a ribosome-inactivating protein (RIP), such as, for
example, saporin, although other cytotoxic agents can also be
5 advantageously used.
The preparation may be produced by chemical means so that the
resulting conjugates are chemical conjugates or using DNA encoding
chimeric molecules to produce fusion proteins. The components of the
conjugates may also be produced by expression of DNA or by chemical
10 synthesis or any other method known to those of skill in this art.
The conjugate can be represented by formula:
(FGF)n-(cytotoxic agent)m,
with the understanding that the FGF and cytotoxic agent may be linked in
any order and through any appropriate linkage, as long as the resulting
15 conjugate binds to an FGF receptor and internalizes the cytotoxic agent(s)
in cells bearing an FGF receptor. FGF refers to the polypeptide reactive
with an FGF receptor, n and m, which in monogenous preparations are
integers, are the same or different, and are 1 to 6, preferably 1 to 4, and
typically 1 or 2, and if m or n, or m and n are greater than 1, then the
20 conjugate may contain more than one cytotoxic agent and more than one
FGF.
Cytotoxic conjugates that contain a plurality of monomers of an FGF
protein linked to the cytotoxic agent are also provided. These conjugates
that contain several, typically two to about six, monomers can be produced
25 by linking multiple copies of DNA encoding the FGF fusion protein, typically
head-to-tail, under the transcriptional control of a single promoter region.
To produce a monogenous preparation of cytotoxic conjugates or
homogeneous compositions of such conjugates, the cytotoxic agent is
linked to the polypeptide that is reactive with an FGF receptor by the
30 methods provided herein. Each member of the resulting preparation of
cytotoxic conjugate contains the same molar ratio of cytotoxic agent to
WO 95/03831 PCT/US94/08511
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polypeptide that is reactive with an FGF receptor. Generally each conjugate
contains one molecule of each of the constituents. In addition, in
preferred embodiments the resulting conjugates do not form aggregates.
Methods for the preparation of the cytotoxic agent, such as a
5 ribosome inactivating protein (RIP), including, but not limited to, saporin,
and the FGF polypeptides and the monogenous preparation of cytotoxic
conjugates that contains a defined molar ratio of each of the constituents
are provided. These methods include chemical conjugation methods and
methods that rely on recombinant production of the cytotoxic conjugates.
10 The methods result in monogenous preparations of cytotoxic conjugates
that can be used, in preferred embodiments, to prepare homogeneous
compositions of monogenous cytotoxic conjugates.
The chemical method relies on several means to reduce the
heterogeneity of the resulting cytotoxic conjugate and to avoid interactions
15 among the conjugates that result in aggregate formation. In preferred
embodiments, the FGF portion of the conjugate is treated so that only one
cysteine is available for reaction with the cytotoxic agent and the cytotoxic
agent, if necessary, is derivatized and only a single species is selected for
reaction with the modified FGF. The cytotoxic agent, may also be modified
20 to include a cysteine residue. The locus of the cysteine residue is selected
such that the cysteine residue is available for conjugation with the available
cysteine in the FGF polypeptide and the resulting conjugate is cytotoxic
upon internalization by targeted eukaryotic cells.
In accordance with this embodiment, modified saporin is provided.
25 Such modifications include, but are not limited to, the introduction of a Cys residue at or near the N-terminus. Saporin is modified by addition of a
cysteine residue at the N-terminus-encoding portion of the DNA by addition
of a Met-Cys. Saporin also has been modified herein by insertion of a
cysteine at position 4 or 10 in place of the wild type residue. The resulting
30 saporin can then be reacted with an available cysteine on an FGF to
WO 95/03831 PCT/US94108511
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produce conjugates that are linked via the added Cys or Met-Cys on
saporin .
In practicing the chemical method, site-directed mutagenesis has
been used to reduce the heterogeneity of the chemical conjugate by
5 replacing one of the reactive cysteines in bFGF with a residue, such as
serine, that does not alter the cytotoxicity of the resulting conjugate, and
leaves only one cysteine available for reaction with the cytotoxic agent. In
preferred embodiments, the cytotoxic agent is a single species of
derivatized SAP. Because there are slight charge differences among
10 different derivatized SAP species that are produced upon the derivatization
of SAP, it has been found herein that it is possible to isolate substantially
pure mono-derivatized SAP. Reaction of mono-derivatized SAP with mono-
reactive cysteine basic FGF produces a monogenous preparation of
cytotoxic conjugates and homogeneous populations of conjugates that are
15 highly cytotoxic to FGF-receptor-bearing cells. In other embodiments, the
saporin is modified at or near the N-terminus to include a cysteine residue,
so that the resulting modified saporin can react with the F~iF protein
without further derivatization.
The recombinant method relies on the expression of DNA that
20 encodes an FGF protein, modified to remove all cysteines that contribute to
aggregate formation, linked to DNA encoding the cytotoxic conjugate.
DNA encoding the FGF polypeptide is mutagenized so that no cysteines are
available in the resulting conjugate for interaction with other conjugates.
The DNA encoding the modified FGF protein is linked directly to the DNA
25 encoding the N-terminus of the saporin polypeptide or via one, preferably
two, or more codons that encode a linking peptide or amino acid. The
number of linking codons is selected such that the resulting DNA encodes a
fusion protein that is cytotoxic to selected cells.
The combination of the modified FGF protein and linked cytotoxic
30 agent is prepared as a chimera, using recombinant DNA techniques. The
fusion protein molecule is designed and produced in such a way that the
WO 95/03831 PCT/US94/08511
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FGF protein portion of the conjugate is available for recognition of its
respective cell-surface receptor and can target the conjugate to cells
containing its respective cell-surface receptor. In a preferred embodiment,
the FGF protein is FGF that has been modified by replacement of the
5 cysteine residues at positions 78 and 96 with serine residues.
The resulting monogenous preparation of conjugates and
homogeneous compositions of conjugates produced by any of the methods
described herein can be used in pharmaceutical compositions to treat
FGF-mediated pathophysiological conditions by specifically targeting to cells
10 having FGF receptors and inhibiting proliferation of or causing death of the
cells. Such pathophysiological conditions include, for example, tumor
development, restenosis, Dupuytren's Contracture, certain complications of
diabetes such as proliferative diabetic retinopathies, and rheumatoid
arthritis. The treatment is effected by administering a therapeutically
15 effective amount of the FGF conjugate, for example, in a physiologically
acceptable excipient. Additionally, the conjugate can be used to target
cytotoxic agents into cells having FGF receptors, and to inhibit the
proliferation of such cells.
The resulting preparations of monogenous FGF conjugates or
20 homogeneous compositions of conjugates may also be administered in
conjunction with anti-tumor agents, such as cis-platin. Such combination
therapy enhances the anti-tumor activity of the FGF-conjugates. In
particular, administration of cis-platin in conjunction with an FGF-cytotoxic
conjugate enhanced the anti-tumor activity of the FGF-cytotoxic conjugate.
25 In particular, a method for inhibiting the proliferation of tumor cells that
bear FGF receptors by administering a proliferation-inhibiting amount of a
cytotoxic conjugate and a cytotoxic amount of cls-platin, in which the
amounts of each are such that the combination of cytoxic conjugate and
cls-platin kills or inhibits the growth of the tumor cells, is provided.
WO 95/03831 PCT/US94/08511
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~IG864
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
' ~ D~ri~ io~s
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood by one of skill
in the art to which the subject matter herein belongs. All U.S. patents and
all publications mentioned herein are incorporated in their entirety by
reference thereto.
The amino acids, which occur in the various amino acid sequences
appearing herein, are identified according to their well-known, three-letter
or one-letter abbreviations. The nucleotides, which occur in the various
DNA fragments, are designated with the standard single-letter designations
used routinely in the art.
As used herein, cytotoxic agents include saporin, the ricins, abrin
and other RlPs, Pseudomonas exotoxin, inhibitors of DNA, RNA or protein
synthesis or other metabolic inhibitors that are known to those of skill in
this art. Saporin is preferred, but other suitable RlPs include, but are not
limite~ to, ricin, ricin A chain, maize RIP, gelonin, diphtheria toxin,
diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviral protein
(PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin,
monordin, bryodin, shiga and others known to those of skill in this art. The
term RIP is used herein to broadly include such cytotoxins, as well as other
cytotoxic molecules that inhibit cellular metabolic process, including
transcription, translation, biosynthetic or degradative pathways, DNA
synthesis and other such process, or that kill cells.
As used herein, saporin (abbreviated herein as SAP) refers to
polypeptides having amino acid sequences found in the natural plant host
SaPonaria officinalis, as well as modified sequences, having amino acid
substitutions, deletions, insertions or additions, which still express
substantial ribosome-inactivating activity. Purified preparations of saporin
are frequently observed to include several molecular isoforms of the
protein. It is understood that differences in amino acid sequences can
WO 95/03831 PCT/US94/08511
.
occur in saporin from different species as well as between saporin
molecules from individual organisms of the same species.
As used herein, N-terminal extension, refers to a peptide region that
is linked to the amino terminus of a biologically active portion of a saporin
5 polypeptide. As demonstrated herein, when saporin is produced by
expressing DNA encoding in a host cell, it is expressed with an N-terminal
extension. The N-terminal extension serves to render the saporin
polypeptide portion of the saporin-containing protein either nontoxic to the
host upon expression of the protein in the host or substantially less toxic to
10 the host than the expression of a saporin polypeptide without an N-terminal
extension. N-terminal extensions having as few as 2 amino acids, and up
to many amino acids, are provided. The length of the N-terminal extension
is not important as long as the resulting cytotoxic conjugate binds to cell
surface receptors, internalizes the cytotoxic agent and is cytotoxic upon
15 internalization, can be employed. The precise number for the upper limit
can be determined empirically, using cytotoxicity assays, such as those
exemplified herein, that are known to those of skill in this art. Presently
preferred N-terminal extension regions are on the order of about 2 to 15
amino acids. Most preferred N-terminal extension regions are in the range
20 of about 2 to about 10 amino acids.
As used herein, a modification that is effected substantially near the
N-terminus of a cytotoxic agent, such as saporin, is generally effected
within the first about ten residues of the protein. Such modifications,
include the addition or deletion of residues, such as the addition of a
25 - cysteine facilitate conjugation between the polypeptide reactive with an
FGF receptor or fragment of the polypeptide and the cytotoxic moiety
portion to form cytotoxic agents that contain a defined molar ratio,
preferably a ratio of 1:1, of cytotoxic agent and polypeptide reactive with
an FGF receptor or fragment of the polypeptide.
As used herein, a mitotoxin is a cytotoxic molecule targeted to
specific cells by a mitogen.
WO 95/03831 PCT/US94/08Sll
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As used herein, the term cytotoxic agent refers to a molecule
capable of inhibiting cell function. The agent may inhibit proliferation or
may be toxic to cells. The term includes agents whose toxic effects are
mediated only when transported into the cell and also those whose toxic
5 effect is mediated at the cell surface. A variety of cytotoxic agents can be
used and include those that inhibit protein synthesis and those that inhibit
expression of certain genes essential for cellular growth or survival.
Cytotoxic agents include those that result in cell death and those that
inhibit cell growth, proliferation and/or differentiation.
As used herein, ligand refers to any polypeptide that is capable of
binding to a cell-surface protein and is capable of facilitating the
internalization of a ligand-containing fusion protein into the cell. Such
ligands include growth factors, antibodies or fragments thereof, hormones,
and other types of proteins.
As used herein, the term "polypeptide reactive with an FGF
receptor" refers to any polypeptide that specifically interacts with an FGF
receptor, preferably the high-affinity FGF receptor, and that is transported
into the cell by virtue of its interaction with the FGF receptor. Polypeptides
reactive with an FGF receptor are also referred to herein as FGF proteins.
20 FGF proteins include members of the FGF family of peptides, including FGF-
1 through FGF-9, chimeras or hybrids of any of FGF-1 through FGF-9, or
FGFs that have deletions (see, e.~., Published International Application No.
W0 90/02800, national stage applications, and patents based thereon) or
insertions of amino acids, as long as the resulting peptide or protein
25 specifically interacts with an FGF receptor and is internalized by virtue of
this interaction.
As used herein, FGF refers to polypeptides having amino acid
sequences of native FGF proteins, as well as modified sequences, having
amino acid substitutions, deletions, insertions or additions in the native
30 protein but retaining the ability to bind to FGF receptors and to be
internalized. Such polypeptides include, but are not limited to, FGF-1 -
WO 95/03831 PCT/US94/08511
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FGF-9. For example, bFGF should be generally understood to refer to
polypeptides having substantially the same amino acid sequences and
receptor-targeting activity as that of bovine bFGF or human bFGF or an
acidic FGF. It is understood that differences in amino acid sequences can
5 occur among FGFs of different species as well as among FGFs from
individual organisms or species and that not all FGFs bind to all FGF
receptor subtypes. It is only required that the FGF bind to at least one FGF
receptor.
Reference to FGFs is also intended to encompass proteins isolated
10 from natural sources as well as those made synthetically, as by
recombinant means or possibly by chemical synthesis. FGF also
encompasses muteins of FGF that possess the ability to target saporin to
FGF-receptor expressing cells. Such muteins include, but are not limited to,
those produced by replacing one or more of the cysteines with serine as
15 herein or that have any other amino acids deleted or replaced as long as the
resulting protein has the ability to bind to FGF-receptor bearing cells and
internalize the linked cytotoxic agent. Typically, such muteins will have
conservative amino acid changes, such as those set forth below in Table 1.
DNA encoding such muteins will, unless modified by replacement of
20 degenerate codons, hybridize under conditions of at least low stringency to
DNA encoding bFGF (SEQ ID N0. 12 and 13) or DNA encoding any of the
FGF's set forth in SEQ ID. NOs. 24-32.
As used herein, DNA encoding an FGF peptide or polypeptide
reactive with an FGF receptor refers to any of the DNA fragments set forth
25 herein as coding such peptides, to any such DNA fragments known to
those of skill in the art, any DNA fragment that encodes an FGF that binds
to an FGF receptor and is internalized thereby and may be isolated from a
human cell library using any of the preceding DNA fragments as a probe
any DNA fragment that encodes any of the FGF peptides set forth in SEQ
30 ID NOs. 24-32 (such DNA sequences are available in publicly accessible
databases, such as DNA (July, 1993 release from DNASTAR, Inc.
WO 95/03831 PCT/US94/08511
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Madison, Wl; see, also U.S. Patent No. 4,956,455, U.S. Patent No.
5,126,323, U.S. Patent No. 5,155,217, U.S. Patent No. 4,868.113,
published International Application W0/90/08771 (and the corresponding
U.S. patent, upon its issuance), which is based on U.S. Application Serial
5 No. 07/304,281, filed January 31, 1989, and Miyamoto et al. (1993) Mol.
Cell. Biol. 13:4251 4259), and any DNA fragment that may be produced
from any of the preceding DNA fragments by substitution of degenerate
codons. It is understood that once the complete amino acid sequence of a
peptide, such as an FGF peptide, and one DNA fragment encoding such
10 peptide are available to those of skill in this art, it is routine to substitute
degenerate codons and produce any of the possible DNA fragments that
encode such peptide. It is also generally possible to synthesize DNA
encoding such peptide based on the amino acid sequence.
As used herein, FGF receptors refer to receptors that specifically
15 interact with a member of the FGF family of proteins and transport it into
the cell. Included among these are the receptors described in International
Application No. W0 91/00916, which is based on U.S. Patent Application
Serial No.07/377,033; International Application No. W0 92/00999, which
is based on U.S. Patent Application Serial No.07/549,587; International
20 Application No. W0 90/05522; and International Application No. W0
92/12948; see, also Imamura (1988) Biochem. Biochvs. Res. Comm.
155:583-590 and Moscatelli (1987) J. Cell. PhYSiol. 131: 123-130.
As used herein, to target a cytotoxic agent means to direct it to a
cell that expresses a selected receptor by linking the agent to a polypeptide
2~ reactive with an FGF receptor. Upon binding to the receptor the saporin-
containing protein is internalized by the cell and is cytotoxic to the cell.
As used herein, preparations of monogenous conjugates are
preparations of conjugates in which each conjugate has the same, generally
about 1 :1, though not necessarily, molar ratio of targeting molecule to
30 targeted agent. Monogenous conjugates are substantially identical in that
they possess indistinguishable chemical and physical properties and
WO 95/03831 PCT/US94/08~11
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generally preparations of such conjugates contain only one species of
conjugate. It is, of course understood, that some variability among the
species may be present and will be tolerated to the extent that the activity
of each member of the conjugate is substantially the same. For example,
5 saporin that is expressed in bacterial hosts as provided herein may contain
a mixture of species that differ at their N-terminus. Such recombinantly
produced saporin, however, is suitable for use to produce chemically
conjugated conjugates by the methods herein. The resulting preparation is
monogenous as defined herein in that each conjugate contains the same
10 molar ratio of FGF protein to targeted agent, but each conjugate is not
necessarily identical, but is substantially identical in that each conjugate
has substantially the same biological activity.
As used herein, a homogeneous population or composition of
conjugates means that the constituent members of the population or
15 composition are monogenous and further do not form aggregates.
As used herein, secretion signal refers to a peptide region within the
precursor protein that directs secretion of the precursor protein from the
cytoplasm of the host into the periplasmic space or into the extracellular
growth medium. Such signals may be either at the amino terminus or
20 carboxyl terminus of the precursor protein. The preferred secretion signal is linked to the amino terminus of the N-terminal extension region.
As used herein, vector or plasmid refers to discrete elements that are
used to introduce heterologous DNA into cells for either expression of the
heterologous DNA or for replication of the cloned heterologous DNA.
25 Selection and use of such vectors and plasmids are well within the level of
skill of the art.
As used herein, expression vector includes vectors capable of
expressing DNA fragments that are in operative linkage with regulatory
sequences, such as promoter regions, that are capable of effecting
30 expression of such DNA fragments. Thus, an expression vector refers to a
recombinant DNA or RNA construct, such as a plasmid, a phage,
WO 95/03831 PCT/US94/08511
~l G~64~
recombinant virus or other vector that, upon introduction into an
appropriate host cell, results in expression of the cloned DNA. Appropriate
expression vectors are well known to those of skill in the art and include
those that are replicable in eukaryotic cells and/or prokaryotic cells and
5 those that remain episomal or may integrate into the host cell genome.
As used herein, operative linkage or operative association of
heterologous DNA to regulatory and effector sequences of nucleotides,
such as promoters, enhancers, transcriptional and translational stop sites,
and other signal sequences, refers to the functional relationship between
10 such DNA and such sequences of nucleotides. For example, operative
linkage of heterologous DNA to a promoter refers to the physical and
functional relationship between the DNA and the promoter such that the
transcription of such DNA is initiated from the promoter by an RNA
polymerase that specifically recognizes, binds to and transcribes the DNA in
15 reading frame.
As used herein, a promoter region refers to the portion of DNA of a
gene that controls transcription of DNA to which it is operatively linked. A
portion of the promoter region includes specific sequences of DNA that are
sufficient for RNA poiymerase recognition, binding and transcription
20 initiation. This portion of the promoter region is referred to as the
promoter. In addition, the promoter region includes sequences that
modulate this recognition, binding and transcription initiation activity of the
RNA polymerase. These sequences may be cis acting or may be
responsive to trans acting factors. Promoters, depending upon the nature
25 of the regulation, may be constitutive or regulated. For use herein,
inducible promoters are preferred. The promoters are recognized by an
- RNA polymerase that is expressed by the host. The RNA polymerase may
be endogenous to the host or may be introduced by genetic engineering
into the host, either as part of the host chromosome or on an episomal
30 element, including a plasmid containing the DNA encoding the saporin-
containing polypeptide. Most preferred promoters for use herein are tightly
WO 95/03831 PCT/US94/08511
41 -18-
regulated such that, absent induction, the DNA encoding the saporin-
containing protein is not expressed.
As used herein, a transcription terminator region has either (a) a
subsegment that encodes a polyadenylation signal and polyadenylation site
in the transcript, and/or (b) a subsegment that provides a transcription
termination signal that terminates transcription by the polymerase that
recognizes the selected promoter. The entire transcription terminator may
be obtained from a protein-encoding gene, which may be the same or
different from the gene, which is the source of the promoter. Preferred
transcription terminator regions are those that are functional in E. coli.
Transcription terminators are optional components of the expression
systems herein, but are employed in preferred embodiments.
As used herein, transfection refers to the taking up of DNA or RNA
by a host cell. Transformation refers to this process performed in a manner
such that the DNA is replicable, either as an extrachromosomal element or
as part of the chromosomal DNA of the host. Methods and means for
effecting transfection and transformation are well known to those of skill in
this art (see, e.~., Wigler et ak (1979) Proc. Natl. Acad. Sci. USA 76:1373-
1376; Cohen et ak (1972) Proc. Natl. Acad. Sci. USA 69:2110).
As used herein, the term biologically active, or reference to the
biological activity of a saporin-containing polypeptide or cytotoxicity of a
saporin-containing polypeptide, refers to the ability of such polypeptide to
inhibit protein synthesis by inactivation of ribosomes either in vivo or in
vitro or to inhibit the growth of or kill cells upon internalization of the
saporin-containing polypeptide by the cells. Preferred biologically active
saporin polypeptides are those that are toxic to eukaryotic cells upon
entering the cells. Such biological or cytotoxic activity may be assayed by
any method known to those of skill in the art including, but not limited to,
the in vitro assays that measure protein synthesis and in vivo assays that
assess cytotoxicity by measuring the effect of a test compound on cell
WO 9~;/03831 PCT/US94/08511
.
~1 6$6~1
-19-
proliferation or on protein synthesis. Particularly preferred, however, are
assays that assess cytotoxicity in targeted cells.
As used herein, FGF-mediated pathophysiological condition refers to
a deleterious condition characterized by or caused by proliferation of cells
5 that are sensitive to bFGF mitogenic stimulation. Basic FGF-mediated
pathophysiological conditions include, but are not limited to, certain
tumors, rheumatoid arthritis, restenosis, Dupuytren's Contracture and
certain complications of diabetes, such as proliferative retinopathy.
As used herein, substantially pure means sufficiently homogeneous
10 to appear free of readily detectable impurities as determined by standard
methods of analysis, such as thin layer chromatography (TLC), gel
electrophoresis, high performance liquid chromatography (HPLC), used by
those of skill in the art to assess such purity, or sufficiently pure such that
further purification would not detectably alter the physical and chemical
15 properties, such as enzymatic and biological activities, of the substance.
Methods for purification of the compounds to produce substantially
chemically pure compounds are known to those of skill in the art. A
substantially chemically pure compound may, however, be a mixture of
stereoisomers. In such instances, further purification might increase the
20 specific activity of the compound.
As used herein, isolated, substantially pure DNA refers to DNA
fragments purified according to standard techniques employed by those
skilled in the art (see, e.q., Maniatis et al. (1982) Molecular Cloninq: A
Laboratorv Manual, Cold Spring Harbor Laboratory Press, Cold Spring
25 Harbor, NY and Sambrook et al. (1989) Molecular Cloninq: A Laboratorv
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.).
- As used herein, to hybridize under conditions of a specified
stringency is used to describe the stability of hybrids formed between two
single-stranded DNA fragments and refers to the conditions of ionic
30 strength and temperature at which such hybrids are washed, following
annealing under conditions of stringency less than or equal to that of the
WO 95/03831 PCTIUS94/08511
ZO-
washing step. Typically high, medium and low stringency encompass the
following conditions or equivalent conditions thereto:
1 ) high stringency: 0.1 x SSPE or SSC, 0.1 % SDS, 65C
2) medium stringency: 0.2 x SSPE or SSC, 0.1% SDS, 50C
3) low stringency: 1.0 x SSPE or SSC, 0.1% SDS, 50C.
Equivalent conditions refer to conditions that select for substantially the
same percentage of mismatch in the resulting hybrids. Additions of
ingredients, such as formamide, Ficoll, and Denhardt's solution affect
parameters such as the temperature under which the hybridization should
be conducted and the rate of the reaction. Thus, hybridization in 5 X SSC,
in 20% formamide at 42 C is substantially the same as the conditions
recited above hybridization under conditions of low stringency. The recipes
for SSPE, SSC and Denhardt's and the preparation of deionized formamide
are described, for example, in Sambrook et al. (1989) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 8; see,
Sambrook et al., vol. 3, p. B.13, see, also, numerous catalogs that describe
commonly used laboratory solutions). SSPE is pH 7.4 phosphate-buffered
0.18 NaCI.
As used herein, expression refers to the process by which nucleic
acid is transcribed into mRNA and translated into peptides, polypeptides, or
proteins. If the nucleic acid is derived from genomic DNA, expression may,
if an appropriate eukaryotic host cell or organism is selected, include
splicing of the mRNA.
As used herein, "culture" means a propagation of cells in a medium
conducive to their growth, and all sub-cultures thereof. The term
"subculture" refers to a culture of cells grown from cells of another culture
(source culture), or any subculture of the source culture, regardless of the
number of subculturings that have been performed between the subculture
of interest and the source culture.
As used herein, reference to nucleic acids includes duplex DNA,
single-stranded DNA, RNA in any form, including triplex, duplex or single-
WO 95/03831 PCT/US94/08511
~1 6~
stranded RNA, anti-sense RNA, polynucleotides, oligonucleotides, single
nucleotides and derivatives thereof.
As used herein an effective amount of a compound for treating a
particular disease is an amount that is sufficient to ameliorate, or in some
manner reduce the symptoms associated with the disease. Such amount
may be administered as a single dosage or may be administered according
to a regimen, whereby it is effective. The amount may cure the disease
but, typically, is administered in order to ameliorate the symptoms of the
disease. Repeated administration may be required to achieve the desired
amelioration of symptoms.
As used herein, pharmaceutically acceptable salts, esters or other
derivatives of the conjugates include any salts, esters or derivatives that
may be readily prepared by those of skill in this art using known methods
for such derivatization and that produce compounds that may be
administered to animals or humans without substantial toxic effects and
that either are pharmaceutically active or are prodrugs.
As used herein, a prodrug is a compound that, upon in vivo
administration, is metabolized or otherwise converted to the biologically,
pharmaceutically or therapeutically active form of the compound. To
produce a prodrug, the pharmaceutically active compound is modified such
that the active compound will be regenerated by metabolic processes. The
prodrug may be designed to alter the metabolic stability or the transport
characteristics of a drug, to mask side effects or toxicity, to improve the
flavor of a drug or to alter other characteristics or properties of a drug. By
virtue of knowledge of pharmacodynamic processes and drug metabolism in
vivo, those of skill in this art, once a pharmaceutically active compound is
known, can design prodrugs of the compound (see, e.q., Nogrady (1985)
Medicinal ChemistrY A Biochemical APProach, Oxford University Press,
New York, pages 388-392).
As used herein, treatment means any manner in which the
symptoms of a condition, disorder or disease are ameliorated or otherwise
WO 95/03831 PCT/US94108511
~ b~
-22-
beneficially altered. Treatment also encompasses any pharmaceutical use
of the compositions herein.
As used herein, amelioration of the symptoms of a particular disorder
by administration of a particular pharmaceutical composition refers to any
5 lessening, whether permanent or temporary, lasting or transient that can be
attributed to or associated with administration of the composition.
As used herein, ED50 refers to the concentration at which 50% of
the cells are killed following incubation, generally for 72-hours or other
specified time period, with a toxin, such as FGF-SAP.
As used herein, lDsO refers to the concentration of saporin-containing
protein required to inhibit protein synthesis in treated cells to 50% of the
protein synthesis in the absence of the protein.
A. r~ lioll of poly~-e~ les and cytotoxic agents
1. Polypeptides reactive with an FGF receptor
Any polypeptide that is reactive with an FGF receptor may be used
in the methods herein. Members of the FGF peptide family, including FGF-
1 - FGF-9, are particularly preferred. Modification of the polypeptide may
be effected by any means known to those of skill in this art. The preferred
methods herein rely on modification of DNA encoding the polypeptide and
20 expression of the modified DNA.
DNA encoding the FGF polypeptide may be isolated, synthesized or
obtained from commercial sources (the amino acid sequences of FGF-1 -
FGF-9 are set forth in SEQ ID NOs. 24-32; DNA sequences may be based
on these amino acid sequences or may be those that are known to those of
25 skill in this art (see, e.q., DNA* (July, 1993 release from DNASTAR, Inc.
Madison, Wl); see, also U.S. Patent No. 4,956,455, U.S. Patent No.
5,126,323, U.S. Patent No. 5,155,217, U.S. Patent No. 4,868.113,
published International Application W0/90/08771 (and the corresponding
U.S. patent, upon its issuance), which is based on U.S. Application Serial
30 No. 07/304,281, filed January 31, 1989, and Miyamoto et al. (1993) Mol.
Cell. Biol. 13:4251-4259)) Expression of a recombinant bFGF protein in
WO 95/03831 PCT/US94/08511
~ ~1 6$6~1
yeast and E. coli is described in Barr et al., J. Biol. Chem.
263:16471-16478 (1988), in copending International PCT Application
Serial No. PCT/US93/05702 and co-pending United States Application
Serial No. 07/901,718. Expression of recombinant FGF proteins may be
5 performed as described herein; and DNA encoding FGF proteins may be
used as the starting materials for the methods herein.
Mutation may be effected by any method known to those of skill in
the art, including site-specific or site-directed mutagenesis of DNA encoding
the protein and the use of DNA amplification methods using primers to
10 introduce and amplify alterations in the DNA template. Site-specific
mutagenesis is typically effected using a phage vector that has single- and
double-stranded forms, such as M13 phage vectors, which are well-known
and commercially available. Other suitable vectors that contain a single-
stranded phage origin of replication may be used (see, e.~., Veira et al.
15 (1987) Meth. Enzymol. 15:3). In general, site-directed mutagenesis is
performed by preparing a single-stranded vector that encodes the protein of
interest (I.e., a member of the FGF family or a cytotoxic molecule, such as
a saporin). An oligonucleotide primer that contains the desired mutation
within a region of homology to the DNA in the single-stranded vector is
20 annealed to the vector followed by addition of a DNA polymerase, such as
E. coli polymerase I Klenow fragment, which uses the double stranded
region as a primer to produce a heteroduplex in which one strand encodes
the altered sequence and the other the original sequence. The
heteroduplex is introduced into appropriate bacterial cells and clones that
25 include the desired mutation are selected. The resulting altered DNA
molecules may be expressed recombinantly in appropriate host cells to
produce the modified protein.
Suitable conservative substitutions of amino acids are known to
those of skill in this art and may be made generally without altering the
30 biological activity of the resulting molecule. Those of skill in this art
recognize that, in general, single amino acid substitutions in non-essential
WO 95/03831 PCT/US94/08511
24-
regions of a polypeptide do not substantially alter biological activity (see,
e.q., Watson et ai. Molecular Biology of the Gene, 4th Edition, 1987, The
Bejacmin/Cummings Pub. co., p.224).
Such substitutions are preferably made in accordance with those set
5forth in TABLE 1 as follows:
TABLE 1
Original residue C~ 5l;
Ala (A) Gly; Ser
Arg (R) Lys
10Asn (N) Gln; His
Cys (C) Ser
Gln (Q) Asn
Glu (E) Asp
Gly (G) Ala; Pro
15His (H) Asn; Gln
lle (I) Leu; Val
Leu (L) lle; Val
Lys (K) Arg; Gln; Glu
Met (M) Leu; Tyr; lle
20Phe (F) Met; Leu; Tyr
Ser (S) Thr
Thr (T) Ser
Trp (W) Tyr
Tyr (Y) Trp; Phe
25Val (V) lle; Leu
Other substitutions are also permissible and may be determined empirically
or in accord with known conservative substitutions.
2. The cytotoxic agent
Saporin and other ribosome inactivating proteins (RlPs) are the
preferred cytotoxic agent for use herein. Any cytotoxic agent that, when
internalized inhibits or destroys cell growth, cell proliferation or other
essential cell functions may be used herein. Such cytotoxic agents are
considered to be functionally equivalent to the RlPs described herein, and
include, but are not limited to, saporin, the ricins, abrin and other RlPs,
Pseudomonas exotoxin, inhibitors of DNA, RNA or protein synthesis or
other metabolic inhibitors that are known to those of skill in this art.
Saporin is preferred, but other suitable RlPs include, but are not limited to,
ricin, ricin A chain, maize RIP, gelonin, diphtheria toxin, diphtheria toxin A
WO 95/03831 PCT/US94/08511
~l 6~6~7
-25-
chain, trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis
antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin, bryodin,
shiga and others known to those of skill in this art (see, e.q., Barbieri et al.(1982) Cancer SurveYs 1:489-520 and European published patent
5 application No. 0466 222, incorporated herein by reference, which provide
lists of numerous RlPs and their sources; see, also, U.S. Patent No.
5,248,608 to Walsh et al., which provides a RIP from maize).
The selected cytotoxic agent is, if necessary, derivatized to produce
a group reactive with a cysteine on the selected FGF. If derivatization
10 results in a mixture of reactive species, a mono-derivatized form of the
cytotoxic agent is isolated and is then conjugated to the mutated FGF.
a. lsolalio" of s&,.oli., and DNA encoding s&"or;"
Saporin is preferred herein. The saporin polypeptides include any of
the isoforms of saporin that may be isolated from SaPonaria officinalis or
15 related species or modified form that retain cytotoxic activity. In particular,
such modified saporin may be produced by modifying the DNA encoding
the protein ~see, e.~., International PCT Application Serial No.
PCT/US93/05702, filed on June 14, 1993, which is a continuation-in-part
of United States Application Serial No. 07/901,718; see, also, copending
20 U.S. Patent Application No. 07/885,242 filed May 20, 1992, and Patent
No. 1231914, granted in Italy on January 15, 1992) by altering one or
more amino acids or deleting or inserting one or more amino acids, such as
a cysteine that may render it easier to conjugate to FGF or other cell
surface binding protein. Any such protein, or portion thereof, that, when
25 conjugated to FGF as described herein, that exhibits cytotoxicity in
standard in vitro or in vivo assays within at least about an order of
magnitude of the saporin conjugates described herein is contemplated for
use herein.
Thus, the SAP used herein includes any protein that is isolated from
30 natural sources or that is produced by recombinant expression (see, e.~.,
copending International PCT Application Serial No. PCT/US93/05702, filed
WO 95/03831 PCT/US94/08511
~ ~864~
-26-
on June 14, 1993, which is a continuation-in-part of United States
Application Serial No. 07/901,718, filed June 16, 1992; see, also Example
1, below).
DNA encoding SAP or any cytotoxic agent may be used in the
5 recombinant methods provided herein. In instances in which the cytotoxic
agent does not contain a cysteine residue, such as instances in which DNA
encoding SAP is selected, the DNA may be modified to include cysteine
codon. The codon may be inserted into any locus that does not reduce or
reduces by less than about one order of magnitude the cytotoxicity of the
10 resulting protein may be selected. Such locus may be determined
empirically by modifying the protein and testing it for cytotoxicity in an
assay, such as a cell-free protein synthesis assay. The preferred loci in
SAP for insertion of the cysteine residue is at or near the N-terminus
(within about 10 residues of the N-terminus).
b. Host cells for e~.,u.essior. of saporin containing
polypeptides
Host organisms include those organisms in which recombinant
production of heterologous proteins have been carried out, such as, but not
limited to, bacteria (for example, E. coli), yeast (for example, Saccharo-
20 myces cerevisiae and Pichia pastoris), mammalian cells, insect cells.Presently preferred host organisms are strains of bacteria. Most preferred
host organisms are strains of E. coli.
c. Methods for reco,-lLi..ant production of saporin
The DNA encoding the cytotoxic agent, such as saporin protein, is
25 introduced into a plasmid in operative linkage to an appropriate promoter
for expression of polypeptides in a selected host organism. The presently
preferred saporin proteins are saporin proteins that have been modified by
addition of a Cys residue or replacement of a non-essential residue at or
near the amino- or carboxyl terminus of the saporin with Cys. Saporin,
3Q such as that of SEQ ID N0. 7 has been modified by insertion of Met-Cys
residue at the N-terminus and has also been modified by replacement of the
WO 95/03831 PCT/US94/08511
~1 6 8G47
-27-
Asn or lle residue at positions 4 and 10, respectively (see EXAMPLE 4).
The DNA fragment encoding the saporin may also include a protein
secretion signal that functions in the selected host to direct the mature
polypeptide into the periplasm or culture medium. The resulting saporin
5 protein can be purified by methods routinely used in the art, including,
methods described hereinafter in the Examples.
Methods of transforming suitable host cells, preferably bacterial
cells, and more preferably E. coli cells, as well as methods applicable for
culturing said cells containing a gene encoding a heterologous protein, are
10 generally known in the art. See, for example, Sambrook et al. (1989)
Molecular Cloninq: A LaboratorY Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
The DNA construct encoding the saporin protein is introduced into
the host cell by any suitable means, including, but not limited to
15 transformation employing plasmids, viral, or bacterial phage vectors,
transfection, electroporation, lipofection, and the like. The heterologous
DNA can optionally include sequences, such as origins of replication that
allow for the extrachromosomal maintenance of the saporin-containing
plasmid, or can be designed to integrate into the genome of the host ~as an
20 alternative means to ensure stable maintenance in the host).
Positive transformants can be characterized by Southern blot
analysis (Sambrook et al. (1989) Molecular Cloninq: A Laboratorv Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) for the site
of DNA integration; Northern blots for inducible-promoter-responsive
25 saporin gene expression; and product analysis for the presence of saporin-
containing proteins in either the cytoplasm, periplasm, or the growth media.
Once the saporin-encoding DNA fragment has been introduced into
the host cell, the desired saporin-containing protein is produced by
subjecting the host cell to conditions under which the promoter is induced,
30 whereby the operatively linked DNA is transcribed. In a preferred
embodiment, such conditions are those that induce expression from the E.
WO 95/03831 PCT/US94/08511~
a-~ b~41
-28 -
coli lac operon. The plasmid containing the DNA encoding the saporin-
containing protein also includes the lac operator (O) region within the
promoter and may also include the lac I gene encoding the lac repressor
protein (see, e.~., Muller-Hill et al. (1968) Proc. Natl. Acad. Sci. USA
59:1259-12649). The lac repressor represses the expression from the lac
promoter until induced by the addition of IPTG in an amount sufficient to
induce transcription of the DNA encoding the saporin-containing protein.
The expression of saporin in E. coli is, thus accomplished in a two-
stage process. In the first stage, a culture of transformed E. coli cells is
grown under conditions in which the expression of the saporin-containing
protein within the transforming plasmid, preferably a encoding a saporin,
such as described in Example 4, is repressed by virtue of the lac repressor.
In this stage cell density increases. When an optimum density is reached,
the second stage commences by addition of IPTG, which prevents binding
of repressor to the operator thereby inducing the lac promoter and
transcription of the saporin-encoding DNA.
In a preferred embodiment, the promoter is the T7 RNA polymerase
promoter, which may be linked to the lac operator and the E. coli host
strain includes DNA encoding T7 RNA polymerase operably linked to the lac
operator and a promoter, preferably the lacUV5 promoter. The presently
preferred plasmid is pET 11a (NOVAGEN, Madison, Wl), which contains the
T71ac promoter, T7 terminator, the inducible E. coli lac operator, and the
lac repressor gene. The plasmid pET 1 5b (NOVAGEN, Madison, Wl), which
contains a His-TagTM leader sequence (Seq. ID No. 36) for use in
purification with a His column and a thrombin cleavage site that permits
cleavage following purification over the column, the T7-lac promoter region
and the T7 terminator, has been used herein for expression of saporin.
Addition of IPTG induces expression of the T7 RNA polymerase and the T7
promoter, which is recognized by the T7 RNA polymerase.
Transformed strains, which are of the desired phenotype and
genotype, are grown in fermentors by suitable methods well known in the
WO 95tO3831 PCT/US94/08511
~1 ~8~7
-29 -
art. In the first, or growth stage, expression hosts are cultured in defined
minimal medium lacking the inducing condition, preferably IPTG. When
grown in such conditions, heterologous gene expression is completely
repressed, which allows the generation of cell mass in the absence of
5 heterologous protein expression. Subsequent to the period of growth under
repression of heterologous gene expression, the inducer, preferably IPTG, is
added to the fermentation broth, thereby inducing expression of any DNA
operatively linked to an IPTG-responsive promoter (a promoter region that
contains lac operator). This last stage is the induction stage.
The resulting saporin-containing protein can be suitably isolated from
the other fermentation products by methods routinely used in the art, e.q.,
using a suitable affinity column as described in Example 1.E-F and 2.D;
precipitation with ammonium sulfate; gel filtration; chromatography,
preparative flat-bed iso-electric focusing; gel electrophoresis, high
15 performance liquid chromatography (HPLC); and the like. A method for
isolating saporin is provided in EXAMPLE 1 (see, also Lappi et aL (1985)
Biochem . BioPhYs. Res. Commun. 1 29:934-942) . The expressed saporin
protein is isolated from either the cytoplasm, periplasm, or the cell culture
medium (see, discussion below B.1.b below and see, e.g., EXAMPLE 4 for
20 preferred methods and saporin proteins).
3. rlasl" -'- for e,cl"ession of the FGF peptide, the cytotoxic
agent and FGF peptide-cytotoxic agent chimeras
The DNA construct is introduced into a plasmid for expression in a
desired host. In preferred embodiments, the host is a bacterial host.
25 The sequences of nucleotides in the plasmids that are regulatory regions,
such as promoters and operators, are operationally associated with one
another for transcription of the sequence of nucleotides that encode a
saporin-containing protein. The sequence of nucleotides encoding the
- saporin-containing protein may also include DNA encoding a secretion30 signal, whereby the resulting peptide is a precursor of saporin. The
resulting processed saporin protein, which if not processed such that the
WO 95/OJ831 PCT/US94/08~ill
G`~ -30-
resulting protein is identical to a native saporin, retains the cytotoXic
activity of the native saporin protein, may be recovered from the
periplasmic space or the fermentation medium.
In preferred embodiments the DNA plasmids also include a
5 transcription terminator sequence. The promoter regions and transcription
terminators are each independently selected from the same or different
genes.
The plasmids used herein preferably include a promoter in operable
association with the DNA encoding the saporin-containing protein and are
10 designed for expression of proteins in a bacterial host. It has been found
that tightly regulatable promoters are preferred for expression of saporin.
Suitable promoters for expression of saporin-containing proteins are widely
available and are well known in the art. Inducible promoters or constitutive
promoters that are linked to regulatory regions are preferred. Such
15 promoters include, but are not limited to, the T7 phage promoter and other
T7-like phage promoters, such as the T3, T5 and SP6 promoters, the trp,
Ipp, and lac promoters, such as the lacUV5, from E. coli; the P10 or
polyhedron gene promoter of baculovirus/insect cell expression systems
and inducible promoters from other eukaryotic expression systems. For
20 expression of the saporin-containing proteins such promoters are inserted in
a plasmid in operative linkage with a control region such as the lac operon.
Preferred promoter regions are those that are inducible and
functional in E. coli. Examples of suitable inducible promoters and
promoter regions include, but are not limited to: the E. coii lac operator
25 responsive to isopropyl ,B-D-thiogalactopyranoside (IPTG; see, et al.
Nakamura et ak (1979) Cell 18:1109-1117); the metallothionein promoter
metal-regulatory-elements responsive to heavy-metal (e.q., zinc) induction
(see, e.~., U.S. Patent No. 4,870,009 to Evans et ak); and the phage
T71ac promoter responsive to IPTG (see, e.q., U.S. Patent No. 4,952,496;
30 and Studier et ak (1990) Meth. Enzvmol. 185:60-89).
WO 95/03831 PCT/US94/08511
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-31 -
The plasmids also preferably include a selectable marker gene or
genes that are functional in the host. A selectable marker gene includes
any gene that confers a phenotype on bacteria that allows transformed
bacterial cells to be identified and selectively grown from among a vast
majority of untransformed cells. Suitable selectable marker genes for
bacterial hosts, for example, include the ampicil!in resistance gene (Ampr),
tetracycline resistance gene (Tcr) and the kanamycin resistance gene (Kanr).
The kanamycin resistance gene is presently preferred.
The preferred plasmids also include DNA encoding a signal for
secretion of the operably saporin-containing protein. Secretion signals
suitable for use are widely available and are well known in the art.
Prokaryotic and eukaryotic secretion signals functional in E. coli may be
employed. The presently preferred secretion signals include, but are not
limited to, those encoded by the following E. coii genes: ompA, ompT,
ompF, ompC, beta-lactamase, and alkaline phosphatase, and the like (von
Heijne (1985) J. Mol. Biol. 184:99-105). In addition, the bacterial pelB
gene secretion signal (Lei Q ai. (1987) J. Bacteriol. 169:4379), the phoA
secretion signal, and the cek2 functional in insect cell may be employed.
The most preferred secretion signal is the E. coli ompA secretion signal.
Other prokaryotic and eukaryotic secretion signals known to those of skill
in the art may also be employed (see, e.q., von Heijne (1985) J. Mol. Biol.
184:99-105). Using the methods described herein, one of skill in the art
can substitute secretion signals that are functional in either yeast, insect or
mammalian cells to secrete saporin-containing proteins from those cells.
Particularly preferred plasmids for transformation of E. coli cells
include the pET expression vectors (see, U.S patent 4,952,496; available
from NOVAGEN, Madison, Wl). Such plasmids include pET 11 a, which
contains the T71ac promoter, T7 terminator, the inducible E. coli lac
operator, and the lac repressor gene; pET 12a-c, which contains the T7
promoter, T7 terminator, and the E. coli ompT secretion signal; and pET
15b (NOVAGEN, Madison, Wl), which contains a His-TagTM leader
WO 95103831 PCTrUS94t08511~
~l ~ 8~1
-32-
sequence (Seq. ID No. 36) for use in purification with a His column and a
thrombin cleavage site that permits cleavage following purification over the
column; the T7-lac promoter region and the T7 terminator.
Other preferred plasmids include the pKK plasmids, particularly pKK
5 223-3, which contains the TAC promoter, (available from Pharmacia; see
also, Brosius et al. (1984) Proc.. Natl. Acad. Sci. 81:6929; Ausubel et al.,
Current Protocols in Molecular Biology; U.S. Patent Nos. 5,122,463,
5,173,403, 5,187,153, 5,204,254, 5,212,058, 5,212,286, 5,215,907,
5,220,013, 5,223,483, and 5,229,279), which contain the TAC promoter.
10 Plasmid pKK has been modified by disruption of the ampicillin resistance
marker gene by digestion with Scal and insertion of a kanamycin resistance
cassette (purchased from Pharmacia; obtained from pUC4K, see, e.q.,
Vieira et al. (1982) Gene 19:259-268; and U.S. Patent No. 4,719,179) cut
with Hincll to remove the EcoRI sticky ends and produce blunt ends.
15 Baculovirus vectors, such as a pBlueBac (also called pJVETL and
derivatives thereof) vector, particularly pBlueBac lll, (see, e.~., 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) may
also be used for expression of the polypeptides in insect cells. The
20 pBlueBaclll vector is a dual promoter vector and provides for the selection
of recombinants by blue/white screening as this plasmid contains the ,~-
galactosidase gene (lacZ) under the control of the insect recognizable ETL
promoter and is inducible with IPTG. A DNA construct is inserted into a
baculovirus vector pBluebac lll (INVITROGEN, San Diego, CA) and then co-
25 transfected with wild type virus into insect cells SPodoPtera fru~iPerda (sf9cells; see, e.~., Luckow et al. (1988) Bio/technolo~v 6:47-55 and U.S.
Patent No. 4,745,051).
Other plasmids include the plN-lllompA plasmids (see, U.S. Patent
No. 4,575,013 to Inouye; see, also, Duffaud et ai. (1987) Meth. Enz.
30 153:492-507), such as plN-lllompA2 . The plN-lllompA plasmids include an
insertion site for the heterologous DNA (the DNA encoding a saporin-
= -- :
WO 95103831 PCT/US94/08511
.
33 l G~6~7
containing protein) linlced for transcriptional expression in reading phasewith four functional fragments derived from the lipoprotein gene of E. coli.
The plasmids also include a DNA fragment coding for the signal peptide of
the ompA protein of E. coli, positioned such that the desired polypeptide is
5 expressed with the ompA signal peptide at its amino terminus, thereby
allowing efficient secretion across the cytoplasmic membrane. The
plasmids further include DNA encoding a specific segment of the E. coli lac
promoter-operator, which is positioned in the proper orientation for
transcriptional expression of the desired polypeptide, as well as a separate
10 functional E. coli lacl gene encoding the associated repressor molecule that, in the absence of lac operon inducer, interacts with the lac
promoter-operator to prevent transcription therefrom. Expression of the
desired polypeptide is under the control of the lipoprotein (Ipp) promoter
and the lac promoter-operator, although transcription from either promoter
15 is normally blocked by the repressor molecule. The repressor is selectively
inactivated by means of an inducer molecule thereby inducing
transcriptional expression of the desired polypeptide from both promoters.
In a preferred embodiment, the DNA fragment is replicated in
bacterial cells, preferably in E. coii. The preferred DNA fragment also
20 includes a bacterial origin of replication, to ensure the maintenance of the
DNA fragment from generation to generation of the bacteria. In this way,
large quantities of the DNA fragment can be produced by replication in
bacteria. Preferred bacterial origins of replication include, but are not
limited to, the f1-ori and col E1 origins of replication. Preferred hosts
25 contain chromosomal copies of DNA encoding T7 RNA polymerase operably
linked to an inducible promoter, such as the lacUV promoter (see, U.S.
Patent No. 4,952,496). Such hosts include, but are not limited to,
Iysogens E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS,
HMS174(DE3) and BL21(DE3). Strain BL21(DE3) is preferred. The pLys
30 strains provide low levels of T7 Iysozyme, a natural inhibitor of T7 RNA
polymerase.
WO 95/03831 PCT/US94/08~11
.
J2,~ b8~ 34-
The DNA fragments provided may also contain a gene coding for a
repressor-protein. The repressor-protein is capable of repressing the
transcription of a promoter that contains sequences of nucleotides to which
the repressor-protein binds. The promoter can be derepressed by altering
5 the physiological conditions of the cell. The alteration can be accomplished
by the addition to the growth medium of a molecule that inhibits, for
example, the ability to interact with the operator or with regulatory proteins
or other regions of the DNA or by altering the temperature of the growth
media. Preferred repressor-proteins include, but are not limited to, the E.
10 coli. Iacl repressor responsive to IPTG induction, the temperature sensitive
c1857 repressor, and the like. The E. coli lacl repressor is preferred.
DNA encoding full-length bFGF or the bFGF muteins has been linked
to DNA encoding the mature saporin protein and introduced into the pET
vectors, including pET-11a and pET-12a expression vectors (NOVAGEN,
15 Madison, Wl), for intracellular and periplasmic expression, respectively, of
FGF-SAP fusion proteins. The resulting fusion proteins exhibit cytotoxic
activity and appear to be at least as potent as the chemically conjugated
FGF-SAP preparations. The resulting bFGF-fusion proteins are highly
cytotoxic when internalized by targeted cells.
20 B. Synthesis of l"ol,ogenous prep3raliGI,s of cytotoxic conjug~tes and
homogeneous populaliGI.s of cytotoxic conjugates
The problem of heterogeneity of compositions and preparations of
cytotoxic FGF conjugates has been addressed in several ways herein. The
first method relies on chemical conjugation and the second method relies
25 on recombinant DNA technology. The methods herein are described with
respect to bFGF and SAP. It is understood, however, that the same
methods may be used to modify and prepare homogeneous populations of
conjugates of any member of the FGF family with SAP, modified SAP, or
any other cytotoxic agent.
WO 95103831 PCT/US94108511
~I G~G~7
-35-
1. Chemical conjugation
To effect chemical conjugation herein, the FGF protein is modified
and then linked to the cytotoxic agent. Chemical conjugation must be used
if the cytotoxic agent is other than a peptide or protein, such as a non-
peptide drug.
a. Selection of the FGF protein
To reduce the heterogeneity of preparations of FGF protein-
containing chemical conjugates, the FGF protein is modified by deleting or
replacing a site(s) on the FGF that causes the heterogeneity. Such sites are
typically cysteine residues that, upon folding of the protein, remain
available for interaction with other cysteines or for interaction with more
than one cytotoxic molecule per molecule of FGF peptide. Thus, such
cysteine residues do not include any cysteine residue that are required for
proper folding of the FGF peptide or for retention of the ability to bind to an
FGF receptor and internalize. For chemical conjugation, one cysteine
residue that, in physiological conditions, is available for interaction, is not
replaced because it is used as the site for linking the cytotoxic moiety. The
resulting modified FGF is conjugated with a single species of cytotoxic
conjugate.
Any protein that is reactive with an FGF receptor may be used
herein. In particular any of FGF-1 - FGF-9 may be modified for use herein
or reacted with a cytotoxic reagent, such that the resulting conjugate is
monogenous. FGF-1 has cysteines at positions 31, 98 and 132; FGF-2 has
cysteines at positions 34, 78, 96 and 101; FGF-3 has cysteines at
positions 50 and 115; FGF-4 has cysteines at positions 88 and 155; FGF-5
has cysteines at positions 19, 93, 160 and 202; FGF-6 has cysteines at
positions 80 and 147; FGF-7 has cysteines at positions 18, 23, 32, 46, 71,
133 and 137; FGF-8 has cysteines at positions 10, 19, 109 and 127; and
FGF-9 has cysteines at positions 68 and 134. The cysteine residues from
each of FGF-1 - FGF-9 that appear to be essential for retention of biological
activity and that should not deleted or replaced are as follows:
WO 95/03831 PCT/US94/08511
.
36-
TABLE 2
FG F- 1 cys93
FGF-2 cyslol
FG F-3 CyS l l 5
FGF-4 cysl55
FGF-5 cys'60
FGF-6 cysl47
FGF-7 cysl37
FGF-8 cysl27
FGF-9 cysl34
The FGF peptides may be modified as described below.
Alternatively, the contribution of each cysteine to the ability to bind to FGF
receptors may be determined empirically. Each cysteine residue may be
systematically replaced with a conservative amino acid change (see Table
15 1, above) or deleted. The resulting mutein is tested for the requisite
biological activity, the ability to bind to FGF receptors and internalize linkedcytotoxic moieties. If the mutein retains this activity, then the cysteine
residue is not required. Additional cysteines are systematically deleted and
replaced and the resulting muteins are tested for activity. In this manner
20 the minimum number and identity of the cysteines needed to retain the
ability to bind to an FGF receptor and internalize may be determined.
For each FGF peptide, the complete amino acid sequence is known
(see, e.q., SEQ ID N0. 24 (FGF-1) and SEQ ID NOs. 26-32 (FGF-3 - FGF-9,
respectively)). The sequence is examined and cysteine residues are
Z5 identified. Comparison among the amino acid sequences of FGF-1 -FGF-9
reveals that one Cys is conserved among FGF family of peptides (see Table
2). These cysteine residues may be required for secondary structure and
should be altered. These residues should not be replaced. Each of the
remaining cysteine residues may be systematically deleted and/or replaced
30 by a serine residue or other residue that would not be expected to alter the
structure of the protein. The resulting peptide is tested for biological
activity. If the cysteine residue is necessary for retention of biological
activity it is not deleted; if it is not necessary, then it is preferably replaced
WO 95/03831 PCT/US94/08511
.
6~6 ,~1
-37-
with a serine or other residue selected so that it does not alter the
secondary structure of the resulting protein.
b. Mo~liricalion of the FGF protein for chemical
conjugation
The polypeptide reactive with an FGF receptor is modified by
removing one or more reactive cysteines that are not required for receptor
binding, but that are available for reaction with appropriately derivatized
cytotoxic agent, so that the resulting FGF protein has only one cysteine
residue available for conjugation with the cytotoxic agent. Other cysteine
residues are removed and, preferably, replaced with an amino acid that
does not substantially alter the biological activity of the resulting mutant
FGF. The resulting mutant FGF is then tested for retention of the ability to
target a cytotoxic agent to a cell that expresses an FGF receptor and to
internalize the cytotoxic agent into such cells. Retention of proliferative
activity is indicative, though not definitive, of the retention of such
activities. Proliferative activity may be measured by any suitable
proliferation assay, such as the assay, exemplified below, that measures
the increase in cell number of adrenal capillary endothelial cells. It is noted,however, that modified or mutant FGFs may exhibit reduced or no prolifera-
tive activity, but may be suitable for use herein, if they retain the ability totarget a linked cytotoxic agent to cells bearing receptors to which the
unmodified FGF binds and result in internalization of the cytotoxic moiety.
Since FGF-3, FGF-4 and FGF-6 have only two cysteines, for
purposes of chemical conjugation, preferably neither cysteine is deleted or
replaced, unless another residue, preferably one near either terminus, is
replaced with a cysteine. With respect to the other FGF family members,
at least one cysteine must remain available for conjugation with the
cytotoxic conjugate and probably two cysteines, but at least the cysteine
residues set forth in Table 2. A second cysteine may be required to form a
disulfide bond. Thus, any FGF peptide that has more than three cysteines
is be modified for chemical conjugation by deleting or replacing the other
WO 95/03831 PCT/U.,, 1/Q8~11
6~1 -38-
cysteine residues. FGF peptides that have three cysteine residues are
modified by elimination of one cysteine, conjugated to a cytotoxic moiety
and tested for the ability to bind to FGF receptors and internalize the
cytotoxic moiety.
In accord with the methods herein, two muteins of basic FGF for
chemical conjugation have been produced (preparation of muteins for
recombinant expression of the conjugate is described below). DNA,
obtained from pFC80 (see, copending International PCT Application Serial
No. PCT/US93/05702, which is a continuation-in-part of United States
Application Serial No. 07/901,718; see also, SEQ ID N0. 12) encoding
basic FGF has been mutagenized. Mutagenesis of cysteine 78 of basic FGF
to serine ([C78S]FGF) or cysteine 96 to serine ([C96S]FGF) produced two
mutants that retain virtually complete proliferative activity of native basic
FGF as judged by the ability to stimulate endothelial cell proliferation in
culture. The activities of the two mutants and the native protein do not
significantly differ as assessed by efficacy or maximal response. Sequence
analysis of the modified DNA verified that each of the mutants has one
codon for cysteine converted to that for serine.
The resulting mutein FGF or unmodified FGF is reacted with a single
species of cytotoxic agent. The bFGF muteins have been reacted with a
single species of derivatized saporin (mono-derivatized saporin) thereby
resulting in monogenous preparations of FGF-SAP conjugates and
homogeneous compositions of FGF-SAP chemical conjugates. The
resulting chemical conjugate does not aggregate and retains the requisite
biological activities.
c. Preparation of saporin
SOIal;GII of mono-derivatized SAP.
For chemical conjugation, the SAP may be derivatized or modified
such that it includes a cysteine residue for conjugation to the FGF protein.
Typically, SAP is derivatized by reaction with SPDP. This results in a
heterogeneous population. For example, SAP that is derivatized by SPDP
WO 95/03831 PCT/US94/08511
G~
-39-
to a level of 0.9 moles pyridine-disulfide per mole of SAP includes a
population of non-derivatized, mono-derivatized and di-derivatized SAP.
Ribosome-inactivating proteins, which are overly derivatized with SPDP,
may lose activity because of reaction with sensitive Iysines (Lambert et al.
5 (1988) Cancer Treat. Res. 37:175-209). The quantity of non-derivatized
SAP in the preparation of the non-purified material can be difficult to judge
and this may lead to errors in being able to estimate the correct proportion
of derivatized SAP to add to the reaction mixture.
Because of the removal of a negative charge by the reaction of SPDP
10 with Iysine, the three species, however, have a charge difference. The
methods herein rely on this charge difference for purification of mono-
derivatized SAP by Mono S cation exchange chromatography. The use of
purified mono-derivatized SAP has distinct advantages over the non-purified
material. The amount of basic FGF that can react with SAP is limited to
15 one molecule with the mono-derivatized material, and it is seen in the
results presented herein that a more homogeneous conjugate is produced.
There are still sources of heterogeneity with the mono-derivatized SAP used
here. Native SAP as purified from the seed itself is a mixture of four
isoforms, as judged by protein sequencing (see, e.q., International PCT
20 Application Serial No. PCT/US93/05702 and copending United States
Application Serial No. 07/901,718; see also, Montecucchi et al. (1989) Int.
J. Pe~t. Prot. Res. 33:263-267; Maras et al. (1990) Biochem. Internat.
21:631-638; and Barra et al. (1991) Biotechnol. APPI. Biochem. 13:48 53).
This creates some heterogeneity in the conjugates, since the reaction with
25 SPDP probably occurs equally with each isoform This source of
heterogeneity can be overcome, for example, by use of SAP expressed in
E. coli.
(2) Recombinant expression of saporin
DNA provided herein includes a sequence of nucleotides encoding a
30 saporin polypeptide and an N-terminal extension sequence linked to the
amino terminus of the saporin. The N-terminal extension permits
WO 95/03831 PCT/US94/08511
a~41 40
expression of saporin in a bacterial host. If saporin is linked to DNA
encoding an FGF peptide, then the N-terminal extension is not necessary,
but may be included and contain from about one up to 20-30 amino acid
residues or more, if desired, and as long as the resulting saporin peptide
5 retains cytotoxic activity.
Suitable N-terminal extension regions may be substantially neutral
and lack any biological function other than rendering the saporin
polypeptide nontoxic or less toxic to the host in which it is expressed. The
specific amino acid makeup of the N-terminal extension region does not
10 appear to be critical for rendering the saporin-containing protein nontoxic or
less toxic to the host upon expression of the protein.
In a preferred embodiment, the N-terminal extension region is
susceptible to cleavage by eukaryotic intracellular proteases, either by
general intracellular degradation or by site-specific proteolytic processing of
15 a proteolytic signal sequence such that, upon internalization, the N-terminalextension region of the saporin-containing fusion protein is cleaved or
degraded by a cellular eukaryotic protease, which renders the
single-fragment saporin protein biologically active, resulting in cell death
(see, e.g., copending U.S. Application 08/ , , filed concurrently
20 herewith).
The DNA molecules provided herein encode saporin that has
substantially the same amino acid sequence and ribosome-inactivating
activity as that of saporin-6 (S0-6), including any of four isoforms, which
have heterogeneity at amino acid positions 48 and 91 ~see, e.a., Maras et
25 al. (1990) Biochem. Internat. 21:631-638 and Barra et ai. (1991)
Biotechnol. APPI. Biochem. 13:48-53 and SEQ ID NOs. 3-7). Other
suitable saporin polypeptides include other members of the multi-gene
family coding for isoforms of saporin-type RlP's including S0-1 and S0-3
(Fordham-Skelton et al. (1990) Mol. Gen. Genet. 221:134-138), S0-2
30 (see, e.g., U.S. Application Serial No. 07/885,242, which corresponds to
GB 2,216,891; see, also, Fordham-Skelton et al. (1991) Mol. Gen. Genet.
WO 95/03831 PCT/US94/08511
-
~ ~1 6 ~
-41 -
229:460-466), S0-4 (see, e.a., GB 2,194,241 B; see, also, Lappi et al.
(1985) Biochem. BioPhYs. Res. Commun. 129:934-942) and S0-5 (see,
e.~., GB 2,194,241 B; see, also, Montecucchi et al. (1989) Int. J. Pe~tide
Protein Res. 33:263-267). S0-4, which includes the N-terminal 40 amino
5 acids set forth in SEQ ID N0. 33, is isolated from the leaves of SaPonaria
officinalis by extraction with 0.1 M phosphate buffer at pH 7, followed by
dialysis of the supernatant against sodium borate buffer, pH 9, and
selective elution from a negatively charged ion exchange resin, such as
Mono S (Pharmacia Fine Chemicals, Sweden) using gradient of 1 to 0.3 M.
10 NaCI and first eluting chromatographic fraction that has SAP activity. The
second eluting fraction is S0-5.
The saporin polypeptides exemplified herein include those having
substantially the sam,e amino acid sequence as those listed in SEQ ID NOs
3-7. The isolation and expression of the DNA encoding these proteins is
15 described in Example 1.
(3) MG~liric~liol) of sal~G,i,.
Because more than one amino group on SAP may react with the
succinimidyl moiety, it is possible that more than one amino group on the
surface of the protein is reactive. This creates the potential for
20 heterogeneity in the mono-derivatized SAP. This source of heterogeneity
has been solved by the conjugating modified SAP expressed in E. coli that
has an additional cysteine inserted, as described above, in the coding
sequence.
Thus, in other embodiments, instead of derivatizing saporin to
25 introduce a sulfhydryl, the saporin can be modified by the introduction of a
cysteine residue into the SAP such that the resulting modified saporin
protein reacts with the FGF protein to produce a monogenous cytotoxic
conjugate that binds to FGF receptors on eukaryotic cells and is cytotoxic
upon internalization by such cells. Preferred loci for introduction of a
30 cysteine residue include the N-terminus region, preferably within about one
to twenty residues from the N-terminus of the cytotoxic agent, such as
WO 95/03831 PCT/US94/08511
.
41 -42-
SAP. For expression of SAP in the bacterial host systems herein, it is also
desirable to add DNA encoding a methionine linked to the DNA encoding
the N-terminus of the saporin protein. DNA encoding SAP has been
modified by inserting a DNA encoding Met-Cys (ATG TGT or ATG TGC) at
5 the N-terminus immediately adjacent to the codon for first residue of the
mature protein.
Muteins in which a cysteine residue has been added at the N-
terminus and muteins in which the amino acid at position 4 or 10 has been
replaced with cysteine have been prepared by modifying the DNA encoding
10 saporin (see, EXAMPLE 4). The modified DNA may be expressed and the
resulting saporin protein purified, as described herein for expression and
purification of the resulting SAP. The modified saporin can then be reacted
with the modified FGF to form disulfide linkages between the single
exposed cysteine residue on the FGF and the cysteine residue on the
15 modified SAP.
The modified DNA may be expressed and the resulting saporin
protein purified, as described herein for expression and purification of the
resulting SAP. The modified saporin can then be reacted with the modified
FGF to form disulfide linkages between the single exposed cysteine residue
20 on the FGF and the cysteine residue on the modified SAP.
Using either methodology (reacting mono-derivatized SAP with the
FGF peptide or introducing a cys residue into SAP), the resulting
preparations of FGF-SAP chemical conjugates are monogenous;
compositions containing the conjugates also appear to be free of
25 aggregates.
The above-described sources for heterogeneity also can be avoided
by producing the cytotoxic conjugate as a fusion protein by expression of
DNA encoding the modified FGF protein linked to DNA encoding the
cytotoxic agent, as described below.
WO 95/03831 PCT/USs4/08511
686~7
-43 -
2. Recombinant production of cytotoxic conjugates containing
r modified FGF
The problem of heterogeneity has also been addressed herein by
preparing the conjugates as fusion proteins using recombinant DNA
5 technology. Preparations containing the fusion proteins may be rendered
more homogeneous by modifying the FGF and/or the targeted agent to
prevent interactions between each conjugate, such as via unreacted
cysteines. Expression of DNA encoding a fusion of an FGF protein linked
to the cytotoxic agent results in a monogenous preparation of cytotoxic
10 conjugates. Such population may, however, form aggregates. Preparations
containing the fusion proteins may be rendered more homogeneous by
modifying the FGF and/or the cytotoxic agent to prevent interactions
between each conjugate, such as via unreacted cysteines. Aggregate
formation has been eliminated by preparing mutein constructs in which the
15 cysteine residues on the FGF are deleted or replaced. Cytotoxic conjugates
containing bFGF in which the cysteines at positions 78 and 96 have been
replaced by serines have been prepared. The resulting preparations of
cytotoxic conjugates retain cytotoxic activity, are monogenous and are free
of aggregates.
a. Pre~ar~liG" of muteins for recombinant production of
the conjugates
For recombinant expression using to the methods herein, all of the
cysteines of the FGF peptide that are not required for biological activity are
deleted or replaced; and for use in the chemical conjugation methods
25 herein, all except for one of these cysteines, which will be used for
chemical conjugation to the cytotoxic agent ,are deleted or replaced. In
practice, it appears that only two cysteines (including each of the cysteine
residues set forth in Table 2), and perhaps only the cysteines set forth in
Table 2, are required for retention of the requisite biological activity of the
30 FGF peptide. Thus, FGF peptides that have more than two cysteines are
modified by replacing the remaining cysteines with serines. The resulting
muteins may be tested for the requisite biological activity.
-
WO 95/03831 PCT/US94/08511
-44-
FGF peptides, such as FGF-3, FGF-4 and FGF-6, that have two
cysteines can be modified by replacing the second cysteine, which is not
listed in Table 2, and the resulting mutein used as part of a construct
containing DNA encoding the cytotoxic agent linked to the FGF-encoding
5 DNA. The construct is expressed in a suitable host cell and the resulting
protein tested for the ability to bind to FGF receptors and internalize the
cytotoxic agent.
As exemplified below, conjugates containing bFGF muteins in which
CyS78 and Cys96 have been replaced with serine residues have been
10 prepared. The resulting conjugates are at least as active as recombinant
conjugates that have wild type FGF components and at least as active as
chemical conjugates of FGF. In addition, it appears that the recombinantly
produced conjugates are less toxic, and thus, can, if necessary, be
administered in higher dosages.
b. DNA constructs and expression of the DNA constructs
To produce monogenous preparations of cytotoxic conjugates using
recombinant means, the DNA encoding the FGF protein is modified so that,
upon expression, the resulting FGF portion of the fusion protein does not
include any cysteines available for reaction. In preferred embodiments,
20 DNA encoding an FGF polypeptide is linked to DNA encoding a saporin
polypeptide. The DNA encoding the FGF polypeptide is modified in order to
remove the translation stop codon and other transcriptional or translational
stop signals that may be present and to remove or replace DNA encoding
the available cysteines. The DNA is then ligated to the DNA encoding the
25 saporin polypeptide directly or via a spacer region of one or more codons
between the first codon of the saporin and the last codon of the FGF. The
size of the spacer region is any length as long as the resulting conjugate
exhibits cytotoxic activity upon internalization by a target cell. Presently,
spacer regions of from about one to about seventy-five to ninety codons
30 are preferred.
WO 95/03831 PCTIUS94/08511
~16~64,?
-45-
DNA encoding FGF peptides and/or the amino acid sequences FGFs
are known to those of skill in this art (see, e , SEQ ID NOs. 24-32). DNA
may be prepared synthetically based on the amino acid sequence or known
DNA sequence of an FGF or may be isolated using methods known to those
of skill in the art or obtained from commercial or other sources known to
those of skill in this art. For example, DNA encoding virtually all of the FGF
family of peptides is known. For example human aFGF (Jaye et ak (1986)
Science 233:541-545), bovine bFGF (Abraham et al. (1986) Science
233:545-548), human bFGF (Abraham et ak (1986) EMB0 J. 5:2523-
2528; and Abraham et ak (1986) Quant. Biol. 51:657-668) and rat bFGF
(see Shimasaki et ak (1988) Biochem. BioPhvs. Res. Comm. and Kurokawa
et al. (1988) Nucleic Acids Res. 16:5201), FGF-3, FGF-7 and FGF-9 are
known (see, also, U.S. Patent No. 5,155,214; U.S. Patent No. 4,956,455;
U.S. Patent No. 5,026,839; and U.S. Patent No. 4,994,559, the
DNASTAR database, and references discussed above and below). The
amino acid sequence of an exemplary mammalian bFGF isolated from
bovine pituitary tissue is also known (see, e.q., in Esch et ak (1985)
Proc.Natl. Acad. Sci. USA 82:6507-6511; and U.S. Patent No.
4,956,455).
The isolated mammalian basic FGF protein is typically a 146-residue
polypeptide having a molecular weight of about 16 kD, and a pl of about
9.6; it may be expressed with an amino terminal extension of about 9
residues so that the resulting protein has a molecular weight of
about 18 kD.
Such DNA may then be mutagenized using standard methodologies
to delete or delete and replace any cysteine residues, as describe herein,
that are responsible for aggregate formation. If necessary, the identity of
cysteine residues that contribute to aggregate formation may be determined
empirically, by deleting and/or deleting and replacing a cysteine residue and
ascertaining whether the resulting FGF with the deleted cysteine form
WO 95/03831 PCT/US94/08511
46-
aggregates in solutions containing physiologically acceptable buffers and
salts.
As discussed above, any FGF protein, in addition to basic FGF
(bFGF) and acidic FGF (aFGF), including HST, INT/2, FGF-5, FGF-6,
5 KGF(FGF-7), FGF-8, and FGF-9 (see, e.a., Baird et ai. (1989) Brit. Med. Bull
45:438-452; Tanaka et ai. (1992) Proc. Natl. Acad. Sci. USA 89:8928-
8932; Miyamoto et ai. (1993) Mol. Cell. Biol. 13:4251-4259; see, also, the
data base, DNA (July, 1993 release from DNASTAR, Inc. Madison, Wl) for
DNA and amino acid sequences of the FGF family; see SEQ ID NOs. 24-32
10 for amino acid sequences of FGF-1 - FGF-9, respectively), may be modified
and expressed in accord with the methods herein. All of the FGF proteins
induce mitogenic activity in a wide variety of normal diploid mesoderm-
derived and neural crest-derived cells and this activity is mediated by
binding to an FGF cell surface receptor followed by internalization. Binding
15 to an FGF receptor followed by internalization are the activities required for
an FGF protein to be suitable for use herein. A test of such "FGF mitogenic
activity", which reflects the ability to bind to FGF receptors and to be
internalized, is the ability to stimulate proliferation of cultured bovine aortic
endothelial cells, as described in Gospodarowicz et al. (1982) J. Biol.
20 Chem. 257:12266-12278; Gospodarowicz et ai. (1976) Proc. Natl. Acad.
Sci. USA 73:4120-4124.
The DNA encoding the resulting modified FGF-SAP can be inserted
into a plasmid and expressed in a selected host, as described above, to
produce monogenous preparations of FGF-SAP and homogeneous
25 compositions containing monogenous FGF-SAP.
Multiple copies of the modified FGF-SAP chimera or modified FGF-
cytotoxic agent chimera can be inserted into a single plasmid in operative
linkage with one promoter. When expressed, the resulting protein will be
an FGF-SAP multimer. Typically two to six copies of the chimera are
30 inserted, preferably in a head to tail fashion, into one plasmid.
WO 95/03831 PCT/US94/08511
~ 6~7
-47 -
DNA encoding human bFGF-SAP having SEQ ID NO. 12 has been
mutagenized as described in the Examples using splicing by overlap
extension (SOE). Another preferred coding region is set forth in SEQ ID NO
13, nucleotides 1 - 465. In both instances, in preferred embodiments, the
5 DNA is modified by replacing the cysteines at positions 78 and 96 with
serine. The codons encoding cysteine residues at positions 78 and 96 of
FGF in the FGF-SAP encoding DNA (SEQ ID NO. 12) were converted to
serine codons by SOE. Each application of the SOE method uses two
amplified oligonucleotide products, which have complementary ends as
10 primers and which include an altered codon at the locus at which the
mutation is desired, to produce a hybrid product. A second amplification
reaction that uses two primers that anneal at the non-overlapping ends
amplify the hybrid to produce DNA that has the desired alteration.
C. r~G,~erlies of and use of the resulting cl~e~ ' c~, jr~g~tes and fusion
,urotei. .s
Cytotoxic conjugates agents can be prepared either by chemical
conjugation, recombinant DNA technology, or combinations of recombinant
expression and chemical conjugation. The methods herein are described
with particular reference to bFGF and saporin. It is understood, however,
20 that the same methods may be used to prepare and use conjugates of any
member of the FGF family with SAP, modified SAP, or any other cytotoxic
agent as described herein.
Using the methods and materials described above and in the
Examples, chemical conjugates and fusion proteins have been synthesized.
25 These include the following constructs:
WO 9S/03831 PCT/IJS94/08511
48-
TABLE 3
FGF CONJUGATES
DESCRIPTION Protein name
wild type chemical conjugate CCFS1
mutein C78S chemical conjugate CCFS2
mutein C96S chemical conjugate CCFS3
mutein C96S Cys-Sap chemical conjugate CCFS4
wild type fusion protein (FGF-Ala-Met-SAP) FPFS1
mutein C78S fusion proteinFPFS2
mutein C96S fusion proteinFPFS3
mutein C78 & C96S fusion protein FPFS4
wild type fusion protein ~SAP-Ala-Met-FGF) FPSF1
wild type fusion protein (FGF-Ala-Met-SAP-Ala-Met-SAP) FPFS16
Particular details of the syntheses of the constructs are set forth in
the EXAMPLES. The above constructs have been synthesized and have
been or can be inserted into plasmids including pET 11 (with and without
the T7 transcription terminator), pET 12 and pET 15 (NOVAGEN, Madison,
Wl)"IpPL and pKK223-3 (PHARMACIA, P.L.) and derivatives of pKK223-3.
20 The resulting plasmids have been and can be transformed into bacterial
hosts including BL21, BL231(DE3)+pLYS S, HMS175(DE3),
HMS175(DE3)+pLYS S (NOVAGEN, Madison, Wl) and N4830(c1857) (see,
Gottesman et al. (1980) J. Mol. Biol. 140:57-75, commercially available
from PL Biochemicals, Inc, also, see, e.~., U.S. Patent Nos. 5,266,465,
25 5,260,223, 5,256,769, 5,256,769, 5,252,725, 5,250,296, 5,244,797,
5,236,828, 5,234,829, 5,229,273, 4,798,886, 4,849,350, 4,820,631
and 4,780,313). N4830 harbors a heavily deleted phage lambda prophage
carrying the mutant c1857 temperature sensitive repressor and an active N
gene.
WO 95/03831 PCT/US94/08511
I G~6~7
-49-
TABLE 4
Fusion Protein Name Plasmid(s) that Encode the Protein
FPFS1 PZ1A, PZIB, PZIC, PZID, PZIE
FPFS4 PZ2B, PZ2C
FPFS 1 6 PZ1 4B
FPSF1 PZ1 5B
D. Therapeutic use of the FGF conjugates
Mouse xenograft tumor models demonstrate that the FGF conjugates
10 exhibit anti-tumor activity. Weekly intravenous injections in mice, with
established SK-Mel-5 xenografts, of wild-type bFGF-SAP conjugates (total
dose 125 ,ug/kg) over four weeks resulted in a mean tumor volume that
was 49% of the controi volume. Modification of the weekly regiment to
include cis-platin (5 mg/kg intraperitoneally once per week on the day
15 following FGF-SAP treatment) resulted in a mean tumor volume at sixty
days that was 23% of the controls. The combined treatment resulted in
complete tumor remission in 10% of the treated mice.
Conjugates produced herein have been injected into such mice and
appear to be less toxic than heterogeneous preparations of chemical
20 conjugates. Certain of the conjugates provided herein have also been
shown to exhibit anti-tumor activity in such mice.
In particular 5 ,ug/kg/week of FPFS1 and CCFS1 were administered
to mice, with established HT-1197 (a human bladder carcinoma cell line)
xenografts. Each treatment resulted in significant inhibition of tumor
25 growth throughout the 61 days of the study. In another study, 0.1 or 0.5
,ug/kg/week of FPFS1 with and without 0.5 mg/kg cisplatin is administered
to mice with established human prostate carcinoma cell tumors.
The chemical conjugate and fusion protein bFGF-SAP provided herein
may also be used for the treatment of restenosis. FGF conjugates have an
30 anti-proliferative effect on smooth muscle cells in rabbit balloon injury
models of restenosis (see, also U.S. Patent No. 5,308,622, which is based
WO 95/03831 PCT/US94/08511
b~1
-50-
on allowed U.S. Application Serial No. 07/915,056, which describes the
use of FGF-cytotoxic conjugates for the treatment of restenosis).
E. Formulation and administration of pharmaceutical composiliGns
The conjugates herein may be formulated into pharmaceutical
5 compositions suitable for topical, local, intravenous and systemic
application. Effective concentrations of one or more of the conjugates are
mixed with a suitable pharmaceutical carrier or vehicle. The concentrations
or amounts of the conjugates that are effective requires delivery of an
amount, upon administration, that ameliorates the symptoms or treats the
10 disease. Typically, the compositions are formulated for single dosage
admini~ tion. Therapeutically effective concentrations and amounts may
be determined empirically by testing the conjugates in known in vitro and in
vivo systems, such as those described here; dosages for humans or other
animals may then be extrapolated therefrom.
Upon mixing or addition of the conjugate(s) with the vehicle, the re-
sulting mixture may be a solution, suspension, emulsion or the like. The
form of the resulting mixture depends upon a number of factors, including
the intended mode of admi"iiL,~lion and the solubility of the conjugate in
the selected carrier or vehicle. The effective concentration is sufficient for
ameliorating the symptoms of the disease, disorder or condition treated and
may be empirically determined based upon in vitro and/or in vivo data, such
as the data from the mouse xenograft model. If necessary, pharmaceuti-
cally acceptable salts or other derivaives of the conjugates may be
prepared.
Pharmaceutical carriers or vehicles suitable for administration of the
conjugates provided- herein include any such carriers known to those skilled
in the art to be suitable for the particular mode of administration.
In addition, the conjugates may be formulated as the sole pharmaceutically
active ingredient in the composition or may be combined with other active
ingredients.
WO 95/03831 pcTluss4lo85
- 5 1 -
The conjugates can be administered by any appropriate route, for
example, orally, parenterally, intravenously, intradermally, subcutaneously,
or topically, in liquid, semi-liquid or solid form and are formulated in a
manner suitable for each route of administration. Preferred modes of
5 administration depend upon the indication treated. Dermatological and
ophthalmologic indications will typically be treated locally; whereas, tumors
and restenosis, will typically be treated by systemic, intradermal or
intramuscular, modes of administration.
The conjugate is included in the pharmaceutically acceptable carrier
10 in an amount sufficient to exert a therapeutically useful effect in the
absence of undesirable side effects on the patient treated. It is understood
that the number and degree of side effects depends upon the condition for
which the conjugates are administered. For example, certain toxic and
undesirable side effects are tolerated when treating life-threatening
15 illnesses, such as tumors, that would not be tolerated when treating
disorders of lesser consequence.
The concentration of conjugate in the composition will depend on
absorption, inactivation and excretion rates thereof, the dosage schedule,
and amount administered as well as other factors known to those of skill in
20 the art.
Typically a therapeutically effective dosage should produce a serum
concentration of active ingredient of from about 0.1 ng/ml to about 50-100
,ug/ml. The pharmaceutical compositions typically should provide a dosage
of from about 0.01 mg to about 100 - 2000 mg of conjugate, depending
25 upon the conjugate selected, per kilogram of body weight per day. For
example, for treatment of restenosis a daily dosage of about between 0.05
and 0.5 mg/kg (based on FGF-SAP chemical conjugate or an amount of
conjugate provided herein equivalent on a molar basis thereto) should be
sufficient. It is understood that the amount to administer will be a function
30 of the conjugate selected, the indication treated, and possibly the side
effects that will be tolerated.
WO 95/03831 PCT/US94/08511
.
-52-
The active ingredient may be administered at once, or may be
divided into a number of smaller doses to be administered at intervals of
time. It is understood that the precise dosage and duration of treatment is
a function of the disease being treated and may be determined empirically
using known testing protocols or by extrapolation from in vivo or in vitro
test data. It is to be noted that concentrations and dosage values may also
vary with the severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage regimens should
be adjusted over time according to the individual need and the professional
judgment of the person administering or supervising the administration of
the compositions, and that the concentration ranges set forth herein are
exemplary only and are not intended to limit the scope or practice of the
claimed compositions.
Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include any of the following
components: a sterile diluent, such as water for injection, saline solution,
fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic
solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens;
antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents,
such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates,
citrates and phosphates; and agents for the adjustment of tonicity such as
sodium chloride or dextrose. Parental preparations can be enclosed in
ampules, disposable syringes or multiple dose vials made of glass, plastic or
other suitable material.
If administered intravenously, suitable carriers include physiological
saline or phosphate buffered saline (PBS), and solutions containing
thickening and solubilizing agents, such as glucose, polyethylene glycol,
and polypropylene glycol and mixtures thereof. Liposomal suspensions may
also be suitable as pharmaceutically acceptable carriers. These may be
prepared according to methods known to those skilled in the art.
WO 95/03831 PCT/US94/08511
~ G4~
-53-
The conjugates may be prepared with carriers that protect them
against rapid elimination from the body, such as time release formulations
or coatings. Such carriers include controlled release formulations, such as,
but r ot limited to, implants and microencapsulated delivery systems, and
5 biodegradable, biocompatible polymers, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and
others. Methods for preparation of such formulations are known to those
skilled in the art.
The conjugates may be formulated for local or topical application, in
10 the form of gels, creams, and lotions and for intracisternal or intraspinal
application. Such solutions may be formulated as 0.01% -10% isotonic
solutions, pH about 5-7, with appropriate salts. The conjugates may be
formulated as aerosols for topical application (see, e.q., U.S. Patent Nos.
4,044,126, 4,414,209, and 4,364,923).
If oral administration is desired, the conjugate should be provided in
a composition that protects it from the acidic environment of the stomach.
For example, the composition can be formulated in an enteric coating that
maintains its integrity in the stomach and releases the active compound in
the intestine. The composition may also be formulated in combination with
20 an antacid or other such ingredient.
Oral compositions will generally include an inert diluent or an edible
carrier and may be compressed into tablets or enclosed in gelatin capsules.
For the purpose of oral therapeutic administration, the active compound or
compounds can be incorporated with excipients and used in the form of
25 tablets, capsules or troches. Pharmaceutically compatible binding agents
and adjuvant materials can be included as part of the composition.
The tablets, pills, capsules, troches and the like can contain any of
the following ingredients, or compounds of a similar nature: a binder, such
as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such
30 as starch and lactose, a disintegrating agent such as, but not limited to,
alginic acid and corn starch; a lubricant such as, but not limited to,
WO 95/03831 PCT/US94/08511
.
o6~1
magnesium stearate; a glidant, such as, but not limited to, colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring
agent such as peppermint, methyl salicylate, and fruit flavoring.
When the dosage unit form is a capsule, it can contain, in addition to
material of the above type, a liquid carrier such as a fatty oil. In addition,
dosage unit forms can contain various other materials which modify the
physical form of the dosage unit, for example, coatings of sugar and other
enteric agents. The conjugates can also be administered as a component
of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup
may contain, in addition to the active compounds, sucrose as a sweetening
agent and certain preservatives, dyes and colorings and flavors.
The active materials can also be mixed with other active materials
that do not impair the desired action, or with materials that supplement the
desired action, such as cis-platin for treatment of tumors.
Finally, the compounds may be packaged as articles of manufacture
containing packaging material, one or more conjugates or compositions as
provided herein within the packaging material, and a label that indicates the
indication for which the conjugate is provided.
The following examples are included for illustrative purposes only
and are not intended to limit the scope of the invention.
WO 95103831 ~1 6 86~1 PCT/US94/08511
EXAMPLE 1
RECOMBINANT PRODUCTION OF SAPORIN
A. Materi~ls and methods
1. Bacterial Strains:
5E. coli strain JA221 (Ipp- hdsM+ trpE5 leuB6 lacY recA1 F'[laclq
lac+ pro+]) is publicly available from the American Type Culture Collection
(ATCC), Rockville, MD 20852, under the accession number ATCC 33875.
(JA221 is also available from the Northern Regional Research Center
(NRRL), Agricultural Research Service, U.S. Department of Agriculture,
10 Peoria, IL 61604, under the accession number NRRL B-15211; see, also,
U.S. Patent No. 4,757,013 to Inouye; and Nakamura et al. (1979) Cell
18: 1109-1117.) Strain INV1 a is commercially available from Invitrogen,
San Diego, CA.
2~ DNA Manipul~ s
The restriction and modification enzymes employed herein are
commercially available in the U.S. Native saporin and rabbit polyclonal
antiserum to saporin were obtained as previously described in Lappi Q al.
(1985) Biochem. BioDhvs. Res. Comm. 129:934-942. Ricin A chain is
commercially available from SIGMA, Milwaukee, Wl. Antiserum was linked
20 to Affi-gel 10 (BIO-RAD, Emeryville, CA) according to the manufacturer's
instructions. Sequencing was performed using the Sequenase kit of United
States Biochemical Corporation (version 2.0) according to the
manufacturer's instructions. Minipreparation and maxipreparations of
plasmids, preparation of competent cells, transformation, M 13
25 manipulation, bacterial media, Western blotting, and ELISA assays were
according to Sambrook et al. ((1989) Molecular Cloninq: A LaboratorY
- Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
The purification of DNA fragments was done using the Geneclean ll kit (Bio
101 ~ according to the manufacturer's instructions. SDS gel electrophoresis
30 was performed on a Phastsystem (Pharmacia).
WO 95/03~31 PCT/US94/08Sll
-56-
Western blotting was accomplished by transfer of the
electrophoresed protein to nitrocellulose using the PhastTransfer system, as
described by the manufacturer. The antiserum to SAP was used at a
dilution of 1 :1000. Horseradish peroxidase labelled anti-lgG was used as
5 the second antibody (see Davis et ai. (1986) Basic methods in molecular
biolo~Y, New York, Elsevier Science Publishing Co., pp 1-338).
B. Isoldlion of DNA encoding sapu,i"
1.1solaliG., of ~enomic DNA and preparation of poly.-,erase
chain reaction (PCR) primers
SaPonaria officinalis leaf genomic DNA was prepared as described in
Bianchietak (1988) PlantMol. Biol. 11:203-214. Primersforgenomic
DNA amplifications were synthesized in a 380B automatic DNA
synthesizer. The primer corresponding to the "sense" strand of saporin
(SEQ ID NO 1) includes an EcoR I restriction site adapter immediately
upstream of the DNA codon for amino acid -15 of the native saporin N-
terminal leader sequence (SEQ ID NO. 1):
5'-CTGCAGAATTCGCATGGATCCTGCTTCAAT-3' .
The primer 5'-CTGCAGAATTCGCCTCGTTTGACTACTTTG-3' (SEQ ID NO.
2) corresponds to the "antisense" strand of saporin and complements the
coding sequence of saporin starting from the last 5 nucleotides of the DNA
encoding the carboxyl end of the mature peptide. Use of this primer
introduced a translation stop codon and an EcoRI restriction site after the
sequence encoding mature saporin.
2. Amplification of DNA encoding saporin
Unfractionated Sa~onaria officinalis leaf genomic DNA (1,ul) was
mixed in a final volume of 100,ul containing 10 mM Tris-HCI (pH 8.3),
50 mM KCI, 0.01 % gelatin, 2 mM MgCI2, 0.2 mM dNTPs, 0.8,ug of each
primer. Next, 2.5 U Taql DNA polymerase (Perkin Elmer Cetus) was added
and the mixture was overlaid with 30 ~l of mineral oil (Sigma). Incubations
were done in a DNA Thermal Cycler (Perkin Elmer Cetus). One cycle
included a denaturation step (940C for 1 min.), an annealing step (600C for
WO 95/03831 PCT/US94/08511
2 min.), and an elongation step (720C for 3 min.). After 30 cycles, a 10 IJI
aliquot of each reaction was run on a 1.5% agarose gel to verify the
correct structure of the amplified product.
The amplified DNA was digested with EcoRI and subcloned into
5 EcoR l-restricted M13mp18 ~NEW ENGLAND BIOLABS, Beverly, MA; see,
also, Yanisch-Perron et ak (1985), "Improved M13 phage cloning vectors
and host strains: Nucleotide sequences of the M13mp18 and pUC19
vectors", Gene 33:103). Single-stranded DNA from recombinant phages
was sequenced using oligonucleotides based on internal points in the
10 coding sequence of saporin (see, Bennati et al. (1989) Eur. J. Biochem.
183:465-470). Nine of the M13mp18 derivatives were sequenced and
compared. Of the nine sequenced clones, five had unique sequences, set
forth as SEQ ID NOs 3-7, respectively. The clones were designated
M13mp18-G4, -G1, -G2, -G7, and -G9. Each of these clones contains all
15 of the saporin coding sequence and 45 nucleotides of DNA encoding the
native saporin N-terminal leader peptide.
C. pOMPAG4 rlas" ' Construction
M13 mp18-G4, containing the SEQ ID NO. 3 clone from Example
1.B.2., was digested with EcoR 1, and the resulting fragment was ligated
20 into the EcoR I site of the vector plN-lllompA2 (see, e.g., U.S. Patent No.
4,575,013 to Inouye; and Duffaud et al. (1987) Meth. Enz. 153:492-507)
using the methods described in Example 1.A.2. The ligation was
accomplished such that the DNA encoding saporin, including the N-terminal
extension, was fused to the leader peptide segment of the bacterial ompA
25 gene. The resulting plasmid pOMPAG4 contains the Ipp promoter
[Nakamura, K. and Inouye, M. Cell., 18:1109-1117 (1979)], the E. coli lac
promoter operator sequence (lac O) and the E. coli ompA gene secretion
signal in operative association with each other and with the saporin and
native N-terminal leader-encoding DNA listed in SEQ ID NO. 3. The plasmid
30 also includes the E. coli lac repressor gene (lac 1).
WO 95/03831 PCT/US94/08511
58-
The M13 mp18-G1, -G2, -G7, and -G9 clones obtained frorn
Example 1.B.2, containing SEQ ID NOs. 4-7 respectively, are digested with
EcoR I and ligated into EcoR I digested plN-lllompA2 as described for M13
mp1 8-G4 above in this example. The resulting plasmids, labeled
5 pOMPAG1, pOMPAG2, pOMPAG7, pOMPA9, are screened, expressed,
purified, and characterized as described for the plasmid pOMPAG4.
INV1~ competent cells were transformed with pOMPAG4 and
cultures containing the desired plasmid structure were grown further in
order to obtain a large preparation of isolated pOMPAG4 plasmid using
10 methods described in Example 1.A.2.
D. Saporin expression in E. coii:
The pOMPAG4 transformed E. coli cells were grown under
conditions in which the expression of the saporin-containing protein is
repressed by the lac repressor to an O.D. in or at the end of the log phase
15 of growth after which IPTG was added to induce expression of the saporin-
encoding DNA.
To generate a large-batch culture of pOMPAG4 transformed E. coli
cells, an overnight culture (lasting approximately 16 hours) of JA221 E. coli
cells transformed with the plasmid pOMPAG4 in LB broth (see e.q.,
ZO Sambrook et al. (1989) Molecular Cloninq: A Laboratorv Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY) containing 125
mg/ml ampicillin was diluted 1:100 into a flask containing 750 ml LB broth
with 125 mg/ml ampicillin. Cells were grown at logarithmic phase shaking
at 87OC until the optical density at 550 nm reached 0.9 measured in a
25 spectrophotometer.
In the second step, saporin expression was induced by the addition
of IPTG (Sigma) to a final concentration of 0.2 mM. Induced cultures were
grown for 2 additional hours and then harvested by centrifugation (25 min.,
6500 x 9). The cell pellet was resuspended in ice cold 1.0 M TRIS,
30 pH 9.0, 2 mM EDTA (10 ml were added to each gram of pellet). The
resuspended material was kept on ice for 20-60 minutes and then
WO 9S/~1 PCT~S94/08511
~16~47
-59-
centrifuged (20 min., 6500 x 9) to separate the periplasmic fraction of E.
coli, which corresponds to the supernatant, from the intracellular fraction
corresponding to the pellet.
E. Pu,iricalioo of secreted recombinant Saporin
1. Anti-SAP immuno-affinity purification
The periplasmic fraction from Example 1.D. was dialyzed against
borate-buffered saline (BBS: 5 mM boric acid, 1.25 mM borax, 145 mM
sodium chloride, pH 8.5). The dialysate was loaded onto an immunoaffinity
column (0.5 x 2 cm) of anti-saporin antibodies, obtained as described in
Lappi et al., Biochem. BioPhvs. Res. Comm., 129: 934-942 (1985), bound
to Affi-gel 10 and equilibrated in BBS at a flow rate of about 0.5 ml/min.
The column was washed with BBS until the absorbance at 280 nm of the
flow-through was reduced to baseline. Next the column containing the
antibody bound saporin was eluted with 1.0 M acetic acid and 0.5 ml
fractions were collected in tubes containing 0.3 ml of 2 M ammonium
hydroxide, pH 10. The fractions were analyzed by ELISA (see, e.a.,
Sambrook et al. (1989) Molecular Cloninq: A Laboratorv Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The peak
fraction of the ELISA was analyzed by Western blotting as described in
Example 1.A.2 and showed a single band with a slightly higher molecular
weight than native saporin. The fractions that contained saporin protein,
as determined by the ELISA, were then pooled for further purification.
2. Reverse Phase High Performance Liquid
ChrG~ Lography purification
To further purify the saporin secreted into the periplasm, the pooled
fractions from Example 1 .E. 1 . were diluted 1 :1 with 0.1 % trifluoroacetic
acid (TFA) in water and chromatographed in reverse phase high pressure
liquid chromatography (HPLC) on a Vydac C4 column (Western Analytical)
equilibrated in 20% acetonitrile, 0.1% TFA in water. The protein was
eluted with a 20 minute gradient to 60% acetonitrile. The HPLC produced
a single peak that was the only area of immunoreactivity with anti-SAP
WO 95/03831 PCTtUS94/08~i11
-60 -
antiserum when analyzed by a western blot as described in Example 1.E.1.
Samples were assayed by an ELISA.
Sequence analysis was performed by Edman degradation in a
gas-phase sequenator (Applied Biosystems) (see, e.~., Lappi et al. (1985)
Biochem. BioPhYs. Res. Comm.129:934-942). The results indicated that
five polypeptides were obtained that differ in the length, between 7 and 12
amino acids, of the N-terminal saporin leader before the initial amino acid
valine of the mature native saporin (SEQ ID N0 3: residue -12 through -7).
All of the N-terminal extended variants retained cytotoxic activity. The size
10 of the native leader is 18 residues, indicating that the native signal peptide
is not properly processed by bacterial processing enzymes. The ompA
signal was, however, properly processed.
To obtain homogeneous saporin, the recombinantly produced saporin
can be separated by size and one of the five polypeptides used to produce
1 5 the conjugates.
F. Pu.iricaliGr of intrnc~ soluble saporin
To purify the cytosolic soluble saporin protein, the pellet from the
intracellular fraction of Example 1.E. above was resuspended in Iysis buffer
(30 mM TRIS, 2 mM EDTA, 0.1% Triton X-100, pH 8.0, with 1 mM PMSF,
20 10,clg/ml pepstatin A, 10,ug aprotinin, ,ug/ml leupeptin and 100,ug/ml
Iysozyme, 3.5 ml per gram of original pellet). To Iyse the cells, the
suspension was left at room temperature for one hour, then frozen in liquid
nitrogen and thawed in a 370C bath three times, and then sonicated for
two minutes. The Iysate was centrifuged at 11,500 x 9 for 30 min. The
25 supernatant was removed and stored. The pellet was resuspended in an
equal volume of Iysis buffer, centrifuged as before, and this second
supernatant was combined with the first. The pooled supernatants were
dialyzed versus BBS and chromatographed over the immunoaffinity column
as described in Example 1.E.1. This material also retained cytotoxic
30 activity.
WO 95/03831 PCT/US94/08511
6~1
-61 -
G. Assay for cytotoxic activity
The RIP activity of recombinant saporin was compared to the RIP
activity of native SAP in an in vitro assay measuring cell-free protein
synthesis in a nuclease-treated rabbit reticulocyte Iysate (Promega).
Samples of immunoaffinity-purified saporin, obtained in Example 1.E.1.,
were diluted in PBS and 5,ul of sample was added on ice to 35,ul of rabbit
reticulocyte Iysate and 10 ~l of a reaction mixture containing 0.5,ul of
Brome Mosaic Virus RNA, 1 mM amino acid mixture minus leucine, 5,11Ci of
tritiated leucine and 3,ul of water. Assay tubes were incubated 1 hour in a
30OC water bath. The reaction was stopped by transferring the tubes to
ice and adding 5,ul of the assay mixture, in triplicate, to 75,ul of 1 N
sodium hydroxide, 2.5% hydrogen peroxide in the wells of a Millititer HA
96-well filtration plate (Millipore). When the red color had bleached from
the samples, 300,ul of ice cold 25% trichloroacetic acid (TCA) were added
to each well and the plate left on ice for another 30 min. Vacuum filtration
was performed with a Millipore vacuum holder. The wells were washed
three times with 300,ul of ice cold 8% TCA. After drying, the filter paper
circles were punched out of the 96-well plate and counted by liquid
scintillation techniques.
The IC50 for the recombinant and native saporin were approximately
20 pM. Therefore, recombinant saporin-containing protein has full protein
synthesis inhibition activity when compared to native saporin.
EXAMPLE 2
RECOMBINANT PRODUCTION OF FGF-SAP FUSION PROTEIN
A. General Descriptions
1. Bacterial Strains and Plasr,~ids
E. coli strains BL21 (DE3), BL21 (DE3)pLysS, HMS174(DE3) and
HMS174(DE3)pLysS were purchased from NOVAGEN, Madison, Wl.
Plasmid pFC80, described below, has been described in the WIPO
International Patent Application No. WO 90/02800, except that the bFGF
coding sequence in the plasmid designated pFC80 herein has the sequence
WO 9~;/03831 PCT/US94/08511
~ G~
-62-
set forth as SEQ ID NO 12, nucleotides 1-465. The plasmids described
herein may be prepared using pFC80 as a starting material or, alternatively,
by starting with a fragment containing the Cll ribosome binding site (SEQ
ID NO 15) linked to the FGF-encoding DNA (SEQ ID NO 12) .
2. DNA ManipulaliG.,s
The restriction and modification enzymes employed here are
commercially available in the U.S. Native SAP, chemically conjugated
bFGF-SAP and rabbit polyclonal antiserum to SAP and FGF were obtained
as described in Lappi et al., Biochem. BioPhYs. Res. Comm.,129: 934-942
(1985) and Lappi et al., Biochem. Bio~hvs., Res. Comm.,160: 917-923
(1989). The pET System Induction Control was purchased from
NOVAGEN, Madison, Wl. The sequencing of the different constructions
was done using the Sequenase kit of United States Biochemical Corporation
(version 2.0). Minipreparation and maxipreparations of plasmids,
preparation of competent cells, transformation, M13 manipulation, bacterial
media and Western blotting were performed using routine methods (see,
e.q.,.Sambrook et al. (1989) Molecular Clonin~: A Laboratorv Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The purification
of DNA fragments was done using the Geneclean ll kit, purchased from Bio
101. SDS gel electrophoresis was performed on a Phastsystem
(Pharmacia) .
B. Construction of plas.., 's encoding FGF-SAP fusion proteins
1. Construction of FGFM13 that contains DNA encoding the
Cl ribosome binding site linked to FGF
A Nco l restriction site was introduced into the SAP-encoding DNA
the M13mpl8-G4 clone, prepared as described in Example 1.B.2. by site-
directed mutagenesis method using the Amersham In vl -mutagenesis
system 2.1. The oligonucleotide employed to create the Nco I restriction
site was synthesized using a 380B automatic DNA synthesizer (Applied
Biosystems) and is listed as:
SEQ ID NO 8 - CAACAACTGCCATGGTCACATC.
WO 9S/03831 ~ 6 PCT/US94/08511
-63-
This oligonucieotide containing the Nco I site replaced the original SAP-
containing coding sequence at SEQ ID N0 3, nts 32-53. The resulting
M13mp18-G4 derivative is termed mpNG4.
In order to produce a bFGF coding sequence in which the stop codon
5 was removed, the FGF-encoding DNA was subcloned into a M13 phage and
subjected to site-directed mutagenesis. Plasmid pFC80 is a derivative of
pDS20 (see, e.a., Duester et al. (1982) Cell 30:855-864; see also U.S.
Patent Nos. 4,914,027, 5,037,744, 5,100,784, and 5,187,261; see, also,
PCT International Application No. W0 90/02800; and European Patent
10 Application No. EP 267703 A1), which is almost the same as plasmid
pKG1800 (see, Bernardi et al. (1990) DNA Sequence 1:147-150; see, also
McKenney et al. (1981) pp. 383-415 in Gene AmPlification and Analvsis 2:
Analvsis of Nucleic Acids bY EnzYmatic Methods Chirikjian et al., eds,
North Holland Publishing Company, Amsterdam) except that it contains an
15 extra 440 bp at the distal end of aalK between nucleotides 2440 and 2880
in pDS20. Plasmid pKG1800 includes the 2880 bp EcoR l-Pvu ll of
pBR322 that contains the contains the ampicillin resistance gene and an
origin of replication.
Plasmid pFC80 was prepared from pDS20 by replacing the entire
20 qalK gene with the FGF-encoding DNA of SEQ ID N0. 12, inserting the trp
promoter (SEQ ID N0. 14) and the bacteriophage lambda Cll ribosome
binding site (SEQ. ID No. 15; see, e.q., Schwarz et al. (1978) Nature
272:410) upstream of and operatively linked to the FGF-encoding DNA.
The Trp promoter can be obtained from plasmid pDR720 (Pharmacia PL
25 Biochemicals) or synthesized according to SEQ ID N0. 14. Plasmid pFC80,
contains the 2880 bp EcoR l-BamH I fragment of plasmid pSD20, a
synthetic Sal l-Nde I fragment that encodes the Trp promoter region (SEQ
ID N0. 14):
EcoRI
AATTCCCCTGTTGACAATTAATCATCGAACTAGTTAACTAGTACGCAGCTTGGCTGCAG
and the Cll ribosome binding site (SEQ ID N0.15)):
WO95/03~1 PCT~S94/08511
.
64-
Sall Ndel
GTCGACCAAGCTTGGGCATACATTCAATCAATTGTTATCTAAGGAAATACTTACATATG
The FGF-encoding DNA was removed from pFC80 by treating it as
follows. The pFC80 plasmid was digested by Haa I and Sal l, which
5 produces a fragment containing the Cll ribosome binding site linked to the
FGF-encoding DNA. The resulting fragment was blunt ended with
Klenow's reagent and inserted into M13mp18 that had been opened by
Sma I and treated with alkaline phosphatase for blunt-end ligation. In order
to remove the stop codon, an insert in the ORI minus direction was
10 mutagenized using the Amersham kit, as described above, using the
following oligonucleotide (SEQ ID NO 9): GCTAAGAGCGCCATGGAGA.
SEQ ID NO 9 contains 1 nucleotide between the FGF carboxy terminal
serine codon and a Nco l restriction site, and it replaced the following wild
type FGF encoding DNA having SEQ ID NO 10:
GCT AAG AGC TGA CCA TGG AGA.
Ala Lys Ser STOP Pro Trp Arg
The resulting mutant derivative of M13mp18, lacking a native stop
codon after the carboxy terminal serine codon of bFGF, was designated
FGFM13. The mutagenized region of FGFM13 contained the correct
20 sequence (SEQ ID NO 11) .
2. ~e~JaraliG" of ~.las"lids pFS92 (PZ1A), PZlB and PZ1C
that e,~co~Je the FGF-SAP fusion protein
a. Plasl.,i I pFS92 (also desig"ated PZlA)
Plasmid FGFM13 was cut with Nco l and Sac I to yield a fragment
25 containing the Cll ribosome binding site linked to the bFGF coding sequence
with the stop codon replaced.
The M13mp18 derivative mpNG4 containing the saporin coding
sequence was also cut with restriction endonucleases Nco l and Sac 1, and
the bFGF coding fragment from FGFM13 was inserted by ligation to DNA
30 encoding the fusion protein bFGF-SAP into the M13mp18 derivative to
produce mpFGF-SAP, which contains the Cll ribosome binding site linked to
the FGF-SAP fusion gene. The sequence of the fusion gene is set forth in
WO 95/03831 ~/~6~64~ PCT/US94/08511
-65-
SEQ ID NO 12 and indicates that the FGF protein carboxy terminus and the
saporin protein amino terminus are separated by 6 nucleotides (SEQ ID NOs
12 and 13, nts 466-471) that encode two amino acids Ala Met.
Plasmid mpFGF-SAP was digested with Xba I and EcoR I and the
5 resuiting fragment containing the bFGF-SAP coding sequence was isolated
and ligated into plasmid pET-11a (available from NOVAGEN, Madison, Wl;
for a description of the plasmids see U.S. Patent No. 4,952,496; see, also
Studier et al. (1990) Meth. Enz. 185:60-89; Studier et al. (1986) J. Mol.
Biol. 189:113-130; Rosenberg et al. (1987) Gene 56:125-135) that had
10 also been treated with EcoR I and Xba 1. The resulting plasmid was
designated pFS92. It was renamed PZ1A.
Plasmid pFS92 (or PZ1A) contains DNA the entire basic FGF protein
(SEQ ID NO 12), a 2-amino acid long connecting peptide, and amino acids
1 to 253 of the mature SAP protein. Plasmid pFS92 also includes the Cll
15 ribosome binding site linked to the FGF-SAP fusion protein and the T7
promoter region from pET-11 a.
E. coli strain BL21 (DE3)pLysS (NOVAGEN, Madison Wl) was
transformed with pFS92 according to manufacturer's instructions and the
methods described in Example 2.A.2.
b. Plas,~'' PZlB
Plasmid pFS92 was digested with EcoR 1, the ends repaired by
adding nucleoside triphosphates and Klenow DNA polymerase, and then
digested with Nde I to release the FGF-encoding DNA without the Cll
ribosome binding site. This fragment was ligated into pET 1 la, which had
25 been BamH I digested, treated to repair the ends, and digested with Nde 1.
The resulting plasmid was designated PZ1 B. PZ1 B includes the T7
transcription terminator and the pET-11 a ribosome binding site.
E. coli strain BL21 (DE3) (NOVAGEN, Madison Wl) was transformed
with PZ1 B according to manufacturer's instructions and the methods
30 described in Example 2.A.2.
WO 95103831 PCT/US94/08511
6~ 66-
c. Plasmid PZlC
Plasmid PZ1C was prepared from PZlB by repiacing the ampicillin
resistance gene with a kanamycin resistance gene.
d. Plasmid PZ1D
Plasmid pFS92 was digested with EcoR I and Nde I to release the
FGF-encoding DNA without the Cll ribosome binding site and the ends were
repaired. This fragment was ligated into pET 1 2a, which had been BamH I
digested and treated to repair the ends. The resulting plasmid was
designated PZ1 D. PZ1 D includes DNA encoding the OMP T secretion signal
10 operatively linked to DNA encoding the fusion protein.
E. coli strains BL21(DE3), BL21(DE3)pLysS, HMS174(DE3) and
HMS174(DE3)pLysS (NOVAGEN, Madison Wl) were transformed with
PZ1 D according to manufacturer's instructions and the methods described
in Example 2.A.2.
C. Ex~ress;G" of the recombinant bFGF-SAP fusion prot~i.,s
The two-stage method described above was used to produce
recombinant bFGF-S~P protein (hereinafter bFGF-SAP fusion protein).
pr~ss~Gr, of rbFGF-SAP from pFS92 (PZ1A)
Three liters of LB broth containing ampicillin (50,ug/ml) and
20 chloramphenicol (25 ~g/ml) were inoculated with pFS92 plasmid-containing
bacterial cells (strain BL21 (DE3)pLysS) from an overnight culture
(1:100 dilution) that were obtained according to Example 2.B. Cells were
grown at 37 C in an incubator shaker to an OD600 of 0.7. IPTG (Sigma
Chemical, St. Louis, MO) was added to a final concentration of 0.2 mM
25 and growth was continued for 1.5 hours at which time cells were
centrifuged. Subsequent experiments have shown that growing the
BL21 (DE3)pLysS cells at 30 C instead of 37 C improves yields. When the
cells are grown at 30 C they are grown to an OD600 of 1.5 prior to
induction. Following induction, growth is continued for about 2 to 2.5
30 hours at which time the cells are harvested by centrifugation.
WO 951Q~831 PCT/US94/08511
~16~b47
The pellet was resuspended in Iysis solution (45-60 ml per 16 9 of
pellet; 20 mM TRIS, pH 7.4, 5 mM EDTA, 10% sucrose, 150 mM NaCI,
Iysozyme, 100 ,ug/ml, aprotinin, 10 ,ug/ml, leupeptin, 10 ,ug/ml, pepstatin A,
10,l,rg/ml and 1 mM PMSF) and incubated with stirring for 1 hour at room
temperature. The solution was frozen and thawed three times and
sonicated for 2.5 minutes. The suspension was centrifuged at 12,000 X g
for 1 hour; the resulting first-supernatant was saved and the pellet was
resuspended in another volume of Iysis solution without Iysozyme. The
resuspended material was centrifuged again to produce a second-
supernatant, and the two supernatants were pooled and dialyzed against
borate buffered saline, pH 8.3.
2. Ex,~.re~s;o,~ of bFGF-SAP fusion protein from PZ1B and
PZlC
Two hundred and fifty mls. of LB medium containing ampicillin
(100,ug/ml) were inoculated with a fresh glycerol stock of PZ1B. Cells
were grown at 30 C in an incubator shaker to an OD600 of 0.7 and stored
overnight at 4 C. The following day the cells were pelleted and
resuspended in fresh LB medium (no ampicillin). The cells were divided into
5 1-liter batches and grown at 30 C in an incubator shaker to an OD600 of
1.5. IPTG (SIGMA CHEMICAL, St. Louis, M0) was added to a final
concentration of 0.1 mM and growth was continued for about 2 to
2.5 hours at which time cells were harvested by centrifugation.
In order to grow PZ1 C, prior to induction, the cells are grown in
medium containing kanamycin (50,ug/ml) in place of ampicillin.
3. Expression of bFGF-SAP fusion protein from PZlD
Two hundred and fifty mls of LB medium containing ampicillin
(100,ug/ml) were inoculated with a fresh glycerol stock of PZ1B. Cells
were grown at 30 C in an incubator shaker to an OD600 of 0.7 and stored
overnight at 4 C. The following day the cells were pelleted and
resuspended in fresh LB medium (no ampicillin). The cells were used to
inoculate a 1 liter batch of LB medium and grown at 30 C in an incubator
WO 95/03831 PCT/US94108511
.
~1 ~6~ 68-
shaker to an OD600 of 1.5. IPTG (SIGMA CHEMlCAL, St. Louis, M0) was
added to a final concentration of 0.1 mM and growth was continued for
about 2 to 2.5 hours at which time cells were harvested by centrifugation.
The cell pellet was resuspended in ice cold 1.0 M Tris pH 9Ø 2 mM
EDTA. The resuspended material is kept on ice for another 20-60 minutes
and then centrifuged to separate the periplasmic fraction (supernatant) from
the intracellular fraction (pellet).
D. Affinity pu.iric~liG" of bFGF-SAP fusion protein
Thirty ml of the dialyzed solution containing the bFGF-SAP fusion
protein from Example 2.C. was applied to HiTrap heparin-Sepharose column
(Pharmacia, Uppsala, Sweden) equilibrated with 0.15 M NaCI in 10 mM
TRlS, pH 7.4 (buffer A). The column was washed: first with equilibration
buffer; second with 0.6 M NaCI in buffer A; third with 1.0 M NaCI in
buffer A; and finally eluted with 2 M NaCI in buffer A into 1.0 ml fractions.
Samples were assayed by the ELISA method.
The results indicate that the bFGF-SAP fusion protein elutes from the
heparin-Sepharose column at the same concentration (2 M NaCI) as native
and recombinantly-produced bFGF. This indicates that the heparin affinity
is retained in the bFGF-SAP fusion protein.
E. Cl~a.aclc~ liol- of the bFGF-SAP fusion protein
1. Western blot of affinity-purified bFGF-SAP fusion protein
SDS gel electrophoresis was performed on a Phastsystem utilizing
20% gels (Pharmacia). Western blotting was accomplished by transfer of
the electrophoresed protein to nitrocellulose using the PhastTransfer system
(Pharmacia), as described by the manufacturer. The antisera to SAP and
bFGF were used at a dilution of 1:1000. Horseradish peroxidase labeled
anti-lgG was used as the second antibody (Davis et al. (1986) Basic
Methods in Molecular Biolo~v, New York, Elsevier Science Publishing Co.,
pp 1-338).
WO 95/03831 PCT/US94/08511
~IG~64~
The anti-SAP and anti-FGF antisera bound to a protein with an
approximate molecular weight of 48,000 kd, which corresponds to the sum
of the independent molecular weights of SAP (30,000) and bFGF (18,000).
2. Assays to assess the cytotoxicity of the FGF-SAP fusion
protein
a. Effect of bFGF-SAP fusion protein on cell-free
~rol~i., s~,.ll,esis
The RIP activity of bFGF-SAP fusion protein compared to the FGF-
SAP chemical conjugate was assayed as described in Example 1.G. The
10 results indicated that the IC50 Of the bFGF-SAP fusion protein is about 0.2
nM and the IC50 of chemically conjugated FGF-SAP is about 0.125 nm.
b. Cytotoxicity of bFGF-SAP fusion protein
Cytotoxicity experiments were performed with the Promega
(Madison, Wl) CellTiter 96 Cell Proliferation/Cytotoxicity Assay. About
15 1,500 SK-Mel-28 cells (available from ATCC), a human melanoma cell line,
were plated per well in a 96 well plate in 90,ul HDMEM plus 10% FCS and
incubated overnight at 37C, 5% CO2. The following morning 10,u1 of
media alone or 10,ui of media containing various concentrations of the
rbFGF-SAP fusion protein, basic FGF or saporin were added to the wells.
20 The plate was incubated for 72 hours at 370C. Following the incubation
period, the number of iiving cells was determined by measuring the
incorporation and conversion of the commonly available dye MTT supplied
as a part of the Promega kit. Fifteen,ul of the MTT solution was added to
each well, and incubation was continued for 4 hours. Next, 100 IJI of the
25 standard solubilization solution supplied as a part of the Promega kit was
added to each well. The plate was allowed to stand overnight at room
temperature and the absorbance at 560 nm was read on an ELISA plate
reader (Titertek Multiskan PLUS, ICN, Flow, Costa Mesa, CA).
The results indicated that the chemical FGF-SAP conjugate has an
30 ID50 of 0.3 nM, the bFGF-SAP fusion protein has a similar ID50 of 0.6 nM,
and unconjugated SAP, which is unable to bind to the cell surface, has an
ID50 of 200 nM. Therefore, when internalized, the bFGF-SAP fusion
WO 95103831 PCT/US94/08511
.
6~
protein appears to have approximately the same cytotoxic activity as the
chemically conjugated FGF-SAP.
EXAMPLE 3
PREPAP~ATION OF FGF MUTEINS
A. Materials and Methods
1. Reagents
Restriction and modification enzymes were purchased from BRL
(Gaithersburg, MD), Stratagene (La Jolla, CA) and New England Biolabs
(Beverly, MA). Native SAP, chemically conjugated basic FGF-SAP and
rabbit polyclonal antiserum to SAP and basic FGF were obtained from
Saponaria officinalis (see, e.~., Stirpe et al. (1983) Biochem. J. 216:617-
625). Briefly, the seeds were extracted by grinding in 5 mM sodium
phosphate buffer, pH 7.2 containing 0.14 M NaCI, straining the extracts
through cheesecloth, followed by centrifugation at 28,000 9 for 30 min to
produce a crude extract, which was dialyzed against 5 mM sodium
phosphate buffer, pH 6.5, centrifuged and applied to CM-cellulose (CM 52,
Whatman, Maidstone, Kent, U.K.). The CM column was washed and S0-6
was eluted with a 0-0.3 M NaCI gradient in the phosphate buffer.
Plasmid pFC80, containing the basic FGF coding sequence, was a
gift of Drs. Paolo Sarmientos and Antonella Isacchi of Farmitalia Carlo Erba
(Milan, Italy). Plasmid pFC80, has been described in WIPO International
Patent Application No. WO 90/02800 and co-pending International PCT
Application Serial No. PCT/US93/05702 (published as WO 93/25688),
which are herein incorporated in their entirety by reference. The sequence
of DNA encoding bFGF in pFC80 is that set forth in copending International
PCT Application Serial No. PCT/US93/05702 and in SEQ ID NO. 12. The
construction of pFC80 is set forth above in Example 2.
Plasmid isolation, production of competent cells, transformation and
M13 manipulations were carried out according to published procedures
(Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Purification of
. WO95/03~1 PCT~S94/08511
~ 8~
DNA fragments was achieved using the Geneclean ll kit, purchased from
Bio 101 (La Jolla, CA). Sequencing of the different constructions was
performed using the Sequenase kit (version 2.0) of USB (Cleveland, OH).
2. Sodium JoJecyl sulphate (SDS) gel elecl,~ oresis and
Western blotting.
SDS gel electrophoresis was performed on a PhastSystem utilizing
20% gels (Pharmacia). Western blotting was accomplished by transfer of
electrophoresed protein to nitrocellulose using the PhastTransfer system
(Pharmacia), as described by the manufacturer. The antisera to SAP and
10 basic FGF were used at a dilution of 1:1000. Horseradish peroxidase
labeled anti-lgG was used as the second antibody as described (Davis, L.,
Dibner et al. (1986) Basic Methods in Molecular Biology, p. 1, Elsevier
Science Publishing Co., New York).
B. P~"ar~.liG" of the mutagenized FGF by site-directed
m~ e~6sis
Cysteine to serine substitutions were made by oligonucleotide-
directed mutagenesis using the Amersham (Arlington Heights, lL) in vitro-
mutagenesis system 2.1. Oligonucleotides encoding the new amino acid
were synthesized using a 380B automatic DNA synthesizer (Applied
20 Biosystems, Foster City, CA).
1. m~ e"e~;s
The oligonucleotide used for in vitro mutagenesis of cysteine 78 was
AGGAGTGTCTGCTAACC (SEQ ID NO. 16), which spans nucleotides 225-
241 of SEQ ID NO. 12. The oligonucleotide for mutagenesis of cysteine 96
25 was TTCTAAATCGGTTACCGATGACTG (SEQ ID NO. 17), which spans
nucleotides 279-302 of SEQ ID NO. 12. The mutated replicative form
DNA was transformed into E. coli strain JM109 and single plaques were
picked and sequenced for verification of the mutation. The FGF mutated
gene was then cut out of M13, ligated into the expression vector pFC80,
30 which had the non-mutated form of the gene removed, and transformed
into E. coli strain JM109. Single colonies were picked and the plasmids
sequenced to verify that the mutation was present. Plasmids with correct
WO 95/03831 PCT/US94/08511
-7Z-
mutation were then transformed into the E. coli strain FICE 2 and single
colonies from these transformations were used to obtain the mutant basic
FGFs. An excellent level of expression, approximately 20 mg per liter of
fermentation broth, was achieved.
2. Purification of mutagenized FGF
Cells were grown overnight in 20 ml of LB broth containing 100
,ug/ml ampicillin. The next morning the cells were pelleted and transferred
to 500 ml of M9 medium with 100,ug/ml ampicillin and grown for 7 hours.
The cells were pelleted and resuspended in Iysis solution (10 mM TRIS, pH
7.4, 150 mM NaCI, Iysozyme, 10,ug/mL, aprotinin, 10,ug/mL, leupeptin,
10,ug/mL, pepstatin A, 10,ug/mL and 1 mM PMSF; 45-60 ml per 16 9 of
pellet) and incubated while stirring for 1 hour at room temperature. The
solution was frozen and thawed three times and sonicated for 2.5 minutes.
The suspension was centrifuged; the supernatant saved and the pellet
resuspended in another volume of Iysis solution without Iysozyme,
centrifuged again and the supernatants pooled. Extract volumes (40 ml)
were diluted to 50 ml with 10 mM TRIS, pH 7.4 (buffer A). Pools were
loaded onto a 5 ml Hi-Trap heparin-Sepharose column (Pharmacia, Uppsala,
Sweden) equilibrated in 150 mM sodium chloride in buffer A. The column
was washed with 0.6 M sodium chloride and 1 M sodium chloride in buffer
A and then eluted with 2 M sodium chloride in buffer A. Peak fractions of
the 2 M elution, as determined by optical density at 280 nm, were pooled
and purity determined by gel electrophoresis. Yields were 10.5 mg of
purified protein for the Cys78 mutant and 10.9 mg for the Cys96 mutant.
The biological activity of [C78S]FGF and [C96S]FGF was measured
on adrenal capillary endothelial cells in culture. Cells were plated 3,000 per
well of a 24 well plate in 1 ml of 10% calf serum-HDMEM. When cells
were attached, samples were added in triplicate at the indicated
concentration and incubated for 48 h at 37C. An equal quantity of
samples was added and further incubated for 48 h. Medium was aspirated;
cells were treated with trypsin (1 ml volume) to remove cells to 9 ml of
WO 95/03831 ~6~64~ PCT/US94/0851l
Hematall diluent and counted in a Coulter Counter. The results show that
the two mutants that retain virtually complete proliferative activity of native
basic FGF as judged by the ability to stimulate endothelial cell proliferation
in culture.
EXAMPLE 4
PREPARATION OF SAPORIN: DERIVATIZATION AND PURIFICATION OF
MONO-DERIVATIZED SAPORIN
Saporin (SAP; 49 mg) at a concentration of 4.1 mg/ml was dialyzed
against 0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.5. A 1.1
10 molar excess (563,ug in 156,ul of anhydrous ethanol) of SPDP (Pharmacia,
Uppsala, Sweden) was added and the reaction mixture immediately agita-
ted and put on a rocker platform for 30 minutes. The solution was then
dialyzed against the same buffer. An aliquot of the dialyzed solution was
examined for extent of derivatization according to the Pharmacia instruction
15 sheet. The extent of derivatization was 0.86 moles of SPDP per mole of
SAP. During these experiments, another batch of SAP was derivatized
using an equimolar quantity of SPDP in the reaction mixture with a resulting
0.79 molar ratio of SPDP to SAP.
Derivatized SAP (32.3 mg) was dialyzed in 0.1 M sodium borate,
20 pH 9.0 and applied to a Mono S 16/10 column equilibrated with 25 mM so-
dium chloride in dialysis buffer. A gradient of 25 mM to 125 mM sodium
chloride in dialysis buffer was run to elute SAP and derivatized SAP. The
flow rate was 4.0 ml/min. and 4 ml fractions were collected. Aliquots of
fractions were assayed for protein concentration (BCA Protein Assay,
25 Pierce Chemical, Chicago, IL) and for pyridylthione released by reducing
agent. Individual fractions from 25 to 37 were analyzed for protein con-
centration and pyridyl-disulfide concentration and are presented in Table 5.
Fractions 24-28 correspond to approximately 2 moles of 2-pyridyl disulfide
per mole of SAP, 29-33 corresponds to one mole per mole and 34-37 con-
30 tain non-derivatized SAP. These data indicate a separation according to the
level of derivatization by SPDP. The initial eluting peak is composed of SAP
WO 95/03831 PCT/US94/08511
~\ ~$~
-74-
that is approximately di-derivatized; the second peak is mono-derivatized
and the third peak shows no derivatization. The di-derivatized material
accounts for 20% of the three peaks; the second accounts for 48% and
the third peak contains 32%. Material from the second peak was pooled
5 and gave an average ratio of pyridyl-disulfide to SAP of 0.95. Fraction 33
showed a divergent ratio of pyridine-2-thione to protein, perhaps because
of its low concentration. It was excluded from the pool. The pooled
material was used for the conjugation described here. Fractions that
showed a ratio of SPDP to SAP greater than 0.85 but less than 1.05 were
10 pooled, dialyzed against 0.1 M sodium chloride, 0.1 M sodium phosphate,
pH 7.5 and used for derivatization with basic FGF. A pool of these
materials had a molar ratio SPDP:SAP of 0.9 with a final yield of 4.6 mg.
TABLE 5
LEVELS OF DERIVATIZATION BY SPDP OF FRACTIONS FROM
CHROMATOGRAPHY OF DERIVATIZED SAP
Fra~;Lion Protein Pyridine-2-Dithione Pyridine-2-
NumberConcentration t,uM)Concentration l,uM) Thione/Protein
Ratio
5.8 9.6 1.7
26 13.5 19.4 1.4
27 9.8 17.3 1.8
28 8.6 14.7 1.7
29 10.7 12.2 1.1
22.0 21.0 0.95
31 27.0 25.0 0.93
32 17.8 15.8 0.89
33 4.5 7.4 1.6
34 33.2 0 0
29.2 o o
36 28.3 0 0
37 10.1 0 0
WO 95/03831 PCT/US94/08511
~ 6~
-75-
EXAMPLE 5
PREPARATION OF SAPORIN: PREPARATION OF MODIFIED SAPORIN
Instead of derivatizing SAP, SAP was modified by addition of a
cysteine residueat the N-terminus-encoding portion of the DNA or the
addition of a cysteine at position 4 or 10. The resulting saporin is then
reacted with an available cysteine on an FGF to produce conjugates that
are linked via the added Cys or Met-Cys on saporin.
Modified SAP has been prepared by modifying DNA encoding the
saporin by inserting DNA encoding Met-Cys or Cys at position -1 or by
replacing the lle or the Asp codon within 10 or fewer residues of the N-
terminus. The resulting DNA has been inserted into pET11a and pET15b
and expressed in BL21 cells. The resulting saporin proteins are designated
FPS1 (saporin with Cys at -1), FPS2 (saporin with Cys at position 4) and
FPS3 (saporin with Cys at position 10). A plasmid that encodes FPS1 and
that has been for expression of FPS1 has been designated PZ50B.
Plasmids that encode FPS2 and that have been used for expression of FPS2
have been designated PZ51B (pETlla-based plasmid) and PZ51E (petl5b-
based plasmid). Plasmids that encode FPS3 and that have been used for
expression of FPS3 have been designated PZ52B (pET1 1a-based plasmid)
and PZ52E (pet15b-based plasmid).
A. Materials and Methods
1. Bacterial strains
Novablue (NOVAGEN, Madison, Wl) and BL21(DE3) (NOVAGEN,
Madison Wl).
2. DNA manipulatiGns
DNA manipulations were performed as described in Examples 1 and
2.
Plasmid PZ1 B (designated PZ1B1) described in Example 2 was used as the
DNA template.
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B. Preparation of sapori-, with an added cysteine residue at the N-
terminus
1. Primers
(a) Primer #1 corresponding to the sense strand of
saporin, nucleotides 472-492 of SEQ ID NO. lZ,
incorporates a Ndel site and adds a cys codon 5'
to the first codon of the mature protein
Ibetween Met and Val):
CATATGTGTGTCACATCAATCACATTAGAT (SEQ ID NO. 34)
(b) Primer #2 - Antisense primer co",l~le .,ents the
coding sequence of saporin spanning nucleotides
547-567 of SEQ ID NO. 12 and contains a
BamHI site:
1 5 CAGGTTTGGATCCTTTACGTT (SEQ ID NO . 35)
2. ISOIdliO~I of saporin-encoding DNA
PZIB1 DNA was amplified by PCR as follows using the above
primers. PZ1B DNA (1 ,ul) was mixed in a final volume of 100,ul containing
10 mM Tris-HCI (pH 8.3), 50 mM KCI, 0.01% gelatin, 2 mM MgCI2,
20 0.2 mM dNTPs, 0.8,ug of each primer. Next, 2.5 U Taql DNA polymerase
(Boehringer Mannheim) was added and the mixture was overlaid with 30,ul
of mineral oil (Sigma). Incubations were done in a DNA Thermal Cycler
(Ericomp). One cycle included a denaturation step (940C for 1 min.), an
annealing step (600C for 2 min.), and an elongation step (720C for 3 min.).
25 After 35 cycles, a 10 ~l aliquot of each reaction was run on a 1.5%
agarose gel to verify the correct structure of the amplified product.
The amplified DNA was gel purified and digested with Ndel and
BamHI and subcloned into Ndel and BamHI-digested pZlB1. This digestion
and subcloning step removed the FGF-encoding DNA and 5' portion of SAP
30 up to the BamHI site at nucleotides 555-560 (SEQ ID No. 12) and replaced
this portion with DNA encoding a saporin molecule that contains a cysteine
residue at position -1 relative to the start site of the native mature SAP
protein. The resulting plasmid is designated pZ50B1.
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C. Preparation of saporin with a cysteine residue at position 4 or
- 10 of the native protein
These constructs were designed to introduce a cysteine residue at
position 4 or 10 of the native protein by replacing the isoleucine residue at
5 position 4 or the asparagine residue at position 10 with cysteine.
1. Materials
(a) Bacterial strains
The bacterial strains were Novablue and BL21 (DE3) (NOVAGEN,
Madison, Wl).
(b) DNA manipulations
DNA manipulations as described above.
2. Preparation of modified SAP-encoding DNA
SAP was amplified by polymerase chain reaction (PCR) from the
parental plasmid pZ1 B1 encoding the FGF-SAP fusion protein.
(aJ Primers
(1I The primer corresponding to the sense
strand of saporin, spanning nucleotides
466-501 of SEQ ID NO. 12, incorporates
a Ndel site and replaces the lle codon
. 20 with a Cys codon at position 4 of the
mature protein (SEQ ID NO. 38):
CATATGGTCACATCATGTACATTAGATCTAGTAAAT.
(2) The primer corresponding to the sense
strand of sapGri", nucleotides 466-515 of
SEQ ID NO. 12, incorporates a Ndel site
and replaces the Asp codon with a cys
codon at position 10 of the mature
protein (SEQ ID NO. 39)
CATATGGTCACATCAATCACATTAGATCTAGTATGTCCGACCGCGGGTCA
(3) Primer #2 - Antisense primer
complements the coding sequence of
saporin spanning nucleotides 547-567 of
SEQ ID NO. 12 and contains a BamHI site
(SEQ ID NO. 35):
CAGGTTTGGATCCTTTACGTT.
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b~ (b) Amplification
The PCR reactions were performed as described above, using the
following cycles: denaturation step 94C for 1 min, annealing for 2 min at
60C, and extension for 2 min at 72C for 35 cycles. The amplified DNA
5 was gel purified, digested with Ndel and BamHI, and subcloned into Ndel
and BamHI digested pZ1B1. This digestion removed the FGF and 5' portion
of SAP (up to the newly added BamHI) from the parental FGF-SAP vector
(pZ1 B1) and replaced this portion with a SAP molecule containing a CYS at
position 4 or 10 relative to the start site of the native mature SAP protein.
10 The resulting plasmids are designated pZ51B1 and pZ52B1, respectively.
D. Cloning of DNA encoding SAP mutants in vector pET15b
The initial step in this construction was the mutagenesis of the
internal BamHI site at nucleotides 555-560 (SEQ ID NO. 12) in pZ1 B1 by
PCR using a sense primer corresponding to nucleotides 543-570 (SEQ ID
15 NO. 12) but changing the G at nucleotide 555 (the third position in the Lys
codon) to an A. The complement of the sense primer was used as the
antisense primer. The PCR reactions were conducted as in B above. One,ul
of the resulting PCR product was used in a second PCR reaction using the
same sense oligonucleotide as in B., above, in order to introduce a Ndel site
20 and a Cys codon onto the 5' end of the saporin-encoding DNA. The
antisense primer was complementary to the 3' end of the saporin protein
and encoded a BamHI site for cloning and a stop codon (SEQ ID NO. 37):
GGATCCGCCTCGTTTGACTACTT .
The resulting plasmid was digested with Ndel/BamHI and inserted
25 into pET15b (NOVAGEN, Madison, Wl), which has a His-TagTM leader
sequence (SEQ ID NO. 36), that had also been digested Ndel/BamHI.
The SAP-Cys-4 and Sap-Cys-10 mutants were similarly inserted into
pET15b using SEQ ID Nos. 38 and 39, respectively as the sense primers
and SEQ ID NO. 37 as the antisense primer.
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DNA encoding unmodified SAP (EXAMPLE 1 ) can be similarly
inserted into a pet1 5b or pet1 1 A and expressed as described below for the
modified SAP-encoding DNA.
E. Expression of the modified saporin-encoding DNA
BL21 (DE3) cells were transformed with the resulting plasmids and
cultured as described in Example 2, except that all incubations were
conducted at 30 C instead of 37 C. Briefly, a single colony was grown in
LB AMP1oo to and OD600 of 1.0-1.5 and then induced with IPTG (final
concentration 0.1mM) for 2 h. The bacteria were spun down.
F. Purification of modified saporin
Lysis buffer (20 mM NaP04, pH 7.0, 5 mM EDTA, 5 mM EGTA,
1 mM DTT, 0.5,ug/ml leupeptin, 1 ,ug/ml aprotinin, 0.7,ug/ml pepstatin)
was added to the rSAP cell paste (produced from pZ50B1 in BL21 cells, as
described above) in a ratio of 1.5 ml buffer/g cells. This mixture was
evenly suspended via a Polytron homogenizer and passed through a
microfluidizer twice.
The resulting Iysate was centrifuged 50,000 rpm for 45 min. The
supernatant was diluted with SP Buffer A (20 mM NaP04, 1 mM EDTA, pH
7.0) so that the conductivity was below 2.5 mS/cm. The diluted Iysate
supernatant was then loaded onto a SP-Sepharose column, and a linear
gradient of 0 to 30% SP Buffer B (1 M NaCI, 20 mM NaPO4, 1 mM EDTA,
pH 7.0) in SP Buffer A with a total of 6 column volumes was applied.
Fractions containing rSAP were combined and the resulting rSAP had a
purity of greater than 90%.
A buffer exchange step was used here to get the SP eluate into a
buffer containing 50 mM NaB03, 1 mM EDTA, pH 8.5 (S Buffer A). This
sample was then applied to a Resource S column (Pharmacia, Sweden) pre-
equilibrated with S Buffer A. Pure rSAP was eluted off the column by 10
column volumes of a linear gradient of 0 to 300 mM NaCI in SP Buffer A.
The final rSAP was approximately 98% pure and the overall yield of rSAP
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was about 50% (the amount of rSAP in crude Iysate was determined by
ELISA) .
In this preparation, ultracentrifugation was used to clarify the Iysate;
other methods, such as filtration and using floculents also can be used. In
addition, Streamline S (PHARMACIA, Sweden) may also be used for large
scale preparations.
EXAMPLE 6
A. Cytotoxicity assays of co,~ tes
Cytotoxicity experiments were performed with the Promega
(Madison, Wl) CellTiter 96 Cell Proliferation/Cytotoxicity Assay. Cell types
used were SK-Mel-28, human melanoma Swiss 3T3 mouse fibroblasts
(from Dr. Pamela Maher, La Joila, CA), B16F10, mouse melanoma, PA-1,
human ovarian carcinoma (from Dr. Julie Beitz, Roger Williams Hospital,
Providence Rl), and baby hamster kidney (BHK) [obtained from the
American Type Culture Collection (ATCC)]. 2500 cells were plated per
well.
B. Coupling of FGF muteins to SAP
1. Chemical Synthesis of [C78S]FGF-SAP(CCFS2) and
[C96S]FGF-SAP(CCFS3)
[C78S]FGF or [C96S]FGF (1 mg; 56 nmol) that had been dialyzed
against phosphate-buffered saline was added to 2.5 mg mono-derivatized
SAP (a 1.5 molar excess over the basic FGF mutants) and left on a rocker
platform overnight. The next morning the ultraviolet-visible wavelength
spectrum was taken to determine the extent of reaction by the release of
pyridylthione, which adsorbs at 343 nm with a known extinction
coefficient. The ratio of pyridylthione to basic FGF mutant for [C78S]FGF
was 1.05 and for [C96S]FGF was 0.92. The reaction mixtures were
treated identically for purification in the following manner: reaction mixture
was passed over a HiTrap heparin-Sepharose column (1 ml) equilibrated
with 0.15 M sodium chloride in buffer A at a flow rate of 0.5 ml/min. The
column was washed with 0.6 M NaCI and 1.0 M NaCI in buffer A and the
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product eluted with 2.0 M NaCI in buffer A. Fractions (0.5 ml) were
analyzed by gel electrophoresis and absorbance at 280 nm. Peak tubes
were pooled and dialyzed versus 10 mM sodium phosphate, pH 7.5 and
applied to a Mono-S 5/5 column equilibrated with the same buffer. A
5 10 ml gradient between 0 and 1.0 M sodium chloride in equilibration buffer
was used to elute the product. Purity was determined by gel
electrophoresis and peak fractions were pooled. The yield for
[C78S]FGF-SAP was 1.6 mg (60% with respect to starting amount of
[C78S]FGF) and was 0.96 mg [C96S]FGF-SAP (35%).
Virtually 100% of the mutant FGFs reacted with mono-derivatized
SAP ([C78S]FGF: 105%, [C96S]FGF: 92%). Because the free surface
cysteine of each mutant acts as a free sulfhydryl, it was unnecessary to
reduce cysteines after purification from the bacteria. The resulting product
was purified by heparin-Sepharose (data not shown), thus establishing that
15 heparin binding activity of the conjugate is retained.
Coomassie staining and Western blotting of the purified proteins
showed a prominent band at a molecular weight of about 48,000,
corresponding to the combined molecular weights of SAP and bFGF. A
much lighter band at a slightly lower molecular weight was detected and
20 attributed to the described mobility of an artifact produced by the high
isoelectric point (10.5) (Gelfi et al. (1987) J. Biochem. BioPhvs. Meth.
15:41-48) of SAP that causes a smearing in SDS gel electrophoresis ~see,
e.~., Lappi et al. (1985) Biochem. BioPhvs. Res. Commun. 129:934-942).
No higher molecular weight bands, corresponding to conjugates containing
25 more than one molecule of SAP per molecule of basic FGF or more than
one molecule of basic FGF per molecule of SAP were detected on
- Coomassie-stained gels of [C78S]FGF-SAP) and of ([C96S]FGF-SAP). Such
bands were present in lanes on the gel in which an equal quantity (by
weight) of heterogeneous FGF-SAP, synthesized from wild-type bFGF and
30 non-purified derivatized SAP, had been loaded.
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Western blotting using antibodies to SAP or basic FGF revealed that,
while 480 ng of either [C78S]FGF-SAP or [C96S]FGF-SAP results in a well-
visualized band (with the additional slight lower molecular weight band) the
same quantity of conjugate produced by the previous procedure is almost
5 undetectable. As in the Coomassie staining, the Western blotting of the
mutant FGF-SAPs reveals much greater homogeneity than with
heterogeneous FGF-SAP synthesized with non-mutagenized basic FGF and
non-purified derivatized SAP.
2. Preparation of [C96S]FGF-rSAP (CCFS4)
Recombinant saporin that has the cys added at the N-terminus (SAP-
CYS-(-1)) that was cloned and expressed in BL21 cells and isolated as
described in EXAMPLE 4 was coupled to [C96S]FGF using (5,5'-dithiobis-
(2-nitrobenzoic acid)) DTNB also called Ellman's reagent. The rSAP and
[C96S]FGF were each treated with 10 mM dithiothreitol (DTT), incubated
15 for 1 h at room temperature, and the DTT was removed by gel filtration in
conjugation buffer (0.1 M NaP04, 100 NaCI and 1 mM EDTA, pH 7.5). A
100-fold molar excess of DTNB was added to the rSAP, incubated for 1 h
at room temperature. Unreacted DTNB was removed by gel filtration. The
[C96S]FGF was added to DTNB-treated SAP (3:1 molar ratio of
20 [C96S]FGF:SAP) and incubated at room temperature for about 1 hr or for
16 hrs at 4 C. The mixture was loaded on heparin sepharose in 10 mM
NaP04, 1 mM EDTA, pH 6 and the conjugate and free [C96S]FGF were
eluted with 2 M NaCI in 10 mM NaP04, 1 mM EDTA, pH 6. The free
[C96S]FGF was removed by gel filtration on Sephacryl S100 (Pharmacia).
25 The resulting conjugate was designated CCFS4.
C. Cytotoxicity of [C78SlFGF-SAP (CCFS2), [C96S]FGF-SAP
(CCFS3) and [C96S]FGF-rSAP (CCFS4)
Cytotoxicity of the two mutant FGF-SAPs to several cell types has
been tested. Heterogeneous FGF-SAP (CCFS1 ) is very cytotoxic to
30 SK-MEL-28 cells, human melanoma cells, with an ED50 of approximately
8 ng/ml. The mutant FGF-SAPs are also potently cytotoxic to these cells.
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[C78S]FGF-SAP and [C96S]FGF-SAP each have an ED50 comparable to the
heterogeneous chemically conjugates, indicting that mutant FGFs are able
to internalize SAP to virtually the same extent as the heterogeneous
FGF-SAP.
Similar results were obtained with an ovarian carcinoma cell type,
PA-1, Swiss 3T3 cells, B16F10, a mouse melanoma and BHK cells
(Table 6).
CCFS4 was tested in the in vitro cytotoxicity assay and its activity
is at ieast as good to the wild-type chemical conjugate (CCFS1).
TABLE 6
CYTOTOXICITY OF HOMOGENEOUS AND HETEROGENOUS
FGF-SAPs TO CELL LINES
ED50s (ng/ml)
Cell Type Heterogeneous
[C96SIFGF-SAP[C78S]FGF-SAP FGF-SAP
SK-MEL-28 8 12 8
Swiss 3T3 60 100 40
PA-1 70 100 40
B16F10 2 3 2
BHK 20 25 15
D. Preparation of homogeneous mixtures of FGF-SAP muteins by
splicing by overlap extension (SOE)
1. Conversion of Cys 78 to Ser 78
(a) Materials
(1) Plasmids
Plasmid PZ1B (designated PZ1B1) described in Example 2 was used
as the DNA template. The primers were prepared as follows:
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(2) Primers
(a) Primer #1 spanning the Ndel site at
the 5' end of the FGF-encoding
DNA from IJlasmE J pZlB:
AAATACTTACATATGGCAGCAGGATC (SEQ ID NO.18).
(b) Primer #Z - A,.lise.,se primer to
nucleotides spa.~ the Cys 78
(nucleotides 220-Z49 of SEQ ID
N0. 12 with base change to
generate Ser 78):
CAGGTAACGGTTAGCAGACACTCCTTTGAT (SEQ ID NO.19).
(cJ Primer #3 - Sense primer to
nucleotides spanning the Cys 78
(nucieotides 220-249 of SEQ ID
N0. 12 with base change to
generate Ser 78):
ATCAAAGGAGTGTCTGCTAACCGTTACCTG (SEQ ID NO. 20).
(d) Primer #4 - A..li:.ense primer to
spanning the Ncol site of FGF in
pZlB (cor-espo,-din~ to nucleotides
456-485 of SEQ ID N0. 12):
GTGATTGATGTGACCATGGCGCTCTTAGCA (SEQ ID NO. 21).
(b) Reactions
(1 ) Reaction A
PZ1B1 DNA (100 ng) was mixed (final volume of 100,u1 upon
addition of the Taq polymerase) with primer #1 (50,uM); primer #2(50
,uM), 10 mM Tri-HCI (pH 8.3), 50 mM KCI, 0.01% gelatin, 2 mM MgCI2,
0.2 mM dNTPs.
(2) Reaction B
Same as above except that primer #3 (50 ,~IM) and primer #4 (50
,uM) were used in place of primers #1 and #2.
Each reaction mixture was heated to 95 C for 5 min, 0.5 U Taql
DNA polymerase (1 ,ul; Boehringer Mannheim) was added and the mixture
was overlaid with 100,ul of mineral oil (Perkin Elmer Cetus). Incubations
were done in a DNA Thermal Cycler (Ericomp). Each cycle included a
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denaturation step (9~oC for 1 min.), an annealing step (600C for 1.5 min.),
and an elongation step (750C for 3 min.). After 20 cycles, the reaction
mixture was incubated at 75 C for 10 minutes for a final elongation. The
products were resolved on a 2% agarose gel and DNA of the correct size
(247 bp and 250 bp) was purified. The ends were repaired by adding
nucleoside triphosphates and Klenow DNA polymerase.
~3) Reaction C
One,ul of each product of reactions A and B were mixed (final
volume of 100 ~L upon addition of Taq polymerase) with primers #1 and
#4 (final concentration of each was 50,uM); 10 mM Tri-HCI (pH 8.3),
50 mM KCI, 0.01% gelatin, 2 mM MgCI2, 0.2 mM dNTPs.
The resulting reaction mixture was heated to 95 C for 5 min, 0.5 U
Taql DNA polymerase (1 ,LII; Boehringer Mannheim) was added and the
mixture was overlaid with 100,ul of mineral oil (Perkin Elmer Cetus).
Incubations were done in a DNA Thermal Cycler (Erricomp). Each cycle
includled a denaturation step (950C for 1 min.), an annealing step (600C for
1.5 min.), and an elongation step (750C for 3 min.), followed, after 20
cycles, by a final elongation step at 75 C for 10 minutes.
The amplified product was resolved on a 1.5% agarose gel and the
correct size fragment (460 bp), designated FGFC78S-SAP was purified.
2. Generation of DNA encoding FGFC78/C96S-SAP
(a) Materials
(1 ) Te,..~,lale
DNA encoding FGFC78S-SAP.
(2) Primers
(a) Primer #5-Sense primer spanning
the Cys 96 (nucleotides 275-300
of SEQ ID NO. 12 with base
change to generate Ser 96)
TGGCTTCTAAATCTGTTACGGATGAG (SEQ ID NO. 22).
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(b) Primer #6-Antisense prim~r
spz.."i"g the Cys 96 (nucleotides
275-300 of SEQ ID N0. 12 with
base change to generate Ser 96):
CTCATCCGTAACAGATTTAGAAGCCA (SEQ ID N0. 23).
(b) Reactions
(1) Rea-;liGn D
FGFC78S-SAP-encoding DNA (100 ng) was mixed (final volume of
lOO,ul upon addition of the Taq polymerase) with primer #1 (50,uM);
primer #5 (50,uM), 10 mM Tri-HCI (pH 8.3), 50 mM KCI, 0.01% gelatin,
2 mM MgCI2 and 0.2 mM dNTPs.
(2) Reaction E
Same as above, except that primers #4 and #6 (50 ,uM final
concentration of each) were used instead of primers #1 and #5.
Each reaction mixture was heated to 95 C for 5 min, 0.5 U Taql
DNA polymerase (1 ~I;Boehringer Mannheim) was added and the mixture
was overlaid with 100 ~ul of mineral oil (Perkin Elmer Cetus). Incubations
were done in a DNA Thermal Cycler (Ericomp). Each cycle included a
denaturation step (950C for 1 min.), an annealing step (600C for 1.5 min.),
and an elongation step (750C for 3 min.) for 20 cycles, followed by a final
elongation step at 75 C for 10 minutes. The products were resolved on a
2% agarose gel and DNA of the correct size (297 bp and 190 bp) was
purified. The ends were repaired by adding nucleoside triphosphates and
Klenow DNA polymerase.
(3) Reaction F
The product of reactions D and E (100 ng of each) were mixed (final
volume of 100 ,uL upon addition of Taq polymerase) with primers #1 and
#4 and amplified as described above. The amplified product resolved on a
1.5% agarose gel and the correct size fragment (465 bp) was purified. The
resulting product, DNA that encodes FGFC78/96S-SAP, had Ndel and Ncol
ends. It was digested with Ndel and Ncol and ligated into Ndel/Ncol-
digested PZlB1 and into Ndel/Ncol-digested PZlC1 (PZIC described in
WO 95/03831 PCTIUS94/08511
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Example 2 above). The resulting constructs were designated PZ2B1 and
PZ2C1, respectively.
E. E~.r~ssio,. of the recor, ' ~a..l FGFC78/96S-SAP fusion
proteins (FPFS4) from PZ2B1 and PZ2C1
The two-stage method described above for production of FPFS1 was
used to produce recombinant FGFC78/96S-SAP protein (hereinafter FPFS4).
Two hundred and fifty mls. of LB medium containing ampicillin
(100 ~g/ml) were inoculated with a fresh glycerol stock of PZ1 B. Cells
were grown at 30 C in an incubator shaker to an OD600 of 0.7 and stored
overnight at 4 C. The following day the cells were pelleted and
resuspended in fresh LB medium (no ampicillin). The cells were divided into
5 1-liter batches and grown at 30 C in an incubator shaker to an OD600 of
1.5. IPTG (SIGMA CHEMICAL, St. Louis, MO) was added to a final
concentration of 0.1 mM and growth was continued for about 2 to
2.5 hours at which time cells were harvested by centrifugation.
In order to grow PZ2C1, prior to induction, the cells were grown in
medium containing kanamycin (50,ug/ml) in place of ampicillin.
F. Biological Activity
The cytotoxicity of the mutein FGF-SAP produced from PZ2B1
~FPFS4) was assessed on SK MEL 28 cells and was at least equivalent to
the activity of the wild type FGF-SAP chemical conjugate, and recombinant
FGF-SAP produced from PZ1 B1 .
The in vivo activity of the mutein FGF-SAP produced from PZ2B1
has been tested in animals, and it appears to be less toxic than FGF-SAP
from PZ1B1 (FPFS1).
EXAMPLE 7
THERAPEUTIC ACTIVITY OF THE WILD-TYPE CHEMICAL CONJUGATE
AND FUSION PROTEIN bFGF-SAP IN THE MOUSE TUMOR XENOGRAFT
MODEL
A. Materials and methods
The methods set forth below were performed substantially as
described in Beitz et ai. (1992) Cancer Research 52:227-230).
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l1) Study Design
Sixty-three athymic mice bearing subcutaneous tumors received four
weekly bolus IV injections of the test materials. Tumor volumes were
measured twice weekly for 61 days.
(2) Test Materials
Wild-type chemical conjugate bFGF-SAP was supplied in Dulbecco's
phosphate buffered saline (PBS) at a concentration of 1.0 mg/ml. Fusion
protein bFGF-SAP in E. coli was supplied in Dulbecco's PBS at a
concentration of 9.0 mg/ml. Basic FGF was supplied in Dulbecco's PBS at a
concentration of 1.0 mg/ml. Saporin was supplied in Dulbecco's PBS
(0.01 M Phosphate, 0.14 M NaCI, pH 7.4) at a concentration of 1.0 mg/ml.
Ail dilutions were made in Dulbecco's PBS with 0.1% bovine serum
albumin (NB 1005-18).
(3) ~pecies
Female Balb/c nu/nu athymic mice (Roger Williams Hospital Animal
Facility, Providence, Rl), 8-12 weeks old, were maintained in an aseptic
environment. Sixty-three animals were selected for the study, and body
weights ranged from 25-30 grams the day prior to dosing.
(4) HuslL an.l~ y
Animals were maintained in a quarantined room and handled under
aseptic conditions. Food and water were supplied ad libitum throughout
the experiment.
(5) Tumor Cells
PA-1 human ovarian teratocarcinoma cells were obtained from the
American Type Culture Collection (Rockville, MD; ATCC accession no.
CRL1572) were grown in modified Eagle's medium supplemented with
10% fetal calf serum.
(6) Turnor Implantation
Five days prior to injection of the test material, mice received a
subcutaneous injection of tumor cells (approximately 2 x 106 PA-1 human
ovarian teratocarcinoma cells/mouse) in the right rear flank.
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(7) Tumor Size Measurements
Calipers were used to measure the dimensions of each tumor.
Measurements (mm) of maximum and minimum width were performed prior
to injection of the test material and at bi-weekly intervals for 61 days.
Tumor volumes (mm3) were computed using the formula
Volume=[(minimum measurement)2(maximum measurement)]/2.
(8) Dose Pre~ar~lio,.
Dosing material was prepared by mixing the test material with
appropriate volumes of PBS/0.1 % BSA to achieve the final doses.
(9) Dosing Procedures
Individual syringes were prepared for each animal. Mice received
four weekly IV injections (250-300 ul) into the tail vein on days 5, 12, 19
and 26 with day 1 designated as the day that the tumor cells were injected
into the mice. Doses were individualized for differences in body weight.
B. Results - Inhibition of tumor growth
In all animals, tumors were measured prior to injection of the test
material and at bi-weekly intervals for 61 days. Tumors from animals in all
groups were approximately 55-60 mm3 on day 5 when treatment began.
The vehicle-treated group (PBS with 0.1 % BSA) showed a 50-fold increase
in tumor volume over the 61 days of the study. The other control groups
demonsL-aled similar levels of tumor growth: the SAP control group
showed a 30-fold increase, the bFGF control group showed a 50-fold
increase, and the bFGF plus SAP group showed a 50-fold increase in tumor
volume. In all the control groups, the rate of growth of the tumor was
fairly consistent over the 61-day period. In the treated groups, with wild-
type chemical conjugate bFGF-SAP and fusion protein bFGF-SAP, there
appeared to be a statistically significant dose-related suppression in tumor
growth compared to controls over the first 30 days; however, tumor
volumes increased again after this period such that there was no longer a
statistical difference between the treated and control groups.
WO 95/03831 PCT/US94/08511
~ ~b~1
The 50 ,ug/kg/week fusion protein bFGF-SAP-treated groups
exhibited tumor volumes that were 29% of controls, but a statistical
comparison to controls was not done because only two animals in the
treated group survived to 30 days. The fusion protein bFGF-SAP 5.0
,ug/kg/week dose achieved significant suppression of tumor growth, with
tumor volumes at 48% of control values. The 0.5 ,ug/kg/week fusion
protein bFGF-SAP group showed significant suppression of tumor growth to
day 26 when tumors were at 71% of controls. There was no statistical
difference between tumor volumes in the 0.5 ,ug/kg/week wild-type
chemical conjugate bFGF-SAP and fusion protein bFGF-SAP groups at 30
days. A statistical comparison of the two 50,ug/kg/week treatment groups
was not done because there were only two surviving animals in the fusion
protein bFGF-SAP group.
All seven animals survived the 61-day study in all groups with the
exception of the 50,ug/kg/week chemical conjugate bFGF-SAP group (3 of
7 survived to 61 days) and the 50,ug/kg/week fusion protein bFGF-SAP
group (1 of 7 survived to 61 days).
Since modifications will be apparent to those of skill in this art, it is
intended that this invention be limited only by the scope of the appended
claims.
WO 95/03831 PCT/US94/08511
~l ~86~7
-91-
8~ ~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
tA) NAME: Prizm Pharmaceuticals
(B) STREET: 10655 Sorrento Valley Road, Suite 200
(C) CITY: San Diego
(D) STATE: California
(E) COUN'1'~: USA
(F) POSTAL CODE (ZIP): 92121
(i) APPLICANT:
(A) NAME: The Whittier Institute for Diabetes and
Endocrinology
(B) STREET: 9894 Genesee Avenue
(C) CITY: La Jolla
(D) STATE: California
(D) C'~UN'1'~: USA
(E) POSTAL CODE (ZIP): 92037
(ii) TITLE OF 1N~N'1'10N: MONOGENOUS PREPARATIONS
OF CYTOTOXIC CONJUGATES
(iii) NUMBER OF S~QU~N~'~S: 39
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(V) ~UKR~N1 APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/145,829
(B) FILING DATE: 29-OCT-1993
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/099,924
(B) FILING DATE: 02-AUG-1993
(2) INFORMATION FOR SEQ ID NO:1:
(i) 8~U~h CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) sT~Nn~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/ Æ Y: misc_recomb
(B) LOCATION: 6..11
(D) OTHER INFORMATION: /standard_name= "EcoRI Restriction Site"
WO 95/03831 PCT/US94/08511
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 12..30
(D) OTHER INFORMATION: /function= ~N-terminal extension"
/product= "Native saporin signal peptide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CTGCAGAATT CGCATGGATC CTGCTTCAAT30
(2) lN~ORISATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: 6..11
(D) OTHER INFORMATION: /standard_name= ~EcoRI Restriction Site"
(ix) FEATURE:
(A) NAME/XEY: terminator
(B) LOCATION: 23..25
(D) OTHER INFORMATION: /note= ~Anti-sense ~top codonl'
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 26..30
(D) OTHER INFORMATION: /note= ~Anti-sense to carboxyl
terminus of mature peptide"
(xi) S~UU~N~ DESCRIPTION: SEQ ID NO:2:
CTGCAGAATT CGC~lC~LLl GACTACTTTG30
(2) INFORMATION FOR SEQ ID NO:3:
(i) s~u~N~: CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEAluKE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= ~Nucleotide sequence
corresponding to the clone M13 mpl8-G4 in Example I.B.2."
WO 9S/03831 PCT/US94/08511
-
-
~ 686~7
-93-
(ix) FEATURE:
- (A) NAME/KEY: mat_peptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= ""Saporin""
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48
Ala Trp Ile Leu Leu Gln Phe Ser Ala Trp Thr Thr Thr Asp Ala Val
-15 -10 -5
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96
Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gln Tyr Ser
5 10 15
TCT TTT GTG GAT AAA ATC CGA AAC AAT GTA AAG GAT CCA AAC CTG AAA 144
Ser Phe Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys
20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA A~A 192
Tyr Gly Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu Lys
35 40 45
TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240
Phe Leu Arg Ile Asn Phe Gln Ser Ser Arg Gly Thr Val Ser Leu Gly
50 55 60 65
CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288
Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn
70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC TTC AAA TCA GAA ATT ACT TCC GCC 336
Thr Asn Val Asn Arg Ala Tyr Tyr Phe Ly8 Ser Glu Ile Thr Ser Ala
85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384
Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gln Lys Ala
100 105 110
TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432
Leu Glu Tyr Thr Glu Asp Tyr Gln Ser Ile Glu Lys Asn Ala Gln Ile
115 120 125
ACA CAG GGA GAT AAA AGT AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA 480
Thr Gln Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu
130 135 140 145
CTT TTG ACG TTC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528
Leu Leu Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys
150 155 160
AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA 576
Asn Glu Ala Arg Phe Leu Leu Ile Ala Ile Gln Met Thr Ala Glu Val
165 170 175
GCA CGA TTT AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC 624
Ala Arg Phe Arg Tyr Ile Gln Asn Leu Val Thr Lys Asn Phe Pro Asn
180 185 190
AAG TTC GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT 672
Lys Phe Asp Ser Asp Asn Lys Val Ile Gln Phe Glu Val Ser Trp Arg
195 200 205
W O gS/03831 PCT~US94/08511
~ G~1 -94-
AAG ATT TCT ACG GCA ATA TAC GGG GAT GCC A~A AAC GGC GT& TTT AAT 720
Lys Ile Ser Thr Ala Ile Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn
210 215 220 225
A~A GAT TAT GAT TTC GGG TTT GGA A~A GTG AGG CAG GTG AAG GAC TTG 768
Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gln Val Lys Asp Leu
230 235 240
CAA ATG GGA CTC CTT ATG TAT TTG GGC A~A CCA AAG 804
Gln Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys
245 250
(2) INFORMATION FOR SEQ ID NO:4:
( i ) S ~:~U~N~'~ CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) sTR~N~n~s double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEAlUKE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEATu~E:
(A) NAME/ Æ Y: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= ~Nucleotide sequence
corresponding to the clone M13 mpl8-G1 in Example I.B.2."
(ix) FEATURE:
(A) NAME/KEY: mat ~eptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48
Ala Trp Ile Leu Leu Gln Phe Ser Ala Trp Thr Thr Thr Asp Ala Val
-15 -10 -5
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96
Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gln Tyr Ser
5 10 15
TCT TTT GTG GAT A~A ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG A~A 144
Ser Phe Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys
20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT A~A GAA A~A 192
Tyr Gly Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu Lys
35 40 45
TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240
Phe Leu Arg Ile Asn Phe Gln Ser Ser Arg Gly Thr Val Ser Leu Gly
50 55 60 65
CTA A~A CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288
Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn
WO 95/03831 PCTtUS94/08511
.
-95~ 6*
ACG AAT GTT AAT CGG GCA TAT TAC TTC AGA TCA GAA ATT ACT TCC GCC 336
Thr Asn Val Asn Arg Ala Tyr Tyr Phe Arg Ser Glu Ile Thr Ser Ala
85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384
Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gln Lys Ala
100 105 110
TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GA~ AAG AAT GCC CAG ATA 432
Leu Glu Tyr Thr Glu Asp Tyr Gln Ser Ile Glu Lys Asn Ala Gln Ile
115 120 125
ACA CAG GGA GAT AAA TCA AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA 480
Thr Gln Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu
130 135 140 145
CTT TTG ACG TCC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528
Leu Leu Thr Ser Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys
150 155 160
AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA 576
Asn Glu Ala Arg Phe Leu Leu Ile Ala Ile Gln Met Thr Ala Glu Val
165 170 175
GCA CGA TTT CGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC 624
Ala Arg Phe Arg Tyr Ile Gln Asn Leu Val Thr Lys Asn Phe Pro Asn
180 185 190
AAG TTC GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT 672
Lys Phe Asp Ser Asp Asn Lys Val Ile Gln Phe Glu Val Ser Trp Arg
195 200 205
AAG ATT TCT ACG GCA ATA TAC GGA GAT GCC AAA A~C GGC GTG TTT AAT 720
Lys Ile Ser Thr Ala Ile Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn
210 215 220 225
AAA GAT TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG 768
Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gln Val Lys Asp Leu
230 235 240
CAA ATG GGA CTC CTT ATG TAT TTG GGC AAA CCA AAG 804
Gln Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys
245 250
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRAN~ S: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= ~Nucleotide sequence
W O 95/03831 PCT~US94/08~11
.
6 ~ -96-
corresponding to the clone M13 mpl8-G2 in Example I.B.2.
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48
Ala Trp Ile Leu Leu Gln Phe Ser Ala Trp Thr Thr Thr Asp Ala Val
-15 -10 -5
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACT GCG GGT CAA TAC TCA 96
Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gln Tyr Ser
5 10 15
TCT TTT GTG GAT A~A ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG A~A 144
Ser Phe Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys
20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT A~A GAT A~A 192
Tyr Gly Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Asp Lys
35 40 45
TTC CTT AGA ATT AAT TTC CA~ AGT TCC CGA GGA ACG GTC TCA CTT GGC 240
Phe Leu Arg Ile Asn Phe Gln Ser Ser Arg Gly Thr Val Ser Leu Gly
50 55 60 65
CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288
Leu LYR Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn
70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC TTC A~A TCA GAA ATT ACT TCC GCC 336
Thr Asn Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu Ile Thr Ser Ala
85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG A~A GCT 384
Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gln Lys Ala
100 105 110
TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432
Leu Glu Tyr Thr Glu Asp Tyr Gln Ser Ile Glu Lys Asn Ala Gln Ile
115 120 125
ACA CAG GGA GAT A~A AGT AGA A~A GAA CTC GGG TTG GGG ATC GAC TTA 480
Thr Gln Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu
130 135 140 145
CTT TTG ACG TTC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT A~A 528
Leu Leu Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys
150 155 160
AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA 576
Asn Glu Ala Arg Phe Leu Leu Ile Ala Ile Gln Met Thr Ala Glu Val
165 170 175
GCA CGA TTT AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC 624
Ala Arg Phe Arg Tyr Ile Gln Asn Leu Val Thr Lys Asn Phe Pro Asn
180 185 190
WO 95/03831 PCT/US94/08511
_97_ ~ l 6 $ G ~
AAG TTC GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT 672
Lys Phe Asp Ser Asp Asn Lys Val Ile Gln Phe Glu Val Ser Trp Arg
195 200 205
AAG ATT TCT ACG GCA ATA TAC GGG GAT GCC A~A AAC GGC GTG TTT AAT 720
- Lys Ile Ser Thr Ala Ile Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn
210 215 220 225
AAA GAT TAT GAT TTC GGG TTT GGA A~A GTG AGG CAG GTG AAG GAC TTG 768
Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gln Val Lys Asp Leu
230 235 240
CAA ATG GGA CTC CTT ATG TAT TTG GGC A~A CCA AAG 804
Gln Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys
245 250
(2) INFORMATION FOR SEQ ID NO:6:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) sTR~n~n~s: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= ~Nucleotide sequence
corresponding to the clone M13 mpl8-G7 in Example I.B.2."
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= ~Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48
Ala Trp Ile Leu Leu Gln Phe Ser Ala Trp Thr Thr Thr Asp Ala Val
-15 -10 -5
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96
Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gln Tyr Ser
5 10 15
TCT TTT GTG GAT A~A ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG A~A 144
Ser Pne Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys
20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT A~A GAA AAA 192
Tyr Gly Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu Lys
35 40 45
TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240
Phe Leu Arg Ile Asn Phe Gln Ser Ser Arg Gly Thr Val Ser Leu Gly
W 0 95/03831 PCT~US94/08511
.
98-
CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288
Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn
70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC TTC AGA TCA GAA ATT ACT TCC GCC 336
Thr Asn Val Asn Arg Ala Tyr Tyr Phe Arg Ser Glu Ile Thr Ser Ala
85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG A~A GCT 384
Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gln Lys Ala
100 105 110
TTA GAA TAC ACA GA~ GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432
Leu Glu Tyr Thr Glu Asp Tyr Gln Ser Ile Glu Lys Asn Ala Gln Ile
115 120 125
ACA CAG GGA GAT A~A TCA AGA A~A GAA CTC GGG TTG GGG ATC GAC TTA 480
Thr Gln Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu
130 135 140 145
CTT TTG ACG TCC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT A~A 528
Leu Leu Thr Ser Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys
150 155 160
AAC GAA GCT AGA TTC CTT CTT ATC GCT ATT CAG ATG ACG GCT GAG GCA 576
Asn Glu Ala Arg Phe Leu Leu Ile Ala Ile Gln Met Thr Ala Glu Ala
165 170 175
GCA CGA TTT AGG TAC ATA CAA AAC TTG GTA ATC AAG AAC TTT CCC AAC 624
Ala Arg Phe Arg Tyr Ile Gln Asn Leu Val Ile Lys Asn Phe Pro Asn
180 185 190
AAG TTC AAC TCG GAA AAC A~A GTG ATT CAG TTT GAG GTT AAC TGG A~A 672
Lys Phe Asn Ser Glu Asn Lys Val Ile Gln Phe Glu Val Asn Trp Lys
195 200 205
A~A ATT TCT ACG GCA ATA TAC GGG GAT GCC A~A AAC GGC GTG TTT AAT 720
Lys Ile Ser Thr Ala Ile Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn
210 215 220 225
A~A GAT TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG 768
Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gln Val Lys Asp Leu
230 235 240
CAA ATG GGA CTC CTT ATG TAT TTG GGC A~A CCA AAG 804
Gln Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys
245 250
(2) INFORMATION FOR SEQ ID NO:7:
(i) ~U~ ~ CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATuKE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
WO 95/03831 PCT/US94/08511
~16864?
_99
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= "Nucleotide sequence
corresponding to the clone M13 mpl8-G9 in Example I.B.2.
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= "Saporin"
(xi) ~u~N~ DESCRIPTION: SEQ ID NO:7:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48
Ala Trp Ile Leu Leu Gln Phe Ser Ala Trp Thr Thr Thr Asp Ala Val
-15 -10 -5
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96
Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gln Tyr Ser
5 10 15
TCT TTT GTG GAT A~A ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG A~A 144
Ser Phe Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys
20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT A~A GAA A~A 192
Tyr Gly Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu Lys
35 40 45
TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240
Phe Leu Arg Ile Asn Phe Gln Ser Ser Arg Gly Thr Val Ser Leu Gly
50 55 60 65
CTA A~A CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288
Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn
70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC TTC AGA TCA GAA ATT ACT TCC GCC 336
Thr Asn Val Asn Arg Ala Tyr Tyr Phe Arg Ser Glu Ile Thr Ser Ala
85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384
Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gln Lys Ala
100 105 110
TTA GAA TAC ACA GAA GAT TAT CAG TCG ATT GAA AAG AAT GCC CAG ATA 432
Leu Glu Tyr Thr Glu Asp Tyr Gln Ser Ile Glu Lys Asn Ala Gln Ile
115 120 125
ACA CAA GGA GAT CAA AGT AGA A~A GAA CTC GGG TTG GGG ATT GAC TTA 480
Thr Gln Gly Asp Gln Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu
130 135 140 145
CTT TCA ACG TCC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528
Leu Ser Thr Ser Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys
150 155 160
GAC GAA GCT AGA TTC CTT CTT ATC GCT ATT CAG ATG ACG GCT GAG GCA 576
Asp Glu Ala Arg Phe Leu Leu Ile Ala Ile Gln Met Thr Ala Glu Ala
165 170 175
WO 95/03831 PCT/US94/08~11
.
6~ 100-
GCG CGA TTT AGG TAC ATA CAA AAC TTG GTA ATC AAG AAC TTT CCC AAC 624
Ala Arg Phe Arg Tyr Ile Gln Asn Leu Val Ile Lys Asn Phe Pro Asn
180 185 190
AAG TTC AAC TCG GAA AAC AAA GTG ATT CAG TTT GAG GTT AAC TGG AAA 672
Lys Phe Asn Ser Glu Asn Lys Val Ile Gln Phe Glu Val Asn Trp Lys
195 200 205
AAA ATT TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT 720
Lys Ile Ser Thr Ala Ile Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn
210 215 220 225
A~A GAT TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG 768
Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gln Val Lys Asp Leu
230 235 240
CAA ATG GGA CTC CTT ATG TAT TTG GGC AAA CCA AAG 804
Gln Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys
245 250
(2) INFORMATION FOR SEQ ID NO:8:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: 10..I5
(D) OTHER INFORMATION: /standard_name= "Nco I restriction enzyme
recognition site"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 15..22
(D) OTHER INFORMATION: /product= ~N-terminus of Saporin
protein"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CAACAACTGC CATGGTCACA TC 22
(2) INFORMATION FOR SEQ ID NO:9:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: 11. 16
(D) OTHER INFORMATION: /standard_name= "Nco I restriction enzyme
recognition site."
WO 95/03831 PCT/US94/08511
.
~1 6 86~7
-101-
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: l..l0
(D) OTHER INFORMATION: /product= ~Carboxy terminus of
mature FGF protein"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCTAAGAGCG CCATGGAGA l9
(2) INFORMATION FOR SEQ ID NO:l0:
U~N~ CHARACTERISTICS:
(A) LENGTH: 2l base pairs
(B) TYPE: nucleic acid
(C) STR~ )N~:~S: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: l..12
(D) OTHER INFORMATION: /product= ~'Carboxy terminus of
wild type FGF"
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: l3..18
(D) OTHER INFORMATION: /standard_name= "Nco I restriction enzyme
recognition site"
(Xi) 8h'~U~ DESCRIPTION: SEQ ID NO:l0:
GCT AAG AGC TGACCATGGA GA 2l
Ala Lys Ser
(2) INFORMATION FOR SEQ ID NO:ll:
( i ) ~QU ~:N~ CHARACTERISTICS:
(A) LENGTH: 102 base pairs
(B) TYPE: nucleic acid
(C) STRPN~ S: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEAlu~E:
(A) NAME/KEY: CDS
(B) LOCATION: l..96
(D) OTHER INFORMATION: /product= "pFGFNcoI"
/note= ~E~uals the plasmid pFC80 wih native FGF
stop codon removed."
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: 29..34
(D) OTHER INFORMATION: /standard_name= "Nco I restriction enzyme
recognition site"
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:ll:
W O 95/03831 PCT~US94/08511
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~t h~ -102-
CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GAG ATC CGG CTG AAT 48
Leu Phe Leu Pro Met Ser Ala LYB Ser Ala Met Glu Ile Arg Leu Asn
1 5 10 15
GGT GCA GTT CTG TAC CGG TTT TCC TGT GCC GTC TTT CAG GAC TCC TGAAATCTT
102
Gly Ala Val Leu Tyr Arg Phe Ser Cys Ala Val Phe Gln Asp Ser
20 25 30
(2) INFORMATION FOR SEQ ID NO:12:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 1230 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~n~s double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1230
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product= "bFGF"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 472..1230
. (D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48
Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly
1 5 10 15
GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96
Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu
20 25 30
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA 144
Tyr Cy8 Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
35 40 45
GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192
Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu
50 55 60
CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC 240
Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn
65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288
Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cy~
85 90 95
GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC 336
Val Thr Asp Glu cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr
100 105 110
WO 95/03831 PCTAus94/08511
6,~
-103-
AAT ACT TAC CGG TCA AGG A~A TAC ACC AGT TGG TAT GTG GCA TTG A~A 384
Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys
115 120 125
CGA ACT GGG CAG TAT A~A CTT GGA TCC A~A ACA GGA CCT GGG CAG A~A 432
Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys
130 135 140
GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GTC ACA TCA 480
Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Val Thr Ser
145 150 155 160
ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA TCT TTT 528
Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gln Tyr Ser Ser Phe
165 170 175
GTG GAT A~A ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG A~A TAC GGT 576
Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly
180 185 190
GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT A~A GAA A~A TTC CTT 624
Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu Lys Phe Leu
195 200 205
AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC CTA A~A 672
Arg Ile Asn Phe Gln Ser Ser Arg Gly Thr Val Ser Leu Gly Leu Lys
210 215 220
CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC ACG AAT 720
Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn Thr Asn
225 230 235 240
GTT AAT CGG GCA TAT TAC TTC A~A TCA GAA ATT ACT TCC GCC GAG TTA 768
Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu Ile Thr Ser Ala Glu Leu
245 250 255
ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG A~A GCT TTA GAA 816
Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gln Lys Ala Leu Glu
260 265 270
TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA ACA CAG 864
Tyr Thr Glu Asp Tyr Gln Ser Ile Glu Lys Asn Ala Gln Ile Thr Gln
275 280 285
GGA GAT A~A AGT AGA A~A GAA CTC GGG TTG GGG ATC GAC TTA CTT TTG 912
Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu Leu Leu
290 295 300
ACG TTC ATG GAA GCA GTG A~C AAG AAG GCA CGT GTG GTT A~A AAC GAA 960
Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys Asn Glu
305 310 315 320
GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA GCA CGA 1008
Ala Arg Phe Leu Leu Ile Ala Ile Gln Met Thr Ala Glu Val Ala Arg
325 330 335
TTT AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC AAG TTC 1056
Phe Arg Tyr Ile Gln Asn Leu Val Thr Lys Asn Phe Pro Asn Lys Phe
340 345 350
W O 9~103831 PCTrUS94/08511
04-
GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT 1104
Asp Ser Asp Asn Lys Val Ile Gln Phe Glu Val Ser Trp Arg Lys Ile
355 360 365
TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT AAA GAT 1152
Ser Thr Ala Ile Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn Lys Asp
370 375 380
TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG CAA ATG 1200
Tyr Asp Phe Gly Phe Gly Lys Val Arg Gln Val Lys Asp Leu Gln Met
385 390 395 400
GGA CTC CTT ATG TAT TTG GGC AAA CCA AAG 1230
Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys
405 410
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1230 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1230
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product= "bFGF~
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 472..1230
(D) OTHER INFORMATION: /product= "Saporin"
(Xi) ~UU~N~ DESCRIPTION: SEQ ID NO:13:
ATG GCT GCT GGT TCT ATC ACT ACT CTG CCG GCT CTG CCG GAA GAC GGT 48
Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly
1 5 10 15
GGT TCT GGT GCT TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96
Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu
20 25 30
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA 144
Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
35 40 45
GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192
Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu
50 55 60
CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC 240
Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn
WO 95/03831 PCT~us94/08~11
.
64~7
-105-
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288
Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys
85 90 95
GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC 336
Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr
100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG G Q TTG AAA 384
Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys
115 120 125
CGA ACT GGG Q G TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG QG AAA 432
Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys
130 135 140
GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GTC A Q T Q 480
Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Val Thr Ser
145 150 155 160
ATC A Q TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA TCT TTT 528
Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gln Tyr Ser Ser Phe
165 170 175
GTG GAT AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA TAC GGT 576
Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly
180 185 190
GGT ACC GAC ATA GCC GTG ATA GGC C Q CCT TCT A~A GAA AAA TTC CTT 624
Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu Lys Phe Leu
195 200 205
AGA ATT AAT TTC CA~ AGT TCC CGA GGA ACG GTC TCA CTT GGC CTA A~A 672
Arg Ile Asn Phe Gln Ser Ser Arg Gly Thr Val Ser Leu Gly Leu Lys
210 215 220
CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT G Q ATG GAT AAC ACG AAT 720
Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn Thr Asn
225 230 235 240
GTT AAT CGG GCA TAT TAC TTC AAA TCA GAA ATT ACT TCC GCC GAG TTA 768
Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu Ile Thr Ser Ala Glu Leu
245 250 255
ACC GCC CTT TTC CCA GAG GCC ACA ACT G Q AAT Q G AAA GCT TTA GAA 816
Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gln Lys Ala Leu Glu
260 265 270
TAC ACA GAA GAT TAT QG TCG ATC GAA AAG AAT GCC CAG ATA ACA CAG 864
Tyr Thr Glu Asp Tyr Gln Ser Ile Glu Lys Asn Ala Gln Ile Thr Gln
275 280 285
GGA GAT AAA AGT AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA CTT TTG 912
Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu Leu Leu
290 295 300
ACG TTC ATG GAA GCA GTG AAC AAG AAG G Q CGT GTG GTT AAA AAC GAA 960
Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys Asn Glu
305 310 315 320
WO 95/03831 PCT/US94/08511
106-
GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA GCA CGA 1008
Ala Arg Phe Leu Leu Ile Ala Ile Gln Met Thr Ala Glu Val Ala Arg
325 330 335
TTT AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC AAG TTC 1056
Phe Arg Tyr Ile Gln Asn Leu Val Thr Lyæ Asn Phe Pro Asn Lys Phe
340 345 350
GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT 1104
Asp Ser Asp Asn Lys.Val Ile Gln Phe Glu Val Ser Trp Arg Lys Ile
355 360 365
TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT A~A GAT 1152
Ser Thr Ala Ile Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn Lys Asp
370 375 380
TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG CAA ATG 1200
Tyr Asp Phe Gly Phe Gly Lys Val Arg Gln Val Lys Asp Leu Gln Met
385 390 395 400
GGA CTC CTT ATG TAT TTG GGC AAA CCA AAG 1230
Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys
405 410
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:14:
AATTCCCCTG TTGACAATTA ATCATCGAAC TAGTTAACTA GTACGCAGCT TGGCTGCAG 59
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GTCGACCAAG CTTGGGCATA CATTCAATCA ATTGTTATCT AAGGA~ATAC TTACATATG 59
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
WO 95/03831 PCT/US94/08511
.
2 1 ~$G~,7
-107-
(xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:16:
AGGAGTGTCT GCTAACC 17
(2) INFORMATION FOR SEQ ID NO:17:
(i) ~U~:N~ CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~SS: 5 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TTCTAAATCG GTTACCGATG ACTG 24
(2) INFORMATION FOR SEQ ID NO:18:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) s~Qu~N~ DESCRIPTION: SEQ ID NO:18:
AAATACTTAC ATATGGCAGC AGGATC 26
(2) INFORMATION FOR SEQ ID No:Is
(i) ~OU~N~ CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CAGGTAACGG TTAGCAGACA CTCCTTTGAT 30
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
ATCAAAGGAG TGTCTGCTAA CCGTTACCTG 30
WO 95/03831 PCT/US94/08511
.
08-
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(xi) ~u~N~ DESCRIPTION: SEQ ID NO:21:
GTGATTGATG TGACCATGGC GCTCTTAGCA 30
(2) INFORMATION FOR SEQ ID NO:22:
(i) S~u~N~ CXARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANnRnNR~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TGGCTTCTAA A~ ACG GATGAG 26
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CEARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genowic)
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CTCATCCGTA ACAGATTTAG AAGCCA 26
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) ~u~ DESCRIPTION: SEQ ID NO:24:
Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe
1 5 10 15
WO 95103831 PCTIUS94/08511
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-109-
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly
Thr Arg Asp Arg Ser Asp Gln Eis Ile Gln Leu Gln Leu Ser Ala Glu
Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu
Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr
100 10S 110
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys
115 120 125
Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala
130 135 140
Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
145 150 155
(2) INFORMATION FOR SEQ ID NO:25:
(i) S~Qu~N~ CHARACTERISTICS:
lA) LENGTH: 155 amino acids
B) TYPE: amino acid
C) STRAN~N~:SS: single
lD) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) S~u~c~ DESCRIPTION: SEQ ID NO:25:
Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly
1 5 10 15
Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu
Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu
Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn
Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys
Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr
100 105 110
Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys
115 120 125
WO 9~;/03831 PCT/US94/08511
bQ0~
-1 1 O-
Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys
130 135 140
Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser
145 150 155
(2) INFORMATION FOR SEQ ID NO:26:
(i) ~U~NC~ CHARACTERISTICS:
(A) LENGTH: 239 amino acids
(B) TYPE: amino acid
(C) STRA-NDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:26:
Met Gly Leu Ile Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp
1 5 10 15
Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg Arg Asp Ala Gly Gly Arg
Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu
Tyr Cys Ala Thr Lys Tyr His Leu Gln Leu His Pro Ser Gly Arg Val
Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala
Val Glu Val Gly Ile Val Ala Ile Arg Gly Leu Phe Ser Gly Arg Tyr
Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr Ser
100 105 110
Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn Thr
115 120 125
Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala Arg
130 135 140
Arg Gln Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val Asn Gly Lys
145 150 155 160
Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser
165 170 175
Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val Arg
180 185 1go
Gln Leu Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gln Pro
195 200 205
Arg Arg Arg Arg Gln Lys Gln Ser Pro Asp Asn Leu Glu Pro Ser His
210 215 220
Val Gln Ala Ser Arg Leu Gly Ser Gln Leu Glu Ala Ser Ala His
225 230 235
WO 95/03831 PCT/US94/08511
~ 7~
-1 1 1 -
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 206 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nN~s single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) ~QU~N~'~ DESCRIPTION: SEQ ID NO:27:
Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu
1 5 10 15
Leu Ala Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro
Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu
Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro
Lys Glu Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile
Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu
Gln Ala Leu Pro Asp Gly Arg Ile Gly Gly Ala His Ala Asp Thr Arg
. 100 105 110
Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser Ile
115 120 125
Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys
130 135 140
Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu Ile
145 150 155 160
Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly
165 170 175
Met Phe Ile Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg
180 185 190
Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu
195 200 205
(2) INFORMATION FOR SEQ ID NO:28:
( i ) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(C) STR~N~ N~:~S: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) ~QukN~ DESCRIPTION: SEQ ID NO:28:
W 0 95/03831 PCTrus94/08511
-112-
Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu Ile Leu
1 5 10 15
Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gln Pro
Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly Ser Ser Ser Arg Gln
Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala
Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln
Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly
Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser
100 105 110
His Glu Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln
115 120 125
Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met
130 135 140
Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys
145 150 155 160
Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser
165 170 175
Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu
180 185 190
Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro
195 200 205
Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln
210 215 220
Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro
225 230 235 240
Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr
245 250 255
Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly
260 265
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nN~s single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
WO 95/03831 PCT/US94/08511
~ ~8 6~7
-113-
Met Ser Arg Gly Ala Gly Arg Leu Gln Gly Thr Leu Trp Ala Leu Val
1 5 10 15
Phe Leu Gly Ile Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Thr
Arg Ala Asn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu
Ser Arg Ser Arg Ala Gly Leu Ala Gly Glu Ile Ala Gly Val Asn Trp
Glu Ser Gly Tyr Leu Val Gly Ile Lys Arg Gln Arg Arg Leu Tyr Cys
Asn Val Gly Ile Gly Phe His Leu Gln Val Leu Pro Asp Gly Arg Ile
Ser Gly Thr His Glu Glu Asn Pro Tyr Ser Leu Leu Glu Ile Ser Thr
100 105 110
Val Glu Arg Gly Val Val Ser Leu Phe Gly Val Arg Ser Ala Leu Phe
115 120 125
Val Ala Met Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gln
130 135 140
Glu Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala
145 150 155 160
Tyr Glu Ser Asp Leu Tyr Gln Gly Thr Tyr Ile Ala Leu Ser Lys Tyr
165 170 175
Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro Ile Met Thr Val Thr
180 185 190
His Phe Leu Pro Arg Ile
195
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~N~s: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) ~U~N~'~ DESCRIPTION: SEQ ID NO:30:
Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg
1 5 10 15
Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser Leu Ala Cys
Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser
Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile
WO 95/03831 PCT/US94/08511
-114-
Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp
Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn
Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly
100 105 110
Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr
115 120 125
Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu
130 135 140
Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly
145 150 155 160
Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly
165 170 175
Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala
180 185 190
Ile Thr
(2) INFORMATION FOR SEQ ID NO:31:
(i) ~U~N-~'~ CHARACTERISTICS:
(A) LENGTH: 215 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:31:
Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu
1 5 10 15
Val Leu Cys Leu Gln Ala Gln Val Thr Val Gln Ser Ser Pro Asn Phe
Thr Gln His Val Arg Glu Gln Ser Leu Val Thr ASp Gln Leu Ser Arg
Arg Leu Ile Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His
Val Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly
Asp Pro Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg
Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys
100 105 110
Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val
115 120 125
WO 95/03831 PCTAus94/08511
-115-
Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala
130 135 140
Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg
145 150 155 160
Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu Val His Phe Met Lys
165 170 175
Arg Leu Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg Phe Glu
180 185 190
Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg
195 200 205
Thr Trp Ala Pro Glu Pro Arg
210 215
(2) INFORMATION FOR SEQ ID NO:32:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 208 amino acids
(B TYPE: amino acid
(C STRAN~N~SS: single
(D, TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(Xi) ~U~'N~ DESCRIPTION: SEQ ID NO:32:
Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala
1 5 10 15
Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu
Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly
Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg
Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly
Thr Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu
Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser
100 105 110
Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu
115 120 125
Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp
130 135 140
Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg
145 150 155 160
Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr
165 170 175
WO 95tO3831 PCT~us94/085ll
~ 116-
Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val
180 185 190
Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile ~eu Ser Gln Ser
195 200 205
(2) INFORMATION FOR SEQ ID NO:33:
(i) ~U~N~ CHARACTERISTICS:
(A LENGTH: 40 amino acids
(B TYPE: amino acid
(C~ STRANDEDNESS: single
(D, TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
~xi) S~Q~N~ DESCRIPTION: SEQ ID NO:33:
al Ile Ile Tyr Glu Leu Asn Leu Gln Gly Thr Thr Lys Ala Gln Tyr
er Thr Ile Leu Lys Gln Leu Arg Asp Asp Ile Lys Asp Pro Asn Leu
20 25 30
Xaa Tyr Gly Xaa Xaa Asp Tyr Ser
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~nN~.~s: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) ~u~N~ DESCRIPTION: SEQ ID NO:34
CATATGTGTG TCACATCAAT CACATTAGAT 30
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) ~Q~N~ DESCRIPTION: SEQ ID NO:35
CAG~lll~A LC~LllACGT T 21
(2) INFORMATION FOR SEQ ID NO:36:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 82 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
WO 9S/03831 PCT/US94/08Sll
-
~ ~1 6~6y7
-117-
(ii) MOLECULE TYPE: DNA tgenomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36
AAGGAGATATACC ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC AGC AGC ~3
Met Gly Ser Ser His His His His His His Ser Ser
l 5 l0
GGC CTG GTG CCG CGC GGC AGC CAT ATG CTC GAG GAT CCG 82
Gly Leu Val Pro Arg Gly Ser His Met Leu Glu Asp Pro
15 20 25
(2) INFORMATION FOR SEQ ID NO:37:
( i ) S ~.~U~N~ CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) sTRANnT~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37
GGATCCGCCT C~Lll~ACTA CTT 23
(2) INFORMATION FOR SEQ ID NO:38:
QU~N~'~ CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) sTR~NnT2n~s single
(D) TOPOLOGY: linear
(ii) ~T.T~TT.T~ TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38
CATATGGTCA CATCATGTAC ATTAGATCTA GTA~AT 36
(2) INFORMATION FOR SEQ ID NO:39:
(i) ~OU~N~ CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) sTR~NnT~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULB TYPE: DNA (genomic)
(Xi) ~ U~N~ DESCRIPTION: SEQ ID NO:39
CATATGGTCA CATCAATCAC ATTAGATCTA GTATGTCCGA CCGCGGGTCA 50
,
