Note: Descriptions are shown in the official language in which they were submitted.
CA 02201944 2002-03-06
WO 96122369 PCTlUS95I12907
- 1 -
ANALOGS OF KERATINOCYTE GROWTH FACTOR HAVING
ENHANCED TEMPERATURE STABILITY
The complex process of healing which follows
injury to tissue, such as by wounding or burning, is
mediated by a number of protein factors sometimes
referred to as soft tissue growth factors. These
factors are required for the growth and differentiation
of new cells to replace the destroyed tissue. Included
within this group of soft tissue growth factors is a
protein family of fibroblast growth factors (FGFs). The
FGFs are mitogenic and chemotactic for a variety of
cells of epithelial, mesenchymal, and neural origins.
In addition, FGFs are angiogenic, that is they are able
to stimulate the formation of blood vessels. Members of
the FGF family include acidic FGF, basic FGF, KGF,
Int-2, HST, FGF-5, and FGF-6.
Acidic FGF (aFGF) and basic FGF (bFGF) are
considered to be two "original".members of the FGF
family. Both aFGF and bFGF are believed to be derived
from the same ancestral gene, with both molecules having
approximately 55% sequence identity in addition to the
same intron/exon structure. Acidic FGF and bFGF are
also known to bind to the same receptor, although the
existence of specific aFGF and bFGF receptors has not
been ruled out. Several molecular weight forms of aFGF
and bFGF are found in different tissues. However,
Southern blotting experiments suggest that there is only
one gene each for aFGF and bFGF, with differences
between these molecules probably being due to post-
translational processing. Both acidic and basic FGF are
mitogens for a wide variety of cell types of mesodermal
and neuroectodermal origin, and are able to induce
CA 02201944 1999-11-26
WO 96122369 PCT/US95/12907
angiogenesis boti-:, in vitro and in viva (see, e.g.,
Gospodarowicz et a1 (1979)), Exp. Eye Res., ~:SO1-514.
The range of biological activities e' the two classes is
near'_y identical, although bFGF is about ten times more
potent than aFGF in most bioassay systems.
F~;GF exhibits potent mitogenic activity for a
variety of cells and it binds to cell surface receptors
on Balb/I~C kerati.nocytes to which aFGF and bFGF may also
bind (Bottaro,et a1 (1990), J. Eiol. Chem., ~: 12767-
12770. However, KGF is distinct from the known FGFs
(e.g., aFGF and bFGF) in that it is not mitogenic for
fibroblasts or endothelial cells. Rubin et a1,(1989),
Proc. Natl. Acad. Sci. USA, ~: 802-806. KGF also has
different receptors on NIH/3T3 fibroblasts from the
receptors f or aFG~F and bFGF which fail to interact with
KGF. (Botta.ro et a1.(1990), J. Biol. Chem.,
265:12767-7.2770)
A shared distinguishing feature of aFGF and
bFGF is the propensity of these factors to bind tightly
to heparin. The affinity of aFGF for heparin appears to
be weaker than for bFGF, with aFGF having an anionic
isoelectric: point. (Thomas et a1 (1984) , Proc. Nat. Acad.
Sci. USA, ,~';~:6409-6413. The unique heparin binding
property of aFGF and bFGF has greatly facilitated T
purification of these factors.
'.the discovery that FGFs have strong affinity
for immobilized heparin has also spurred investigation
into the regulatory role of heparin-like molecules in
the in viva biology of FGFs. Although the full spectrum
of functions for heparin has yet to be determined, it is
known that heparin can regulate FGF function in several
ways (Lobb (1988), Eur. J. CZin. Invest., 1$:321-326.
For example, heparin-like molecules can play a direct
role in FGF function, including the activation, or
CA 02201944 1997-04-04
WO 96/22369 PCTJUS95/12907
- 3 -
potentiation, of aFGFs (Uhllrich et a1 (1986), 9iochem.
Biophys. Res. Comm., ,7:1205-1213.
There is, however, no direct correlation
between the affinity of the FGF for immobilized heparin
and its ability to be potentiated by soluble heparin.
In this respect, the potentiating power of heparin
appears to be selective for aFGF. For example, Uhllrich
et a1. (1986), supra, found the degree of potentiation
of pure aFGF to be about ten times greater than that of
pure bFGF, raising the potency of the aFGF to
approximately the same level as that of bFGF. However,
in the presence of fetal calf serum, the potentiating
effect of heparin was found to decrease significantly
(Uhllrich et a1. (1986) , supra) .
The use of FGF proteins is believed to be
effective in promoting the healing of tissue subjected
to trauma. The unique angiogenic property of FGFs makes
these factors especially valuable in the healing of deep
wounds. The bFGF native proteins have been alleged to
be useful in the treatment of myocardial infarction
(U.S. Patents No. 4,296,100 and 4,378,347). In
addition, human bFGF has-been found to increase neuronal
survival and neurite extension in fetal rat hippocampal
neurons, suggesting that this factor may also be useful
in the treatment of degenerative neurological disorders,
such as Alzheimer's disease and Parkinson's disease
(Wallicke et al (1986), Proc. Natl. Acad. Sci. USA, ~:
3012-3016).
A major stumbling block to the effective use
of aFGF in therapeutic applications appears to be
related to its significantly lower biological activity,
as compared with bFGF. Although studies with heparin
suggest that the observed difference in potency between
CA 02201944 1997-04-04
WO 96/22369 PCTIUS95/12907
- 4 -
aFGF and bFGF can be substantially diminished by using
heparin to boost the activity of aFGF to a level
comparable to that of bFGF, the use of heparin in
pharmaceutical preparations may not always be desirable.
In this regard, it is important to note that heparin, a
highly sulfated glycosaminoglycan of heterogeneous
structure, is known to be an anticoagulant which
functions by accelerating the rate at which antithrombin
III inactivates the proteases of homeostasis (Jacques
(1980) , Pharmacol Rev, ~: 99-166) . It is not known
whether it might be deleterious to use heparin in a
pharmaceutical preparation for the treatment of deep
wounds, where some degree of coagulation may be desired
to achieve proper healing.
In addition, practical considerations can be
expected to arise where heparin is incorporated into a
pharmaceutical preparation for wound healing. Drug
delivery concerns include the matter of controlling the
composition of the pharmaceutical preparation
(containing the combination of aFGF and heparin) upon
entry into the patient's body. Moreover, the negative
effect of fetal calf serum on the potentiating effect of
heparin on aFGF (observed by Uhllrich et al) suggests
that any advantage obtained by including heparin in the
pharmaceutical preparation as an activating or
potentiating factor for aFGF could be completely negated
or lost once contact is made with the patient's own
serum.
It is an object of the present invention to
provide an analog of protein from the FGF family having
enhanced stability as compared to the naturally
occurring form of the protein It is a further object of
the present invention to provide an analog of aFGF which
exhibits enhanced stability and biological activity in
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 5 -
the absence of heparin. It is a further object of the
present invention to provide an aFGF analog for
therapeutic use.
y,mma_ry of the Invention
The present invention provides novel analogs
of proteins in the FGF family. One such analog is an
aFGF analog that is more stable and exhibits greater
biological activity in the absence of heparin than
naturally occurring aFGF. Another such analog is a KGF
analog that has enhanced thermal stability as compared
to naturally occurring KGF. Enhanced stability is
achieved by substituting at least one amino acid having
higher loop-forming potential for an amino acid residue
of lower loop-forming potential in or about the loop-
forming sequence Asn-His-Tyr-Asn-Thr-Tyr of the
naturally occurring protein. In the case of aFGF, this
loop-forming sequence occurs in the area of about amino
acids 92 to 96. In the case of KGF, this loop-forming
region occurs in the area of about amino acids 115-119.
A preferred analog of the present invention incorporates
the substitution of an amino acid having higher loop-
forming potential for the histidine residue in the loop-
forming sequence.
FIG. 1 shows the nucleic acid and amino acid
sequences of recombinant bovine [Ala4~,G1y93] aFGF.
FIG. 2 show the amino acid sequence of
recombinant human [G1y93] aFGF.
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 6 -
FIG. 3 demonstrates the elution profiles for
bovine [Ala4~] and [Ala4~,G1y93] aFGF analogs using
hydrophobic interaction chromatography.
FIGS. 4A and 4B show the circular dichroic
spectra for bovine [Ala4~] and (Ala4~,G1y93] aFGF
analogs.
FIG. 5 shows the second derivative FTIR
spectra of bovine [Ala4~] and [Ala4~,G1y93) aFGF analogs
in the amide I' tC=0 stretch in deuterated proteins)
region.
FIG. 6 is a graph showing a plot of the log of
the concentration of bovine [Ala4~) and [Ala4~,G1y93]
aFGF analogs and human [Ser~O,Ser88] bFGF versus the
percentage of maximal stimulation.
FIG. 7 is a graph showing the loss of activity
over time of bovine [Ala4~) and [Ala4~,G1y93] aFGF
analogs in the absence of heparin as compared with human
[Ser~O,SerBa] bFGF.
FIG. 8 shows the structure of the bovine
[Ala4~,G1y93] aFGF analog of the present invention, as
determined by X-ray crystallography.
FIG. 9 shows the nucleic acid and amino acid
sequences of naturally occurring KGF.
FIG. 10 shows the nucleic acid and amino acid
sequences of recombinant [G1y116] KGF.
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
peta_iled Description of the T_nvention
Novel analogs of the FGF family are provided
in accordance with the present invention. These analogs
exhibit improved stability, as compared with the
corresponding naturally occurring protein. In the case
of the aFGF analog of the present invention, the analog
exhibits enhanced stability and biological activity in
the absence of heparin. In the case of the KGF analog
of the present invention, the analog exhibits enhanced
thermal stability. The analogs of the present invention
have at least one different amino acid residue from the
corresponding naturally occurring protein in or about
the loop-forming sequence Asn-His-Tyr-Asn-Thr-Tyr found
in the naturally occurring form. In the case of aFGF,
the loop forming sequence occurs in the area of about
amino acid residues 92 to 96 (based on the numbering of
the known amino acid sequence for bovine aFGF, as shown
in Fig. 1). In the case of KGF, the loop forming
sequence occurs in the area of about amino acid residues
115-119, as shown in Figs. 9 and 10. The different
amino acids) is selected for its higher loop-forming
potential in order to stabilize this area of the analog.
Amino acids having relatively high loop-forming
potential include glycine, proline, tyrosine, aspartic
acid, asparagine, and serine [Leszcynski et al. (1986),
Science, 24:849-855 (1986) (relative values of loop-
forming potential assigned on the basis of frequency of
appearance in loop structures of naturally occurring
molecules)]. Preferably, a different amino acid having
higher loop-forming potential replaces the histidine
residue in the loop-forming sequence. Still more
preferably, the histidine in the loop-forming sequence
is replaced with a glycine residue.
CA 02201944 1997-04-04
WO 96/22369 PCTJUS95/12907
g _
Other additions, substitutions, and/or
deletions may be made to the analogs of the present
invention. For example, the analog may also optionally
include an amino acid substitution for non-conserved
cysteine residues (e.g., the cysteine residue at
position 47 of the bovine aFGF molecule and the cysteine
residue at position 16 of the human aFGF molecule). In
addition, the analogs of the present invention which are
expressed from E. coli host cells may include an initial
methionine amino acid residue (i.e., at position -1, as
shown in Fig. 1). Alternatively, one or more of the
terminal amino acid residues may be deleted from the DNA
sequence, as is known to those skilled in the art, while
substantially retaining the enhanced biological activity
of the corresponding naturally occurring protein.
DNA sequences coding for all or part of the
analogs of the present invention are also provided.
Such sequences preferably may include the incorporation
of codons "preferred" for expression by selected E. coli
host strains ("E. coli expression codons"), the
provision of sites of cleavage by restriction
endonuclease enzymes, and/or the provision of additional
initial, terminal, or intermediate DNA sequences which
facilitate construction of readily expressed vectors.
These novel DNA sequences include sequences useful in
securing the expression of the analogs of the present
invention in both eucaryotic and procaryotic host cells,
such as E. coli.
More specifically, the DNA sequences of the
present invention may comprise the DNA sequence set
forth in Fig. 1, wherein at least one codon encoding an
amino acid residue in the area of about amino acids 92
to 96 is replaced by a codon encoding a different amino
acid residue having a higher loop-forming potential
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
_ g _
(hereinafter "aFGF analog sequence(s)" or "analog
sequences)"), as well as a DNA sequence which
hybridizes to one of the analog sequences or to
fragments thereof, and, a DNA sequence which, but for
the degeneracy of the genetic code, would hybridize to
one of the analog sequences.
Correspondingly, the DNA sequences of the
present invention may comprise the DNA sequence set
forth in Fig. 10, wherein at least one codon encoding an
amino acid residue in the area of about amino acids 115-
119 is replaced by a codon encoding a different amino
acid residue having a higher loop-forming potential
(hereinafter "KGF analog sequence(s)" or "analog
sequences)"), as well as a DNA sequence which
hybridizes to one of the analog sequences or to
fragments thereof, and, a DNA sequence which, but for
the degeneracy of the genetic code, would hybridize to
one of the analog sequences.
The analogs of the present invention can be
encoded, expressed, and purified by any one of a number
of recombinant technology methods known to those skilled
in the art. The preferred production method will vary
depending upon many factors and considerations,
including the cost and availability of materials and
other economic considerations. The optimum production
procedure for a given situation will be apparent to
those skilled in the art through minimal
experimentation. The analogs of the present invention
can be expressed at particularly high levels using
E. coli host cells, with the resulting expression
product being subsequently purified to near homogeneity
using procedures known in the art. A typical
purification procedure involves first solubilizing the
inclusion bodies containing the analogs, followed by ion
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 10 -
exchange chromatography, then refolding of the protein,
and finally, hydrophobic interaction chromatography.
The analogs of the present invention exhibit a
surprising degree of enhanced stability. Unlike
naturally occurring aFGF, the aFGF analogs of the
present invention demonstrate enhanced stability and
biological activity in the absence of heparin. While it
is known that more stable bFGF analogs can be obtained
through the substitution of serine or other neutral
amino acids in place of certain cysteine residues (for
example, as disclosed in published PCT Patent
Application No. 88/04189), substitution for the non-
conserved cysteine residue at position 47 of naturally
occurring bovine aFGF alone is not believed to be
significant in enhancing the biological activity and/or
stability of an aFGF analog. This is demonstrated by
the lower activity exhibited by a bovine [A1a47] aFGF
analog (cysteine substituted) compared with a bovine
[A1a47,G1y93] aFGF analog (having the desired amino acid
substitution in the residue 92 to 96 region of the aFGF
molecule), as set forth in the examples which follow.
Specifically, the bovine (A1a47,G1y93] analog, although
still less potent compared with the bFGF, was found to
be approximately ten times more potent than the bovine
[Ala4~] aFGF analog. Upon the addition of 45 ~g/ml
heparin, bioactivity of all three forms of FGF was
enhanced, with the bovine [A1a47,G1y93) analog, the
bovine [Ala4~,Gly93J analog, and human [Ser~O,Ser88]
bFGF analog having substantially identical potency.
The reason for the enhanced mitogenic activity
and stability of the bovine (A1a47,G1y93J aFGF analog
relative to bovine [A1a47) aFGF in the absence of
heparin was not immediately clear. The substitution of
glycine for the histidine residue at position 93
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 11 -
appeared to make the aFGF molecule somewhat more
hydrophobic, but it did not appear to drastically alter
its tertiary structure, as determined by circular
dichroism and FTIR spectroscopy. However, the relative
differences in the activities observed in the in vitro
bioassays for the bovine [A1a47,G1y93] aFGF analog and
for the bovine (A1a47] aFGF analog (with substitution at
only position 47) suggested that the glycine-substituted
amino acid 93 position of the bovine [A1a47,G1y93] aFGF
analog might be within or near the region responsible
for receptor binding. Although the receptor binding
region in aFGF has not been determined, position 93 in
aFGF corresponds to a region in bFGF which is reported
to be within or near the receptor binding domain (Baird
et a1 (1988), Proc. Nat. Acad. Sci. USA, $,x:2324-2328.
In addition, the bovine [A1a47,G1y93] aFGF
analog of the present invention, unlike the bovine
[A1a47] analog, exhibited enhanced stability,
maintaining its original mitogenic activity in the
absence of heparin over the course of 250 hours, while
the bovine [A1a47] analog rapidly lost activity.
The bovine [A1a47,G1y93] aFGF analog was
crystallized, and the resulting crystals examined by
X-ray crystallography. The X-ray crystallographic data
obtained from examination of these crystals supports the
suggestion from the hydrophobic interaction
chromatography data that residue 93 is exposed to
solvent, i.e., that the glycine for histidine
substitution at position 93 makes the molecule less
hydrophilic. Detailed examination of the bovine
[Ala9~,G1y93] aFGF analog sequence around residue 93
revealed a clustering of approximately 8 amino acids
with high loop-forming potentials in the region from
about the glutamic acid residue at position 90 to about
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 12 -
the tyrosine residue at position 97. The relative loop-
forming potentials of amino acids have been reported,
with glycine being identified as the amino acid residue
having the highest loop-forming potential among all
amino acids (Leszcynski et a1 supra). Thus, the glycine
for histidine substitution is believed to stabilize the
presumed loop, due to the much higher loop-forming
potential of the glycine residue compared with
histidine.
The KGF analogs of the present invention also
demonstrate that the corresponding region in KGF is a
solvent-exposed loop that may be involved in receptor
binding. Specifically, the [G1y116)KGF analog of the
present invention was found to exhibit an altered,
decreased mitogenic activity compared to naturally
occurring KGF, as set forth in the examples which
follow. The [G1y116J KGF analog was also found to have
5-7°C higher thermal stability relative to naturally
occurring KGF.
Other analogs, in addition to the preferred
[G1y93] aFGF and [G1y116] KGF analog specifically set
forth herein, are contemplated by the present invention.
These other analogs could easily be made by one skilled
in the art by following the teachings provided herein.
For example, there are no fewer than fifteen amino acids
reported to have higher loop-forming potential than
histidine (Leszcynski et al . (1986) , supra) . These
amino acids are (in descending order of loop-forming
potential) glycine, proline or tyrosine, aspartic acid
or asparagine, serine, cysteine, glutamic acid,
threonine, lysine, cystine, glutamine, arginine,
phenylalanine, and tryptophan. Substitution of any of
these residues for the histidine residue in the loop-
forming sequence of the naturally occurring protein
CA 02201944 1997-04-04
WO 96/22369 PCTIUS95/12907
- 13 -
could be expected to result in an analog of the present
invention having an enhanced stability. Of course, it
will be preferred to replace the histidine residue in
the loop-forming sequence with amino acids having the
highest possible loop-forming potential, without
creating any potential negative effects, such as the
formation of undesired disulfide bonds through the
insertion of additional cysteine or cystine residues.
Thus, other preferred amino acid substitutions at the
histidine residue in the loop-forming sequence (i.e., in
addition to glycine) are seen to include proline,
tyrosine, aspartic acid, asparagine, serine, glutamic
acid, threonine, lysine, glutamine, arginine,
phenylalanine, and tryptophan.
The present invention also contemplates the
substitution of an amino acid having high loop-forming
potential for other amino acid residues within the amino
acid 92 to 96 region of naturally occurring aFGF (i.e.,
amino acids 92 and 94-96) and the amino acid 115 to 119
region of naturally occurring aFGF (i.e., amino acids 92
and 115-117-119). The aFGF analogs of the present
invention include, for example, aFGF analogs having the
threonine residue at position 96 of naturally occurring
aFGF replaced with glycine, proline or tyrosine,
aspartic acid or asparagine, serine, or glutamic acid,
in order of preference, although minimal enhancement of
stability and/or biological activity would be expected
with the substitution of glutamic acid for threonine,
due to the similarity of loop-forming potential of these
two amino acids.
The amino acid residues at positions 92. 94,
95, and 97 (asparagine, tyrosine, asparagine, and
tyrosine, respectively) of naturally occurring aFGF have
sufficiently high loop-forming potential such that
CA 02201944 1997-04-04
WO 96/22369 PCTIUS95/12907
- 14 -
minimal benefits are envisioned to arise from
substitution for these particular residues.
The analogs of the present invention are seen
to encompass analogs of both human and animal (e. g.,
bovine) origin, as well as all forms of a protein having
the following loop-forming amino acid sequence:
92 93 94 95 96 (aFGF)
-Asn-His-Tyr-Asn-Thr-
115 116 117 118 119 (KGF)
For example, both the human and bovine forms
of aFGF are known, and have been identified as having
the identical amino acid sequence (shown above) at
positions 92 to 96. Moreover, there is approximately
92~ sequence identity between human and bovine aFGF, and
a 97$ "similarity" (i.e., 5~ of the total 8~ changes
between the two aFGF forms are "conservative"). Both
the human and bovine forms of naturally occurring aFGF
exhibit substantially the same in vitro mitogenic
activity.
Because of their enhanced stability and
biological activity in the absence of heparin, the novel
biologically active aFGF analogs of the present
invention are particularly well suited for use in
pharmaceutical formulations for the treatment by
physicians and/or veterinarians of many types of wounds
of mammalian species. The KGF analogs of the present
invention, because of their enhanced thermal stability,
may also be well suited for use in pharmaceutical
preparations. The amount of biologically active analog
used in such treatments will, of course, depend upon the
severity of the wound being treated, the route of
administration chosen, and the specific activity or
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 15 -
purity of the analog, and will be determined by the
attending physician or veterinarian. The term "analog
therapeutically effective" amount rafers to the amount
of analog determined to produce a therapeutic response
in a mammal. Such therapeutically effective amounts are
readily ascertained by one of ordinary skill in the art.
The analogs of the present invention may be
administered by any route appropriate to the wound or
condition being treated. Conditions which may be
beneficially treated with therapeutic applications) of
the analog of the present invention include but are not
limited to, the healing of surface wounds, bone healing,
angiogenesis, nerve regeneration, and organ generation
and/or regeneration.
The formulations of the present invention,
both for veterinary and for human use, comprise a
therapeutically effective amount of analog together with
one or more pharmaceutically acceptable carriers
therefore and, optionally, other therapeutic
ingredients. The carriers) must be "acceptable" in the
sense of being compatible with the other ingredients of
the formulation and not be deleterious to the recipient
thereof. The formulations may be conveniently presented
in unit dosage form and may be prepared by any of the
methods well-known in the art. All methods include the
step ef bringing into association the active ingredient
with the carrier which constitutes one or more accessory
ingredients. In general, the formulations are prepared
by uniformly and intimately bringing into association
the analog with liquid carriers or finely divided solid
carriers or both.
The following examples are provided to aid in
the understanding of the present invention, the true
CA 02201944 1997-04-04
WO 96/22369 PCT/US95I12907
- 16 -
scope of which is set forth in the appended claims. It
is understood that modifications can be made in the
procedures set forth without departing from the spirit
of the invention.
Production of fAla41~~1 aFGF Analog
~vnthesis
A [A1a47,G1y93] aFGF analog and a [G1y116] KGF
analog were made using site-directed mutagenesis. It
will be appreciated, however, that these and other
analogs of the present invention can be made by other
methods, including chemical synthesis. In the case of
the [A1a47,G1y93] aFGF analog, a bovine sequence was
used. Specifically, the [A1a47,G1y93] aFGF analog was
prepared as follows:
A bovine aFGF analog according to the present
invention was prepared and examined in the following
examples. This analog, bovine [A1a47,Glyg3] aFGF, was
constructed to contain both a desired amino acid
substitution (glycine for histidine at position 93) in
the residue 92 to 96 loop-forming sequence of the aFGF
molecule and an additional amino acid substitution of
alanine for the non-conserved cysteine residue at
position 47, as shown in Fig. 1. A bovine [A1a47] aFGF
analog, having only the amino acid substitution of
alanine for cysteine, was also prepared for use as a
control for the desired bovine [A1a47,Glyg3] aFGF
analog. Although these examples demonstrate a bovine
aFGF analog of the present invention, the same results
can be achieved for the highly homologous human aFGF
analogs. For example, the amino acid sequence of the
CA 02201944 1999-11-26
WO 96/22369 PCT/US95/12907
- I7 -
corresponding human (G1y93] aFGF analog of the present
invention is displayed in Fig. 2.
A synthetic gene coding for the (A1a47,G1y93]
analog of bovine aFGF was assembled in two sections from
a total of 28 component oligonucleotides. The amino
acid seque:zce of Gimenez-Gallego et a1. (1985), Science,
~Q:1385-1:388 was used as the basis far this gene, with
codon choices.selected to optimize expression of the
analog in .E. coli (Gimenez-Gallego et a1. (1985),
supra). Section I was assembled from 16
oligonucleotides to yield a 287 nucleotide fragment
which could be inserted into a olasmid vector at Xba I
and Xho I :restriction endonuclease sites. Section II
was assemb:Led from 12 oligonucleotides to give a 170
nucleotide fragment bounded by Xho I and gam HI
compatible ends. The two sections were inserted into
the expression plasmid pCFM1156, which had been
previously digested with Xba I and Ham HI in a 3-
component :Ligation, yielding the complete aFGF gene
under the control of the lambda pL promoter.
The pl.asmid pCFM1156 is prepared from the
known plasmid pCFM836. The preparation of plasmid
pCFM836 is described in U.S. Patent No. 4,710,473
particularly in Examples 1 to 7 of the specification.
To prepare pCFM1156 from pCFM836, the two endogenous Nde
I restriction sites are cut, the exposed ends are filled
with T4 po:lymerase, and the filled ends are blunt-end
ligated.
The resulting plasmid is then digested with
C1a I and .Kpn I and the excised DNA fragment is replaced
with a DNA oligonucleotide of the following sequence:
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 18 -
5'-CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC-3'
3'-TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC-5'
E. coli cells transformed with this plasmid
were grown in a 16-liter fermentation vessel (as
described in Fox et a1 (1988), J. Biol. Chem.,
x:18452-18458.
The gene coding for the bovine [G1y93,A1a4~]
aFGF was converted to the [Ala4~] form using oligo site-
directed mutagenesis. The aFGF gene was first
transferred into the phage vector M13mp18, and single-
stranded DNA to serve as a template for the mutagenesis
reaction was prepared. Approximately 0.5 ~g of this DNA
was mixed with 5 picomoles each of the mutagenic primer
(5'-GAAGAAAACCATTACAACAC-3') and the M13 universal
primer used for DNA sequencing, heated to 65°C for
3 minutes, and allowed to slow cool. The annealed
template-primer was mixed with ATP, a dNTP mixture, DNA
polymerase I large fragment, and T4 DNA ligase, then
incubated at 15°C for 4 hours. Aliquots of this
reaction mixture were added to competent E. coli JM101
cells and plated in 0.7~ L-agar. The resulting plaques
were replicated onto nitrocellulose filters and the
filters were hybridized with 32P-labeled mutagenic
primer. DNA prepared from phage which hybridized was
sequenced to verify successful completion of the desired
mutagenesis event. The resultant gene was then
transferred back to the pCFM1156 vector for expression
of the recombinant protein.
Both the bovine [G1y93,Ala4~J and [Ala4~] aFGF
analogs were purified from the insoluble fraction
obtained from centrifugation of mechanically lysed
CA 02201944 1997-04-04
WO 96/22369 PCT/US95112907
- 19 -
E. coli cells expressing the recombinant protein. The
pellet fraction was solubilized in 8 M urea, 0.1 M
glycine, pH 2.5, and centrifuged t~ remove insoluble
materials. The supernatant was loaded onto an S-
Sepharose~ (Pharmacia, Uppsala, Sweden) column
equilibrated with 6 M urea, 10 mM glycine, pH 3.0, and
washed with 6 M urea, 20 mM sodium citrate, pH 6.5.
Proteins which bound to the column were eluted with a
linear 0 to 0.5 M sodium chloride gradient in 20 mM
sodium citrate, pH 6.5. The fractions containing the
aFGF were pooled, diluted 20-fold with 20 mM sodium
citrate, 0.1 M ammonium sulfate, and centrifuged to
remove any precipitate. The supernatant was mixed with
one volume of 20 mM sodium citrate, 2 M ammonium
sulfate, and loaded onto a phenyl-Sepharose~ column
equilibrated with 20 mM sodium citrate, 1 M ammonium
sulfate, pH 6.5. The bound proteins were eluted from
the column with a linear descending gradient (1 M to
0 M) of ammonium sulfate. The aFGF-containing fractions
were pooled and dialyzed against 20 mM sodium citrate,
pH 6.5. This product was essentially homogeneous, as
demonstrated by the fact that no other bands in
Coomassie blue appeared in the SDS gel, as shown in
Fig. 4.
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 20 -
~xa~~ple 2
Gel filtration was performed at room
temperature using a Superose~-12 column on a Pharmacia
FPLC system (Pharmacia, Uppsala, Sweden). The column
was run at 0.5 ml/min in 20 mM sodium citrate, 0.2 M
sodium chloride, pH 6.5.
Gel filtration chromatography showed that the
purified bovine [A1a47] and [A1a47,G1y93] aFGF analogs
eluted as single peaks at an elution position identical
to that of 'ribonuclease A (Mr = 13,700). This indicated
that both proteins are monomeric and have the same
hydrodynamic radius, although there is a possibility
that both forms of the protein interact with the column
matrix and give a retarded elution from the column.
Exam 1p a 3
Hydrophobic interaction chromatography was
performed at room temperature using a phenyl-Superose~
column on a Pharmacia FPLC system. The sample, in 2 M
ammonium sulfate, 20 mM sodium citrate, pH 6.5, was
loaded onto the column which had been equilibrated with
2 M ammonium sulfate. After a 2 M ammonium sulfate
wash, the remaining protein was eluted with an ammonium
sulfate gradient descending from 2 M to 0 M, followed by
a final wash with 20 mM sodium citrate, pH 6.5.
Because the elution position of a protein in
hydrophobic interaction chromatography (HIC) is strongly
dependent upon the exposure of hydrophobic regions in
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 21 -
the folded state, this technique provides a sensitive
probe of the conformational homogeneity of similar
proteins. Fig. 3 presents the elution profiles for the
bovine [Ala4~] and [Ala4~,G1y93] aFGF analogs. The
[Ala4~] aFGF showed a major peak eluting at 0.25 M
ammonium sulfate, while the [Ala4~,G1y93] aFGF analog
showed a single peak at 0.13 M ammonium sulfate,
suggesting that both proteins exist primarily in a
single distinct conformation. The elution at lower salt
concentration by the [Ala4~,G1y93] aFGF indicates that
it is slightly more hydrophobic than the [Ala4~] form.
This observation is consistent with the replacement of
the histidine residue at position 93 by glycine if the
conformation of the protein is such that this residue is
exposed to the solvent. Alternatively, the change in
this residue could induce an overall change in the
conformation of the molecule to produce a more
hydrophobic structure.
Examgle 4
Circular dichroic spectra were determined at
room temperature on a Jasco Model J-500C
spectropolarimeter (Jasco, Tokyo, Japan) equipped with
an Oki If 800 Model 30 computer (Oki, Tokyo, Japan).
Measurements were carried out at a band width of 1 nm
using cuvettes of
1 and 0.02 cm for the near and far ultraviolet ranges,
respectively. The data were expressed as the mean
residue ellipticity, [A], calculated using the mean
residue weight of 113 for both forms of aFGF.
CA 02201944 1997-04-04
WO 96!22369 PCT/LTS95/12907
- 22 -
Circular dichroism (CD) spectra of the bovine
[A1a47,G1y93] and [A1a47] aFGF analogs were nearly
identical in both the far and near ultraviolet regions,
as shown in Figs. 4A and 4B, respectively. The CD of
the analogs were also very similar to the spectrum
reported for human bFGF (Arakawa, et a1 (1989), E9RC,
x:335-341. The similarity of the spectra in the near
ultraviolet region is consistent with similar tertiary
structures for the FGFs.
The thermal transition of proteins was
determined on a Response II spectrophotometer (Gilford,
Medfield, Massachusetts) equipped with thermal
programming and a thermal cuvette holder. Samples were
heated at an increment of 0.1°C/min or 0.5°C/min and
their absorbance monitored at 287 nm. Protein
concentrations were determined spectrophotometrically
using an extinction coefficient of 0.98 for bFGF and
1.04 for both bovine aFGF analogs at 280 nm for 0.1~
protein.
Thermal denaturation of the aFGF analogs was
examined in the presence and absence of heparin at both
pH 6.5 and 7.0, 20 mM sodium citrate. In all cases, the
proteins precipitated as the temperature was increased.
The. temperature at which the abrupt absorbance increase
occurred was taken as the denaturation temperature. In
the absence of heparin, this temperature was about 10°C
higher for the bovine [A1a47,G1y93] aFGF analog than for
the bovine [A1a47) aFGF analog. Addition of either
1.4-fold or 8-fold (w/w) excess of heparin increased the
denaturation temperature for both forms by 14-20°C,
depending upon the rate of temperature increase used.
The difference between the denaturation temperature of
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 23 -
the two forms remained at about 10°C. There was no
apparent effect of 1.4-fold or 8-fold (w/w) excess
heparin on the CD spectra of either protein in the 240
to 340 nm range, although in the case of 8-fold excess
heparin, the aFGF spectrum in the 240-260 nm region was
masked by the absorbance of the heparin itself.
~ot?r~Pr-transform Infrared (FTIR) Spectroscopy
Fourier-transform infrared (FTIR) spectra were
determined to further examine the similarity in
conformation of both aFGFs. For FTIR spectroscopy, the
proteins were thoroughly dialyzed against water. Each
protein was prepared as a 2$ solution in a 20 mM
imidizole buffer made in D20 (Sigma Chemical Co., 99.9
isotopic purity). Solutions were placed in IR cells
with CaF2 windows and 100 Eun spacers. For each
spectrum, 1500 interferograms were collected and coded
on a Nicolet 800 FTIR system equipped with a germanium-
coated KBr beam splitter and a DTGS detector. The
optical bench was continuously purged with dry nitrogen
gas. Second derivative spectra were calculated (as
described in Susi et a1 (1988), Biochem. Biophys. Res.
Comm., ~1,~:391-397). A 9-point smoothing function was
applied to the water vapor-subtracted spectra.
Fig. 5 shows the second derivative spectra of
the [A1a47] and [A1a47,Glyg3] bovine aFGF analogs in the
amide I' (C=0 stretch in deuterated proteins) region.
For polypeptides and proteins, the frequencies of the
component bands in this region are related to secondary
structure content. Surewicz et al, (1988), Biochem.
Biophys. Acta,~7:115-130. The spectra show strong
bands at 1630 and 1685 cm-1 which are indicative of a
significant amount of ~-structures in the two proteins.
A strong band near 1647 cm-1 is indicative of the
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 24 -
presence of irregular or disordered structures. The
weaker peaks near 1666 and 1673 cm-1 arise from turn
structures. A small peak is present near 1651 cm-1 in
the spectra of both proteins. Amide I' components near
this frequency are typically assigned to a-helices.
However, it was recently shown that this band may arise
from loop structures. Wilder et a1, (1990), Abstracts
of the Fourth Symposium of the Protein Society, San
Diego. As shown in Fig. 5, the highly resolved FTIR
spectra, unlike CD, clearly demonstrate the presence of
~-structures and turns, and the spectra for bovine
[A1a47] aFGF analog and bovine [A1a47,G1y93] aFGF analog
are nearly superimposable, again suggesting that these
two proteins have similar conformation.
The second derivative spectra showed
apparently no difference in conformation between the two
aFGF analogs. However, it was evident that deuteration
of exchangeable protons occurred faster for the bovine
[A1a47] aFGF analog than for the [A1a47,G1y93] analog
during equilibration of lyophilized protein with D20
solution. Since the two proteins have a similar
conformation, the observed difference in H-D exchange
rate cannot be explained from differences in the extent
of exposure of exchangeable protons between them. It is
more likely that the [A1a47] aFGF analog has a more
flexible structure, which renders amide protons more
accessible to the solvent.
Exam 1,p a 5
H~rarin Chromatography of fAla~ZiGlv~31 aFGF Analocr
Heparin-Sepharose~ (Pharmacia) was packed into
a 1 x S cm column and equilibrated with 10 mM Tris~HC1,
pH 7.2. The column was loaded, washed with 10 mM
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 25 -
Tris~HC1, pH 7.2 and eluted with a linear gradient from
0 to 2.8 M sodium chloride in the same buffer at a flow
rate of 0.5 ml/min using a Pharmacia FPLC system.
Acidic and basic FGF are distinguished by
their avid binding to heparin and heparin-like
molecules. Both the bovine [Ala4~,Glyg3] and [Ala4~]
aFGF analogs showed a single peak eluting at 1.54 M
sodium chloride in 10 mM Tris~HC1, pH 7.2.
15 In Vitro Bioassays
The mitogenic activity on NIH 3T3 cells of the
aFGF analogs from the previous examples was determined
as described below. In addition, a human [Ser~O,Ser88]
bFGF analog, prepared as described in published PCT
Patent Application No. 88/04189, was also examined in
the bioactivity assays, alongside the aFGF analogs.
NIH 3T3 cells were obtained from ATCC. The
cells were grown in DME supplemented with 10~ calf
serum,
10 units/ml penicillin, 2 mM glutamine and 10 units/mI
streptomycin. Cells were passaged at a ratio of 1:40
two times per week. On day 1 of the assay, subconfluent
cultures were trypsin dispersed and plated into 24-well
plates at a concentration of 20,000 cells/ml, 1 ml per
well in the above media. On day 5, the media was
replaced with 1 ml/well DMEM without serum but
containing penicillin, streptomycin, and glutamine at
the above concentrations. On day 6, experimental
samples were added to the media in volumes no greater
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 26 -
than 100 ~1. Eighteen hours later, cells were pulsed
for 1 hour with 1 ml of the above media containing 2-10
~.Ci of tritiated thymidine at 37°C. After the pulse,
cells were washed once with media, then 250 mM sucrose,
10 mM sodium phosphate, 1 mM EDTA, pH 8 was added and
the plates incubated at 37°C for 10 minutes to release
the cells. Cells were harvested on a Skatron harvester
(Skatron, Inc., Sterling, Virginia). Filters were
dried, placed in scintillation fluid, and counted in a
Beckman scintillation counter (Beckman Instruments,
Inc., Fullerton, California).
The mitogenic activity of the bovine
[A1a47,Glyg3] and [A1a47] aFGF analogs on NIH 3T3 cells
was examined as shown in Fig. 6. In the absence of
heparin, the [A1a47] aFGF analog produced a dose
dependent stimulation of 3H-thymidine uptake in the
range of 1 to 100 ng/ml, with half-maximal stimulation
of 25 ng/ml. Under the same assay conditions, the
[A1a47,Glyg3] aFGF analog was able to produce the same
mitogenic effect at a much lower protein concentration,
the half-maximal dose being about 1 ng/ml. Recombinant
bFGF was 4-5 times more potent than the [A1a47,G1y93]
aFGF, with a half-maximal dose of 220 pg/ml. When 4.5
~g/ml heparin was added to both analogs, their in vitro
activity was increased with the [A1a47,G1y93] aFGF
analog remaining more potent. In the presence of 45
~g/ml heparin, the activities were enhanced such that
the dose response of all three molecules were nearly
identical, with a half-maximal dose of 90 pg/ml.
The stability of the aFGF analogs, as
determined by retention of their respective mitogenic
activity, was examined by incubation of a 0.1 mg/ml
solution of each FGF analog in 20 mM sodium citrate, pH
7 at 37°C, both in the presence and absence of 1 mg/ml
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 27 -
heparin. In the absence of heparin, the bovine [A1a47]
aFGF analog rapidly lost activity, with a half-life of
about 13 hours, as shown in Fig. 7. However, in the
presence of heparin, bovine [A1a47] aFGF lost no
biological activity over the 250 hour course of the
experiment. In contrast, neither bovine [A1a47,G1y93]
aFGF analog nor the human [Ser70,Ser88] bFGF analog
exhibited any loss of activity over the 250 hours,
whether or not heparin was present.
Exam 1~7
~",,rys a~ oa~~y of fAla41"~9~1 aFGF Analog
Crystals of bovine [A1a47,G1y93] aFGF analog
were grown by vapor diffusion against 0.2 M NHqSOq, 2 M
NaCl, 0.099 M sodium citrate, and 0.02 M sodium
potassium phosphate, pH 5.6. The protein droplet
contained equal volumes of the reservoir solution and a
10 mg/ml protein solution. The crystals were trigonal
(space group P3121, a = 78.6 ~, c = 115.9 ~) and
diffracted to 2.5 ~ resolution. Intensity data were
collected with a Siemens (Madison, Wisconsin) multiwire
area detector mounted on an 18 kw rotating anode
generator. The Siemens suite of processing programs was
used for data reduction. Multiple isomorphous
replacement (mir) phases were calculated to 3 ~
resolution from two derivatives, with a figure of merit
of 0.68. After solvent flattening, regions
corresponding to two independent aFGF molecules in the
asymmetric unit were identified. The general non-
crystallographic symmetry relationships between these
molecules were determined from rotation function, real-
space translation function, and density correlation
studies. A molecular envelope was defined around an
averaged aFGF molecule with a modified B.C. Wang
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 28 -
algorithm. The phases were iteratively refined by
molecular averaging and solvent flattening.
Initial maps revealed extended regions of
sheet structure that were truncated at the loops, due to
a small molecular envelope, as shown in Fig. 8. The
final map for model building was calculated with mir
phases (from heavy atom parameters re-refined against
averaged phases, as described in Rould et a1, (1989),
Science, 2:1135-1142) and iteratively averaged with a
molecular envelope generated by placing 6 ~ spheres
about the atomic positions in the initial model.
Averaging at 3 ~ resolution converged to a final R-
factor of 17.8 between the observed structure factors
and structure factors calculated from the averaged and
solvent flattening map. The graphic program TOM/FRODO,
implemented for a Silicon Graphics 4D80 by C. Cambillau,
was used to build residues 10 to 136 of the aFGF
sequence into an averaged electron density map.
The crystallography results supported the
hypothesis that the 90-97 region is involved in a loop
structure. If this region is, in fact, involved in
receptor binding (as suggested by Baird et al, (1988),
supra) any amino acid substitution which stabilizes the
loop may stabilize and/or enhance the biological
activity of the molecule. This is presumably the
mechanism for the observed activity enhancement attained
with the bovine [A1a47,G1y93] aFGF.
CA 02201944 1997-04-04
WO 96/22369 PCTJUS95/12907
- 29 -
ProdLCtion of ~G1v11Sz1 KGF Analoa
~,vnthesis
In order to make the [G1y116~ KGF analog, a
coding sequence was first obtained for naturally
occurring KGF and then altered at the codon for amino
acid 116 in order to achieve a coding sequence for the
analog.
The coding sequence for naturally occurring
KGF was obfained using RNA isolated from human
fibroblast cells (cell line AG1523) as a starting
material from which to make cDNA for KGF using standard
techniques known in the art. The KGF cDNA was then used
as a template in polymerase chain reactions (PCR) to
amplify the KGF gene. Due to the presence of an
internal NdeI site in the KGF gene, the PCR DNA was made
as two fragments that were then linked together at a
unique BsmI site. Oligonucleotides 238-21 and 238-24
(shown below) were used to make a DNA product that was
subsequently cut with BsmI and BamHI and then isolated
to yield a 188 base pair fragment of KGF.
Oligonucleotides 238-22 and 238-24 were used to make a
DNA product that was subsequently cut with Ndel and BsmI
to yield a 311 base pair fragment of KGF. The two DNA
fragments, when ligated together create the gene for
naturally occurring KGF, shown in Fig. 9.
In order to obtain a coding sequence for the
desired (G1y116~ KGF analog, it was necessary to
substitute a glycine codon for the His116 codon in the
KGF gene. This was achieved using PCR overlapping
oligonucleotide mutagenesis with oligonucleotides 315-17
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 30 -
and 315-18, encoding the KGF DNA sequence corresponding
to the His116 region with the appropriate base pair
changes to encode the [G1y116] KGF analog. The KGF DNA
template for the PCR was the same as shown in Fig. 9,
except that the DNA sequence between the KpnI and EcoRI
sites was replaced by chemically synthesized DNA as
shown in Fig. 10. Any convenient oligonucleotides
corresponding to KGF DNA regions 5'- and 3'- of the
site-directed mutational change, such as
oligonucleotides 238-22 and 238-24 could be used to
provide the outside primers for the overlapping
mutagenesis PCR. An EcoRI to BamHI DNA fragment
containing the (G1y116] KGF analog coding sequence was
then ligated into the previously described expression
plasmid pCFM1156 already containing the KGF gene, so as
to replace the corresponding region of the KGF gene with
a region of the coding sequence containing the necessary
changes to encode the [G1y116] KGF analog (Fig. 10).
The ligation DNA was then transformed into an FMS
(ATCC$ 53911) host and colonies were isolated that
contained the pCFM1156 [G1y116] KGF analog plasmid. The
FMS/pCFM1156 KGF [G1y116) KGF analog strain was then
fermented and cell paste harvested using standard
fermentation techniques.
oligo 238-21 5'-ACAACGCGTGCAATGACATGACTCCA-3'
oligo 238-22 5'-ACACATATGTGCAATGACATGACTCCA-3'
oligo 238-24 5'-ACAGGATCCTATTAAGTTATTGCCATAGGAA-3'
oligo 315-17 5'-GGAAAACGGTTACAACACATATGCA-3'
oligo 315-18 5'-GTGTTGTAACCGTTTTCCAGAATTAG-3'
Purification of fGlvll~l KGF Analoor
The cells containing the (G1y116] KGF analog
from the fermentation described above were first broken
by suspending 665 g of the E. coli cell paste in ca. 4 L
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 31 -
20 mM sodium phosphate, pH 6.8, 0.2 M NaCl and then
passing the suspension 3 times through a Gaulin
Homogenizer at 9,000 psi. The suspension was then
centrifuged in a Beckman JA-10 rotor (Beckman
Instruments, Fullerton, California) at 10,000 rpm, for
30 minutes at 4°C .
Ion exchange chromatography was performed by
applying supernatant from the centrifuged suspension to
an S-Sepharose~ Fast Flow (Pharmacia, Uppsala, Sweden)
column (5 x 23 cm., 450 ml total volume) equilibrated
with 20 mM sodium phosphate, pH 7.5, 0.2 M NaCl, at a
flow rate of 25 ml/min. The column was then washed with
2 L of 20 mM sodium phosphate, pH 7.5, 0.4 M NaCl, and
the [G1y116J KGF analog eluted with a linear gradient of
0.4 M to 0.6 M NaCl in 20 mM sodium phosphate, pH 7.5.
Total gradient volume was 7 L (about 16 times column
volume). Fractions containing KGF were pooled and
concentrated about 22-fold over a YM~-10 membrane
(Amicon) in a 400 ml stirred cell.
Size exclusion chromatography (SEC) was
performed by applying half of the volume of the
concentrated KGF (total volume of 80 ml) obtained from
ion exchange chromatography to a Sephadex~ G-75
(Pharmacia) column (4.4 x 85 cm, total volume of 1300
ml) equilibrated with 20 mM sodium phosphate, pH 6.8,
O.S M NaCl and developing the column with this buffer.
The process was then repeated with the second half of
the concentrated KGF preparation.
A second ion exchange chromatography procedure
was then performed by first pooling SEC fractions
corresponding to the monomeric form of [G1y116J KGF
analog, then diluting the pooled fractions with five
volumes of 20 mM sodium phosphate, pH 6.8, 0.2 M NaCl
CA 02201944 1999-11-26
WO 96!22369 PC'T/US95/12907
- 32 -
and applying the diluted fractions to an S-Sepharosem
Fast Flow (Pharmacia) column (5 x 23 cm., 450 ml total
volume) eqsilibrated with 20 mM sodium phosphate, pH
6.8, 0.4 M NaCl. This column was then washed with about
1.5 L of 20 mM sodium phosphate, pH 6.8, 0.4 M NaCl.
The purifi~=_d [G1y116~ KGF analog was eluted with a
linear gra~3ient of 0.4 M to 0.6 M NaC1 in 20 mM sodium
phosphate, pH 6.8. Total gradient volume was 10 L
(about 22 1_imes column volume). Samples containing the
(G1y116] KGF analog, as ascertained by SDS-PAGE, were
pooled and the KGF content was determined by W
absorption.
~nectroscoov of KGF Anal,~q
Samples were prepared for infrared
spectroscopy by diafiltering protein solutions in 20 mM
sodium phosphate, 0.5 M NaCl, pH = 6.8 into a 20 mM
sodium phosphate, 0.15 M NaCl, pD = 6.8 buffer prepared
in D20 (Sigma, 99.9%+ isotopic purity). The pD values
were determined :by adding 0.45 to the pH reading from a
glass electrode pH meter, according to Covington et a1.
(1968), Anal. Chem., ~Q:700-706. The final protein
concentrat:Lon was 30 mg/ml. Protein solutions were
placed in :CR cells with CaF2 windows and 100 ~cm TeflonTM
spacers.
For structural characterizations, 256 double-
sided interferograms were co-added and Fourier-
transformed after application of a Happ-genzel
apodization function using a Nicolet 800 FTIR system.
The resolution was set at 2 cm-1. Derivative spectra
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/1290?
- 33 -
and Fourier self-deconvolutions were performed according
to Susi and Byler (1983), Biochme. Biophys Res. Comm.,
x:391-397, using the Nicolet software. Curve fitting
was performed using program PeakfitT" (Jandel Scientific
Co.). The infrared spectra of KGF showed strong
similarity to those of bFGF and aFGF, indicating similar
structures for these three proteins.
Thermal stability studies of the naturally
occurring KGF and the [G1y116~ KGF analog Were performed
by placing the IR cells in an electric heating jacket
controlled by an automatic temperature controller
(Specac Inc., Fairfield, Connecticut). The temperature
was increased at a rate of 0.5°C/minute. IR spectra
were collected at 8 cm-1 resolution.
At temperatures below 50°C, the spectra
appeared to change little from the spectrum at ambient
temperature. At temperatures greater than 50°C, the
spectra indicated that the naturally occurring KGF
undergoes a thermotropic transition at this point.
Peaks near 1616 and 1685 cm-1 were evident in the
spectra and, by 65°C, these peaks dominated the spectra
with a corresponding loss of intensity at 1643 cm-1.
This spectral transition represents the cooperative
unfolding of naturally occurring KGF. The observed
thermal transition was not reversible, most likely due
to aggregation of the unfolded protein. The melting
temperature, Tm, for naturally occurring KGF was
estimated to be 60°C, while the Tm for the [G1y116] KGF
analog was estimated to be 65°C, 5°C higher than that of
naturally occurring KGF, indicating that the [G1y116~
KGF analog has a higher relative thermal stability than
naturally occurring KGF.
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 34 -
L11_travio~Ar ~ ectrosc~py
The thermal denaturation of both the [G1y116)
KGF analog and naturally occurring KGF was studied using
a Response II W spectrophotometer (Gilford, Medfield,
Massachusetts) with a Peltier temperature controller and
thermal programmer. KGF solutions in 20 mM sodium
phosphate, pH 6.8, were mixed with 8 M guanidine HC1 or
20 mM sodium phosphate for a final protein concentration
of about 0.5 mg/ml and a final guanidine HC1
concentration of 0 to 2 M. The thermal scan rate was
set at 0.5°C/minute, and the wavelength monitored was
286 nm. Addition of a small amount of guanidine HC1
eliminated-precipitation of the proteins during thermal
denaturation, but did not make the denaturation
reversible. Therefore, samples to be compared were run
simultaneously.
Thermal denaturation experiments using W
spectroscopy were unsuccessful in the presence of 0 and
1 M guanidine HC1, due to the development of turbidity
upon heating. Nevertheless, turbidity developed at a
lower temperature for the naturally occurring KGF than
for the [G1y116] KGF analog, suggesting that the former
protein denatures and, consequently, aggregates at a
lower temperature. In 1.5 M guanidine HC1, the
absorbance at 287 nm decreased as the protein denatured
and then increased due to aggregation. The temperatures
at which the absorbance decrease and increase occurred
were respectively 25 and 44°C for the [G1y116] KGF
analog. Addition of 2 M guanidine HC1 resulted in
partial denaturation for both the naturally occurring
KGF and the [G1y116] KGF analog at 25°C. Increasing the
temperature resulted in complete denaturation for the
proteins, denaturation ending around 37°C for naturally
occurring KGF and 46°C for the [G1y116] KGF analog.
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 35 -
These results indicate that the [G1y116] KGF analog is
more thermally stable by several degrees in melting
temperature, and are in agreement with the infrared
analysis indicating an approximately 5°C increase in Tm
of the [G1y116] KGF analog relative to naturally
occurring KGF.
M~ ocrenic Activity of ~Glvllfil KGF analn~
Mitogenic activity of the [G1y116] KGF analog
was analyzed using the mitogenesis assay of Rubin et al.
(1989), Proc. Natl. Acad. Sci. USA, $x:802-806. The
[G1y116] KGF analog demonstrated less 3H-thymidine
incorporation than was observed for naturally occurring
KGF, indicating a lower specific activity for the
[G1y116] KGF analog.
CA 02201944 1999-11-26
WO 96/22369 PCT/US95/12907
- 36 -
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Amgen Inc.
(ii) TITLE OF INVENT:CON: Analogs of Acidic Fibroblast Growth
Factor Having Enhanced Stability and
Biological Activity
(iii) NUMBER OF' SEQUE1JCES: 17
( iv) CORRESPOD1DENCE i~DDRESS
(A) ADDRESSEE: Amgen Inc
(B) STREET: 1840 DeHavilland Drive
(C) CITS.': Thousand Oaks
(D) STATE: Cal:Lfornia
( E ) COUrITRY : U:iA
(F) ZIP: 91320--1789
(v) COMPUTER F;EADABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COME~UTER: IBM PC'~" compatible
(C) OPERATING :SYSTEM: PC-DOST"'/MS-DOST"'
(D) SOFTWARE: ;?atentInT" Release #1.0, Version #1.25
(vi) CURRENT APPLICA'PION DATA:
(A) APPLICATI01A NUMBER:
(B) FILC:NG DATE:
(C) CLA:iSIFICA'PION:
(2) INFORMATION F'OR SEQ ID NO:1:
(i) SEQUENCE CHARAC'PERISTICS:
(A) LENGTH: 463 base pairs
(B) TYPE: nucleic acid
(C) STR~~NDEDNE,SS : unknown
(D) TOPOLOGY: 'unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCI: DESCRIPTION: SEQ ID N0:1:
TCTAGAAAAA ACCAAGGAGG TAATAAATAA TGTTCAACCT GCCGCTGGGT AACTACAAAA 60
AACCTAAGCT TCTGTACTGC TCTAACGGCG GTTACTTCCT GCGCATTCTC CCGGATGGCA 120
CTGTAGACGG TACCAA:~GAT CGTTCCGACC AGCACATTCA GCTCCAGCTC GCTGCAGAAT 180
CTATCGGTGA AGTTTACATC AAATCCACCG AAACTGGTCA GTTCCTGGCT ATGGATACTG 240
ATGGTCTCCT CTACGG'PTCT CAGACTCCGA ACGAAGAGTG CCTGTTCCTC GAGCGTCTGG 300
AAGAAAACGG TTACAACACC TACATCTCCA AAAAACACGC TGAAAAACAC TGGTTCGTTG 360
GTCTGAAAAA AAACGG'PCGT TCTAAACTGG GTCCGCGCAC TCACTTCGGT CAGAAAGCTA 420
TCCTGTTCCT CCCTCTGCCG GTTTCTTCCG ATTAATAGGA TCC 463
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 37 -
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Phe Asn Leu Pro Leu Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr
1 5 10 15
Cys Ser Asn Gly Gly Tyr Phe Leu Arg Ile Leu Pro Asp Gly Thr Val
20 25 30
Asp Gly Thr Lys Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ala
35 40 45
Ala Glu Ser Ile Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln
50 55 60
Phe Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro
65 70 75 80
Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn Gly Tyr Asn
85 90 95
Thr Tyr Ile Ser Lys Lys His Ala Glu Lys His Trp Phe Val Gly Leu
100 105 110
Lys Lys Asn Gly Arg Ser Lys Leu Gly Pro Arg Thr His Phe Gly Gln
115 120 125
Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
130 135 140
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe
1 5 10 15
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Ala Ser
20 25 30
CA 02201944 1997-04-04
WO 96/22369 PCTIUS95/12907
- 38 -
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly
35 40 45
Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu
50 55 60
Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu
65 70 75 80
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu
85 90 95
Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn Gly Tyr Asn Thr Tyr
100 105 110
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 N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 502 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
TATGTGCAAT GACATGACTC CAGAGCAAAT GGCTACAAAT GTGAACTGTT CCAGCCCTGA 60
GCGACACACA AGAAGTTATG ATTACATGGA AGGAGGGGAT ATAAGAGTGA GAAGACTCTC 120
TGTCGAACAC AGTGGTACCT GAGGATCGAT AAAAGAGGCA AAGTAAAAGG GACCCAAGAG 180
ATGAAGAATA ATTACAATAT CATGGAAATC AGGACAGTGG CAGTTGGAAT TGTGGCAATC 240
AAAGGGGTGG AAAGTGAATT CTATCTTGCA ATGAACAAGG AAGGAAAACT CTATGCAAAG 300
AAAGAATGCA ATGAAGATTG TAACTTCAAA GAACTAATTC TGGAAAACCA TTACAACACA 360
TATGCATCAG CTAAATGGAC ACACAACGGA GGGGAAATGT TTGTTGCCTT AAATCAAAAG 420
GGGATTCCTG TAAGAGGAAA AAAAACGAAG AAAGAACAAA AAACAGCCCA CTTTCTTCCT 480
ATGGCAATAA CTTAATAGGA TC 502
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 39 -
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 497 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (1..497)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
CTATTAAGTT ATTGCCATAG GAAGAAAGTG GGCTGTTTTT TGTTCTTTCT TCGTTTTTTT 60
TCCTCTTACA GGAATCCCCT TTTGATTTAA GGCAACAAAC ATTTCCCCTC CGTTGTGTGT 120
CCATTTAGCT GATGCATATG TGTTGTAATG GTTTTCCAGA ATTAGTTCTT TGAAGTTACA 180
ATCTTCATTG CATTCTTTCT TTGCATAGAG TTTTCCTTCC TTGTTCATTG CAAGATAGAA 240
TTCACTTTCC ACCCCTTTGA TTGCCACAAT TCCAACTGCC ACTGTCCTGA TTTCCATGAT 300
ATTGTAATTA TTCTTCATCT CTTGGGTCCC TTTTACTTTG CCTCTTTTAT CGATCCTCAG 360
GTACCACTGT GTTCGACAGA AGAGTCTTCT CACTCTTATA TCCCCTCCTT CCATGTAATC 420
ATAACTTCTT GTGTGTCGCT CAGGGCTGGA ACAGTTCACA TTTGTAGCCA TTTGCTCTGG 480
AGTCATGTCA TTGCACA 497
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 164 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Cys Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys
1 5 10 15
Ser Ser Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly
20 25 30
Asp Ile Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg
35 40 45
Ile Asp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn
50 55 60
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 40 -
Tyr Asn Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile
65 70 75 80
Lys Gly Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys
85 90 95
Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu
100 105 110
Ile Leu Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His
115 120 125
Asn Gly Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val
130 135 140
Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro
145 150 155 160
Met Ala Ile Thr
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 503 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
TATGTGCAAT GACATGACTC CAGAGCAAAT GGCTACAAAT GTGAACTGTT CCAGCCCTGA 60
GCGACACACA AGAAGTTATG ATTACATGGA AGGAGGGGAT ATAAGAGTGA GAAGACTCTT 120
CTGTCGAACA CAGTGGTACC TGCGTATCGA CAAACGCGGC AAAGTCAAGG GCACCCAAGA 180
GATGAAAAAC AACTACAATA TTATGGAAAT CCGTACTGTT GCTGTTGGTA TCGTTGCAAT 240
CAAAGGTGTT GAATCTGAAT TCTATCTTGC AATGAACAAG GAAGGAAAAC TCTATGCAAA 300
GAAAGAATGC AATGAAGATT GTAACTTCAA AGAACTAATT CTGGAAAACG GTTACAACAC 360
ATATGCATCA GCTAAATGGA CACACAACGG AGGGGAAATG TTTGTTGCCT TAAATCAAAA 420
GGGGATTCCT GTAAGAGGAA AAP.AAACGAA GAAAGAACAA AAAACAGCCC ACTTTCTTCC 480
TATGGCAATA ACTTAATAGG ATC 503
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 497 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 41 -
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (1..497)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
CTATTAAGTT ATTGCCATAG GAAGAAAGTG GGCTGTTTTT TGTTCTTTCT TCGTTTTTTT 60
TCCTCTTACA GGAATCCCCT TTTGATTTAA GGCAACAAAC ATTTCCCCTC CGTTGTGTGT 120
CCATTTAGCT GATGCATATG TGTTGTAACC GTTTTCCAGA ATTAGTTCTT TGAAGTTACA 180
ATCTTCATTG CATTCTTTCT TTGCATAGAG TTTTCCTTCC TTGTTCATTG CAAGATAGAA 240
TTCAGATTCA ACACCTTTGA TTGCAACGAT ACCAACAGCA ACAGTACGGA TTTCCATAAT 300
ATTGTAGTTG TTTTTCATCT CTTGGGTGCC CTTGACTTTG CCGCGTTTGT CGATACGCAG 360
GTACCACTGT GTTCGACAGA AGAGTCTTCT CACTCTTATA TCCCCTCCTT CCATGTAATC 420
ATAACTTCTT GTGTGTCGCT CAGGGCTGGA ACAGTTCACA TTTGTAGCCA TTTGCTCTGG 480
AGTCATGTCA TTGCACA 497
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 164 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE DESCRIPTION: N0:9:
SEQ
ID
MetCysAsn AspMet ThrPro GluGlnMet AlaThrAsn ValAsn Cys
1 5 10 15
SerSerPro GluArg HisThr ArgSerTyr AspTyrMet GluGly Gly
20 25 30
AspIleArg ValArg ArgLeu PheCysArg ThrGlnTrp TyrLeu Arg
35 40 45
IleAspLys ArgGly LysVal LysGlyThr GlnGluMet LysAsn Asn
50 55 60
TyrAsnIle MetGlu IleArg ThrValAla ValGlyIle ValAla Ile
65 70 75 80
CA 02201944 1997-04-04
WO 96/22369 PCT/US95I12907
- 42 -
Lys Gly Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys
85 90 95
Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu
100 105 110
Ile Leu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His
115 120 125
Asn Gly Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val
130 135 140
Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro
145 150 155 160
Met Ala Ile Thr
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CGATTTGATT CTAGAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GGTAC 55
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
- 43 -
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
GAAGAAAACC ATTACAACAC 20
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
ACAACGCGTG CAATGACATG ACTCCA 26
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
ACACATATGT GCAATGACAT GACTCCA 27
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
ACAGGATCCT ATTAAGTTAT TGCCATAGGA A 31
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
CA 02201944 1997-04-04
WO 96/22369 PCT/US95/12907
A189-A - 44 -
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
GGAAAACGGT TACAACACAT ATGCA 25
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GTGTTGTAAC CGTTTTCCAG AATTAG 26
