Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02346742 2001-O1-08
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S HYALURONIC ACID MIMICS AND METHODS RELATED THERETO
This application claims priority to U.~i. Provisional Patent
Application Serial Number 60/091,758, filed July 6, 1998.
Fj,F~~[~OF THE IIVVENTI~V
This invention pertains to the fields of biochemistry, specifically
biochemistry related to compounds which interact with hyaluronic acid
receptors.
1S BACKGROUND OF THE I'L~JVI?NTIO~j
Hyaluronic acid (HA) is a large glycoaminoglycan that contains
repeating disaccharide units of N-acetyl glucos~amine and glucuronic acid. It
occurs in the extraceliular matrix and on the cell surface. It has been shown,
among other things, to promote cell mobility, adhesion, and proliferation.
HA has an role in many physiological processe;r, for example, morphogenesis,
wound repair, inflammation, and metastasis. Many of the effects of HA are
mediated through cell surface receptors, several of which have been
molecularly characterized, namely CD44, RHAIvfM (Receptor for Hyaluronan
Mediated Mobility}, and ICAM- I (Intracellular Adhesion Molecule- 1),
2S BEHAB, Link Protein arid TSG-6. Binding of the HA ligand to its receptors
triggers signal transduction events.
Although considerable information on the structure of the HA-related
surface receptors is available, the three-dimensional. structure/biochemistry
of HA which influences the receptor/HA interaction is not known. The
popular hypothesis for receptor-HA interaction is that the HA binding motif
is present in sequences of these receptors and i~~ responsible for the binding
of
these sequences to HA.
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Other journal articles which hypothesize motifs that would bind to HA
binding domains are:
Day, A. j. (1999) The structure and regulation of hyaluronan-binding proteins.
Biochem Soc Trans 27, 115-21.
Knudson, C. B., and Knudson, W. (1993) Hyaluronan-binding proteins in
development, tissue homeostasis, and disease. Faseb J 7, 1233-41
Sherrnan, L., Sleeman, J., Herrlich, P., and Ponta, H. (1994} Hyaluronate
receptors: key players in growth, differentiation, migration and tumor
progression. Curr Opin Cell Biol 6, 726-33.
Toole, B. P. (1990) Hyaluronan and its binding proteins, the hyaladherins.
I5 Curr Opin Cell Biol 2, 839-44.
Ward, A. C., Dowthwaite, G. P., and Pitsillide:>, A. A. (1999) Hyaluronan in
joint cavitation. Biochem Soc Trans 27, 128-35.
Bajorath, J., Greenfield, B., Munro, S. B., Day, A. j., and Aruffo, A. (I998).
Identification of CD44 residues important for hyaluronan binding and
delineation of the binding site. J Biol Chem 273, 338-43.
Kohda, D., Morton, C. j., Parkar, A. A., Hatanaka, H., Inagaki, F. M.,
Campbell,
I. D., and Day, A. J. (1996). Solution strucaure of the link module: a
hyaluronan-binding domain involved in extracellular matrix stability and
cell migration. Cell 86, 767-75.
Maier, R., Wisniewski, H. G., Vilcek, J., and Lotz, M. (1996}. TSG-6
expression
in human articular chondrocytes. Possible implications in joint
inflammation and cartilage degradation. Arthriitis Rheum 39, 552-9.
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Parkar, A. A., Kahrnann, J. D., Howat, S. L., Bayliss, M. T., and Day, A.
J. (1998). TSG-6 interacts with hyaluronan and aggrecan in a pH-dependent
manner via a common functional element: implications for its regulation in
inflamed cartilage. FEBS Lett 428, 171-6.
The following patents discuss modified HAs, none of which are
remotely similar to the present HA mimics:
5,$74,417 & 5;616,568 Functionalized derivatives of hyaluronic acid
5,652,347 Method for making functionalized derivatives of hyaluronic acid
5,631,241 Pharmaceutical compositions containing hyaluronic acid fractions
5,356,883 & 5;502,081 & 5;356,883 & 5,017,229 & 4,937,270 Water-insoluble
derivatives of hyaluronic acid and their methods of preparation and use
5,520,916 Non-woven fabric material comprising hyaluronic acid derivatives
and use
5,503,848 Spongy material consisting essentially of hyaluronic acid or its
derivatives, and its use in microsurgery
5,202,431 Partial esters of hyaluronic acid
4,636,524 & 4,605,691 & 4,582,865 Cross-linked gels of hyaluronic acid and
products containing such gels
The following patents discuss the use of HA for treating various
diseases, which uses are also applicable uses of the present HA mimics:
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5,914,314 Use of a form of hyaluronic acid and a medicinal agent for reducing
rejection of organs transplantation in mammals
5,888,986 & 5,880,108 5,591,724 Method for treating the urinary bladder and
associated structures using hyaluronic acid
5,847,002 Compositions, for inhibition, control and regression of
angiogenesis,
containing hyaluronic acid and NSAID
5,834,444 Hyaiuronic acid and salts thereof inhibit arterial restenosis
5,830,882 Compositions containing a form of hyaluronic acid and a medicinal
agent for treating acne in mammals and methods for administration of such
composition
5,8'27,834 Method of using hyaluronic acid or its pharmaceutically acceptable
salts for the treatment of disease
5,728,391 Hyaluronic acid and its salt for treating skin diseases
5,679,655 Method of treating lesions resuitin;g from genital herpes with
hyaluronic acid-urea pharmaceutical compositions
5,674,857 Use of hyaluronic acid to repair ischernia reperfusion damage
5,646,129 Method of using low molecular 'weight hyaluronic acid for
stimulating bone formation
5,639,738 Treatment of basal cell carcinoma and actinic keratosis employing
hyaluronic acid and NSAIDs
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5,b33,003 Use of intratracheally administered hyaluronic acid to ameliorate
emphysema
5,631,242 Hyaluronic acid-urea pharmaceutical compositions utilized for
treatment of diseases of cutis
5,b24,915 & 5,583,120 & 5,583,119 & 5,550,112 & .5,529;987 Hyaluronic acid-
urea
pharmaceutical compositions and uses
5,614,506 Use of hyaluronic acid and forms to prevent arterial restenosis
5,604,200 Wound therapeutic mixture containing medical grade hyaluronic
acid and tissue culture grade plasma-fibronectin in a delivery system that
i5 creates a moist environment which' simulates in utero healing
5,583,118 Method of treating an anorectal disease using hyaluronic acid-urea
pharmaceutical compositions
4,801,b19 Hyaluronic acid preparation to be used for treating inflammations
of skeletal joints
The following patents have disclosE~d compositions of matter
comprising HA as an ingredient, and which would be useful in making the
present compositions claimed, except that the present HA mimics would be
substituted for HA:
5,847,002 Compositions, for inhibition, control and regression of
angiogenesis,
containing hyaluronic acid and NSAID
5,b79,655 Method of treating lesions resulting from genital herpes with
hyaluronic acid-urea pharmaceutical compositions
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The following patents have disclosed other methods of using HA,
which other uses would be applicable uses of the present HA mimics:
5,772,982 Method of using hyaluronic acid for the detection, location and
diagnosis of tumors
4,804,537 Sperm selection process using a salt of hyaluronic acid
Citation of the above documents is not intended as an admission that any of
the foregoing is pertinent prior art. All sitatements as to the date or
representation as to the contents of these documents is based on subjective
characterization of information available to the applicant, and does not
constitute any admission as to the accuracy of the dates or contents of these
documents.
SUMMARY QF THE I~VEN;TION
The present invention provides HA mirrlics comprising a polypeptide
having alternating acidic / non-acidic residues;, wherein said mimic binds to
the hyaluronic acid binding domain of the receptor for hyaluronan mediated
mobility. Those HA mimics wherein said polypeptide is four to 15 residues
in length are preferred. More preferred are those HA mimics as described,
wherein said residues include at least one dextrorotatory residue. Most
preferred are HA mimics comprising an amino acid sequence selected from
the group consisting of: SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO
4; SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 8; SEQ ID NO 9; SEQ
ID NO 10; SEQ ID NO 11; SEQ ID NO 12; SEQ ID NO 13; SEQ ID NO 14; SEQ ID
NO i5; SEQ ID NO 16; SEQ ID NO 17; SEQ ID NO 18; SEQ ID NO 19; and SEQ
ID NO 20, or a homologue thereof, wherein said homologue binds to the
hyaluronic acid binding domain of the receptor for hyaluronan mediated
mobility.
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In a preferred embodiment of the present invention, the HA mimics
comprising the sequence:
X-A/a-X-A/a-X-a-X-al
wherein (X) can be any amino acid, (A.) is L-glutamate or L-
aspartate and {a) and (al) are D-glutamate or D-aspartate.
Those wherein X is hydrophobic are more preferred and those wherein X is
hydrophobic and al is D-glutamate are most pre~Eerred.
The present invention also provides nucleic acid compounds
comprising a nucleic acid which encodes an amino acid sequence selected
from the group consisting of: SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ
ID NO 4; SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 8; SEQ ID NO
9; SEQ ID NO 10; SEQ ID NO 21; SEQ ID NO 12; SEQ ID NO 13; SEQ ID NO I4;
SEQ ID NO 15; SEQ ID NO 16; SEQ ID NO 17; SEQ ID NO 18; SEQ-ID NO 19;
and SEQ ID NO 20; and homologues thereof, wherein said homologue binds
to the hyaluronic acid binding domain of i:he receptor for hyaluronan
mediated mobility.
Also provided are HA mimics comprising a polypeptide comprising
double hydrophobic residues, wherein said mimic binds to the hyaluronic
acid binding domain of the TSG-b. In particular, provided are HA mimics of
which are selected from the group consisting of: GYYFNVAM; WAYNFLVM;
TQSLNNHM; WWPFINAY; WWKADMVG; WWPFINAY; MALQLPYY;
IIYEEFFV; ISINNRWY; VTPPVYFT; Q:IRNGWFW; SWWFGPLA;
GDWEQILT; PAGFGWNL; NMRFNIEN; and QMTFFDGV, or a homologue
thereof, wherein said homologue binds to hyaluronic acid binding domain of
the TSG-6. Nucleic acids which encode the above compounds are also part of
the present invention.
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The present invention also provides methods to affect a
hyaluronidase-mediated biological response in a patient in need of such
affecting , comprising administering an HA mimic of the present invention.
The present invention also provides methods to inhibit hyaluronidase
activity in a patient in need of such inhibition, comprising administering an
HA mimic of the present invention.
The present invention also provides methods to bind receptors that
bind hyaluronic acid, comprising introducing an HA mimic of the present
invention.
The present invention also provides methods to mimic hyaluronic
acid in a biological system; comprising introducing an HA mimic of the
present invention.
The present invention also provides mef:hods to affect cell signalling
associated with hyaluronic acid/hyaluronic acid receptor interactions,
comprising introducing an HA xnimic of the present invention.
The present invention also provides methods to treat hyaluronic acid-
associated disease in a. patient in need of such treatment, comprising
administering an HA mimic of the present invention. In particular, those
methods wherein said disease is selected from the group consisting of:
inflammation; tumor angiogenesis; skin disease; bone disease; wound
healing; osteoarthritis; rheumatoid arthritis; infectious disease; immune
disease; and cardiovascular disease are preferred.
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The present invention also provides methods to inhibit metastasis in a
patient in need of such inhibition, comprising administering an HA mimic of
the present invention.
The present invention also provides mei~hods to inhibit fertilization in
a patient in need of such inhibition, comprisinf; administering an HA mimic
of the present invention.
The present invention also provides methods to introduce an agent
into a cell which binds hyaluronic acid, comprising administering the agent
in conjunction with an HA mimic of the present invention.
The present invention also provides methods to isolate peptides that
mimic a ligand's binding to a receptor comprising the steps of:
(a) preparing a random library of peptides and binding
said library to a bead;
(b) placing the library in contact wiith the receptor
under conditions for binding;
(c) washing off unbound peptides;
{d) contacting the washed bead-bound :library with anti-
receptor antibody;
{e) detecting and selecting beads having antibody
bound thereto;
(f) eluting the antibody; -~.
(g) repeating steps (a} through (f) and then repeating
steps (a) through (c), and then incubating the beads
in the presence of receptor prior to adding anti-
receptor antibody;
(h) detecting and selecting beads that did not bind antibody;
(i) determining the sequence of the bead-bound
peptide.
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"Protein" means any compound which comprises amino acids, including
peptides , polypeptides, fusion proteins, etc.
"in conjunction with" means any physical interaction that results in co-
localization.
Moreover, for the purposes of the present invention, the term "a" or
"an" entity refers to one or more of that entity; :Eor example, "a protein" or
"a
nucleic acid molecule" refers to one or more of those compounds or at least
one compound. As such, the terms "a" (or "an"), "one or more" and "at least
one" can be used interchangeably herein. It is ~~lso to be noted that the
terms
"comprising", "including", and "having" can be used interchangeably.
Furthermore, a compound "selected from the group consisting of" refers to
one or more of the compounds in the list that follows, including mixtures
(i.e., combinations) of two or more of the compounds. According to the
present invention, an isolated, or biologically pure, protein or nucleic acid
molecule is a compound that has been removed from its natural milieu. As
such, "isolated" and "biologically pure" do not necessarily reflect the extent
to
which the compound has been purified. An isolated compound of the
present invention can be obtained from its natural source, can be produced
using molecular biology techniques or can be produced by chemical synthesis.
BRIEFS ION OF THE D:IZAWINC,S
FIG.I shows a scheme far screening a random phage library for
binding to HA.
FIG. 2 shows binding of two synthetic peptides :to a hyaluronic binding
domain.
FIG. 3 shows affinities of peptides that bind to the HA binding domain.
FIG. 4 shows a table describing abbreviations of i:he amino acids.
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.TE AILED Di~"~[PTION OF THE INVENTION
The present invention provides HA mirxtics comprising a polypeptide
having alternating acidic / non-acidic residues,, wherein said mimic binds to
the hyaluronic acid binding domain of the receptor for hyaluronan mediated
mobility. Those HA .mimics wherein said polypeptide is four to 15 residues
in length are preferred. More preferred are tr~ose HA mimics as described,
wherein said residues include at least one d:extrorotatory residue. Most
preferred are HA mimics comprising an amino acid sequence selected from
IO the group consisting of: SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO
4; SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SE~Q ID NO 8; SEQ ID NO 9; SEQ
ID NO 10; SEQ ID NO 11; SEQ ID NO 12; SEQ ID NO 13; SEQ ID NO 14; SEQ ID
NO 15; SEQ ID NO 16; SEQ ID NO 17; SEQ ID I~fO 18; SEQ ID NO 19; and SEQ
ID NO 20, or a homologue thereof, wherein said homologue binds to the
IS hyaluronic acid binding domain of the receptor for hyaluronan mediated
mobility.
In a preferred embodiment of the present invention, the HA mimics
comprising the sequence:
X-A/a-X-A/a-X-a-X-a1
wherein (X) can be any amino acid, {?~) is L-glutamate or L-
aspartate and (a) and (al) are D-glutamatEr or D-aspartate.
Those wherein X is hydrophobic are more preferred and those wherein X is
hydrophobic and al is D-glutamate are most preferred.
Also provided are HA mimics comprising a polypeptide comprising
double hydrophobic residues, wherein said mimic binds to the hyaluronic
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acid binding domain of the TSG-6. In particular, provided are HA mimics of
which are selected from the group consisting of: GYYFNVAM; WAYNFLVM;
TQSLNNHM; WWPFINAY; WWKADMVG; WWPFINAY; MALQLPYY;
IIYEEFFV; ISINNRWY; VTPPVYFT; QIRNGWFW; SWWFGPLA;
GDWEQILT; PAGFGWNL; NMRFNIEN; and ~)MTFFDGV, or a homologue
thereof, wherein said homologue binds to hyalu.ronic acid binding domain of
the TSG-b. Nucleic acids which encode the above compounds are also part of
the present invention.
The present invention also provides nucleic acid compounds
comprising a nucleic acid which encodes an amino acid sequence selected
from the group consisting of: SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ
ID NO 4; SEQ ID NO 5; SEQ ID NO 6; SEQ iD NO 7; SEQ ID NO 8; SEQ ID NO
9; SEQ ID NO 10; SEQ ID NO 11; SEQ ID NO I2; SEQ ID NO 13; SEQ ID NO 14;
SEQ ID NO 15; SEQ ID NO 16; SEQ ID NO I7; SEQ ID NO 18; SEQ ID NO 19;
and SEQ ID NO 20; and homologues thereof, wherein said homologue binds
to the hyaluronic acid binding domain of the receptor for hyaluronan
mediated mobility.
Also provided are compositions of matter comprising an HA mimic of
the present invention, in particular, those which comprise a non-steroidal
anti-inflammatory drug.
The present invention also provides methods to affect a
hyaluronidase-mediated biological response in a patient in need of such
affecting , comprising administering an HA mimic of the present invention.
The present invention also provides methods to inhibit hyaluronidase
activity in a patient in need of such inhibition, comprising administering an
HA mimic of the present invention.
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The present invention also provides mE~thods to bind receptors that
bind hyaluronic acid, comprising introducing an HA mimic of the present
invention.
The present invention also provides xxiethods to mimic hyaluronic
acid in a biological system, comprising introducing an HA mimic of the
present invention.
The present invention also provides methods to affect cell signalling
associated with hyaluronic acid/hyaluronic acid receptor. interactions,
comprising introducing an HA mimic of the present invention.
The present invention also provides methods to treat hyaluronic acid-
associated disease in a patient in need of such treatment, comprising
administering an HA mimic of the present invention. In particular, those
methods wherein said disease is selected from the group consisting of:
inflammation; tumor angiogenesis; skin disease; bone disease; wound
healing;osteoarthritis; rheumatoid arthritis; infectious disease; immune
disease; and cardiovascular disease are preferred.
The present invention also provides methods to inhibit metastasis in a
patient in need of such inhibition, comprising z~dministering an HA mimic of
the present invention.
The present invention also provides methods to inhibit fertilization in
a patient in need of such inhibition, comprising administering an HA mimic
of the present invention.
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The present invention also provides methods to introduce an agent
into a cell which binds hyaluronic acid, comprising administering the agent
in conjunction with an HA mimic of the present invention.
S The present invention also provides methods to isolate peptides that
mimic a ligand's binding to a receptor comprising the steps of:
(a) preparing a random library of peptides and binding
said libraxy to a bead;
(b} placing the library in contact ~~ith the receptor
under conditions for binding;
(c) washing off unbound peptides;
(d) contacting the washed bead-bound library with anti-
receptor antibody;
(e) detecting and selecting beads having antibody
bound thereto;
(f) eluting the antibody;
(g} repeating steps (a) through (f) anal then repeating
steps (a} through (c), and then inctabating the beads
in the presence of receptor prior to adding anti
receptor antibody;
(h) detecting and selecting beads that dlid not bind antibody;
(i) . determining the sequence of the bead-bound
peptide.
HA mimic homologs of the present invention can be produced using
techniques known in the art including, but not limited to, direct
modifications to the peptide or modifications to the gene encoding the
peptide using, for example, classic or recombinant nucleic acid techniques to
effect random or targeted mutagenesis.
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A HA mimic of the present invention, including a homolog, can be
identified in a straight-forward manner by the protein's ability to inhibit an
HA target enzyme.
One embodiment , of a HA mimic of they present invention is a fusion
protein that includes a HA receptor binding domain attached to one or more
fusion segments. Suitable fusion segmenia for use with the present
invention include, but are not limited to, segments that can: enhance a
protein's stability and/or assist purification of an HA mimic (e.g., by
affinity
chromatography). A suitable fusion segment can be a domain of any size that
has the desired function (e.g.; imparts increased stability, imparts increased
immunogenicity to a protein, and/or simplifies purification of a protein).
Fusion proteins are preferably produced by culturing a recombinant cell
transformed with a fusion nucleic acid molecule that encodes a protein
including the fusion segment attached to either the carboxyl and/or amino
terminal end of an HA receptor binding domain. Preferred fusion segments
include a metal binding domain (e.g., a poly-histidine segment); an
immunoglobulin binding domain (e.g., Protein A; Protein G; T cell; B cell; Fc
receptor or complement protein antibody-binding domains); a sugar binding
domain (e.g., a maltose binding domain); a "tag" domain (e.g., at Least a
portion of Q-galactosidase, a strep tag peptide, other domains that can be
purified using compounds that bind to the domain,. such as monoclonal
antibodies); and/or a linker and enzyme domain (e.g., alkaline phosphatase
domain connected to A HA mimic by a linker). More preferred fusion
segments include metal binding domains, such as a poly-histidine segment; a
maltose binding domain; a strep tag peptide; and a phage T7 S10 peptide.
An HA mimic of the present invention can also be a chimeric
molecule comprising an HA mimic and a se<:ond molecule. In particular,
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there is provided a chimeric molecule that enables the chimeric molecule to
be bound to a surface in such a manner that the chimera inhibits an HA-
target enzyme in essentially the same manner as an HA mimic that is not
bound to a surface. An example of a suitable second molecule includes a
portion of an immunoglobulin molecule or another ligand that has a suitable
binding partner that can be immobilized on a substrate, e.g., biotin and
avidin, or a metal-binding protein and a metal (e:g., His), or a sugar-binding
protein and a sugar (e.g., maltose).
i0 Nucleic acid molecules comprising a nucleic acid molecule which
encodes the present HA mimics are also provided by the present invention.
In particular, there are provided nucleic acids encoding the present HA
mimics, wherein said HA mimics comprise, an amino acid sequence selected
the group consisting of: SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO
4; SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 8; SEQ ID NO 9; SEQ
ID NO 10; SEQ ID NO 11; SEQ ID NO 12; SEQ ID NO 13; SEQ ID NO 14; SEQ ID
NO 15; SEQ ID NO 16; SEQ ID NO 17; SEQ ID NO 18; SEQ ID NO 19; and SEQ
ID NO 20.
The present invention also comprises expression vectors and
recombinant cells comprising the present nucleic acid molecules. Also
provided are fusion constructs using the present: nucleic acid compounds.
One approach is to create fusogenic peptides, in which a short (18-mer)
peptide is attached to the effector peptide that causes endocytotic uptake of
the .
peptide into the cell. j. Hawiger, Noninvasive intracellular delivery of
functional peptides and proteins, Curr. Opinion Chemical Biology 3, 89-94
(1999) and A. Prochiantz, Getting hydrophilic compounds into cells: lessons
from heomeopeptides. Cur. Opin. Neurobioi 6, 629-630 (1996)
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Included within the scope of the present invention, with particular
regard to the nucleic acids above, are degenerate sequences and homologues.
The present invention also includes variants due to laboratory manipulation,
such as, but not limited to, variants produced during polymerase chain
reaction amplification or site-directed mutageoesis. It is also well known
that there is a substantial amount of redundancy in the various codons which
code for specific amino acids. Therefore, this invention is also directed to
those nucleic acid sequences which contain alternative codons which code for
the eventual translation of the identical amino acid. Also included within
the scope of this invention are mutations either in the nucleic acid sequence
or the translated protein which do not substantially alter the ultimate
physical properties of the expressed protein. For example, substitution of
valine for leucine, arginine for lysine, or asparagine for glutamine may not
cause a change in functionality of the polypeptide.
Knowing the nucleic acid sequences of certain HA mimic nucleic acid
molecules of the present invention allows one skilled in the art to, for
example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic
acid molecules including at least a portion of such nucleic acid molecules
(e.g., nucleic acid molecules including full-length genes, full-length coding
regions, regulatory control sequences, truncated coding regions), and (c)
obtain HA mimic nucleic acid molecules. Such nucleic acid molecules can be
obtained in a variety of ways including screening appropriate expression
libraries with antibodies of the present invention; traditional cloning
techniques using oligonucleotide probes of the present invention to screen
appropriate libraries of DNA; and PCR amplification of appropriate libraries
or DNA using oligonucleotide primers of the present invention. Preferred
libraries to screen or from which to amplify nucleic acid molecules include
livestock (cattle, horse, pig) and companion animal (dog and cat) cDNA
libraries as well as genomic DNA libraries. Si~cr~ilarly, preferred DNA
sources
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to screen or from which to amplify nucleic acid molecules include adult
cDNA and genomic DNA. Techniques to clone and amplify genes are
disclosed, for example, in Sambrook et al., ibid.
One embodiment of the present invention includes a recombinant
vector, which includes at least one isolated nucleic acid molecule of the
present invention, inserted into any vector capable of delivering the nucleic
acid molecule into a host cell. Such a vector contains heterologous nucleic
acid sequences, that is, nucleic acid sequences. that are not naturally found
adjacent to nucleic acid molecules of the present invention and that
preferably are derived from a species other than the species from which the
nucleic acid molecules) are derived. The vector can be either RNA or DNA,
either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
Recombinant vectors can be used in the r_loning, sequencing, and/or
otherwise manipulation of the HA mimic nucleic acid molecules of the
present invention.
One type of recombinant vector, referred to herein as a recombinant
molecule, comprises a nucleic acid molecule of the present invention
operatively linked to an expression vector. The phrase "operatively linked"
refers to insertion of a nucleic acid molecule into an expression vector in a
manner such that the molecule is able to be exlrressed when transformed into
a host cell. As used herein, an expression ve~~tor is a DNA or RNA vector
that is capable of transforming a host cell and of effecting expression of a
specified nucleic acid molecule. Preferably, the expression vector is also
capable of replicating within the host cell. Expression vectors can be either
prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression
vectors of the present invention include any vE~ctors that function (i.e.,
direct
gene expression) in recombinant cells of the present invention, including in
bacterial, fungal, endoparasite, insect, other animal, and plant cells.
Preferred
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expression vectors of the present invention can direct gene expression in
bacterial, yeast, insect and mammalian cells and more preferably in the cell
types disclosed herein.
Tn particular, expression vectors of the present invention contain
regulatory sequences such as transcription control sequences, translation
control sequences, origins of replication, and other regulatory sequences that
are compatible with the recombinant cell and that control the expression of
nucleic acid molecules of the present invention. In particular, recombinant
molecules of the present invention include transcription control sequences.
Transcription control sequences are sequences which control the initiation,
elongation, and termination of transcription. Particularly important
transcription control sequences are those which control transcription
initiation, such as promoter, enhancer, operator and repressor sequences.
Suitable transcription control sequences include any transcription control
sequence that can function in at Ieast one of the recombinant cells of the
present invention. A variety of such transcription control sequences are
known to those skilled in the art. Preferred transcription control sequences
include those which function in bacterial, yeast, insect and mammalian cells,
such as, but not limited to; fac, lac, trp, trc, oxy-pro, omp/Ipp, rrnB,
bacteriophage lambda (such as lambda pL and lambda pR and fusions that
include such promoters), bacteriophage T7, T7lac, bacteriophage T3,
bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor,
Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis
virus subgenomic promoters), antibiotic resistance gene, baculovirus,
Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus,
other poxvirus, adenovirus, cytomegalovirus (such as intermediate early
promoters), simian virus 40, retrovirus, actin, retroviral long terminal
repeat,
Rous sarcoma virus, heat shock, phosphate and nitrate transcription control
sequences as well as other sequences capable of controlling gene expression in
19
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prokaryotic or eukaryotic cells. Additional suitable transcription control
sequences include tissue-specific promoters and enhancers as well as
lymphokine-inducible promoters (e.g., promotErrs inducible by interferons or
interleukins). Transcription control sequences of the present invention can
also include naturally-occurring transcription. control sequences naturally
associated with humans: The present invention also comprises expression
vectors comprising a nucleic acid molecule described herein.
Recombinant DNA technologies can be used to improve expression of
transformed nucleic acid molecules by manipulating, for example, the
number of copies of the nucleic acid mole<:ules within a host cell, the
efficiency with which those nucleic acid molecules are transcribed, the
efficiency with which the resultant transcripts are translated, and the
efficiency of post-translational modifications. P.ecombinant techniques useful
IS for increasing the expression of nucleic acid molecules of the present
invention include, but are not limited to, operatively linking nucleic acid
molecules to high-copy number plasmids, integration of the nucleic acid
molecules into one or more host cell chromosomes, addition of vector
stability sequences to plasmids, substitutions or modifications of
transcription
control signals {e.g., promoters, operators, enhancers}, substitutions or
modifications of translational control signals (e.g., ribosome binding sites,
Shine-Dalgarno sequences), modification of nucleic acid molecules of the
present invention to correspond to the codon usage of the host cell, deletion
of sequences that destabilize transcripts, and use of control signals that
temporally separate recombinant cell growth from recombinant enzyme
production during fermentation. The activity of an expressed recombinant
protein of the present invention may be improved by fragmenting,
modifying, or derivatizing nucleic acid molecules encoding such a protein.
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Also provided by the present invention are recombinant cells
transformed with a nucleic acid described herein.
Transformation of a nucleic acid molecule into a cell can be
accomplished by any method by which a nucleic: acid molecule can be inserted
into the cell. Transformation techniques include, but are not limited to,
transfection, electroporation, microinjection, lipofection, adsorption, and
protoplast fusion. A recombinant cell may remain unicellular or may grow
into a tissue, organ or a multiceliular organism. Transformed nucleic acid
molecules of the present invention can remain extrachromosomal or can
integrate into one or more sites within a chromosome of the transformed
(i.e., recombinant) cell in such a manner that their ability to be expressed
is
retained.
Suitable host cells to transform include any cell that can be transformed
with a nucleic acid molecule of the present invention. Host cells can be
either
untransformed cells or cells that are already i:ransformed with at least one
nucleic acid molecule (e.g., nucleic acid molecules encoding one or more
proteins of the present invention and/or other proteins useful in the
production of multivalent vaccines). Host cells of the present invention
either can be endogenously (i.e., naturally) capable of producing HA mimic of
the present invention or can be capable of producing such proteins after being
transformed with at least one nucleic acid molE~cule of the present invention.
Host cells of the present invention can be any cell capable of producing at
least
one protein of the present invention, and include bacterial, fungal (including
yeast), other insect, other animal and plant cells. Preferred host cells
include
bacterial, mycobacterial, yeast, parasite, insect and mammalian cells. More
preferred host cells include Salmonella, Escherichia, Bacillus, Listeria,
Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster
kidney) cells, COS (e.g., COS-7) cells, and Vero cells. Particularly preferred
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host cells are Escherichia coli, including E, coli K-12 derivatives; and
insect
cell systems which utilize baculovirus.
A recombinant cell is preferably produced by transforming a host cell
with one ar more recombinant molecules, each comprising one or more
nucleic acid molecules of the present invention operatively linked to an
expression vector captaining one or more tra~,nscription control sequences.
The phrase "operatively linked" refers to insertion of a nucleic acid molecule
into an expression vector in a manner such that the molecule is able to be
expressed when transformed into a host cell.
A recombinant cell of~ the present :invention includes any cell
transformed with at least one of any nucleic acid molecule of the present
invention. Suitable and preferred nucleic acid molecules as well as suitable
and preferred recombinant molecules with which to transform cells are
disclosed herein.
The translation of the RNA into a peptiide or a protein will result in
the production of the HA mimic protein which can be identified, for example,
by the activity of HA mimic or by immunological reactivity with an anti-HA
mimic antibody. In this method, pools of mRNA isolated from HA mimic-
praducing cells can be analyzed for the presence of an RNA which encodes at
least a portion of the HA mimic protein. Further fractionation of the RNA
pool can be done to purify the HA mimic RNA from non-HA mimic RNA.
The peptide or protein produced by this method may be analyzed to provide
amino acid sequences which in turn are used to provide primers far
production of HA mimic cDNA, or the RNA, used for translation can be
analyzed to provide nucleotide sequences encoding HA mimic and produce
probes for the production of HA mimic cDNA. These methods are known in
the art and can be found in, for example, Sambrook, J., Fritsch, E. F.,
Maniatis,
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T. in Molecadar Cloning: A Laboratory Manual, Second Edition, Coid Spring
Harbor Laboratory Press, Cold Spring Harbor, N.,Y. 1989.
Other types of libraries, as well as libraries constructed from other cells
or cell types, may be useful for isolating HA :mimic-encoding DNA. Other
types of libraries include, but are not limited to, cDNA libraries derived
from
other mammals or cell lines derived from other mammals, and genomic
DNA libraries. Preparation of cDNA libraries c:an be performed by standard
techniques. Well known cDNA library construction techniques can be found
IO in, for example, Sarnbrook, J., et al., ibid.
In one embodiment, an isolated protein of the present invention is
produced by culturing a cell capable of expressing the protein under
conditions effective to produce the protein, and recovering the protein. A
IS preferred cell to culture is a recombinant cell of the present invention.
Effective culture conditions include, but are not limited to, effective media,
bioreactor, temperature, pH and oxygen conditions that permit protein
production. An effective medium refers to any medium in which a cell is
cultured to produce a HA mimic of the present invention. Such a medium
20 typically comprises an aqueous medium having assimilable carbon, nitrogen
and phosphate sources, and appropriate salt;, minerals, metals and other
nutrients, such as vitamins. Cells of the present invention can be cultured in
conventional fermentation bioreactors, shake flasks, test tubes, microtiter
dishes, and petri plates. Culturing ran be carried out at a temperature, pH
and
25 oxygen content appropriate for a recombinant cell. Such culturing
conditions
are within the expertise of one of ordinary skill in the art.
Depending on the vector and host system used for production,
resultant proteins of the present invention rrtay either remain within the
30 recombinant cell; be secreted into the fermentation medium; be secreted
into
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a space between two cellular membranes, such as the periplasmic space in E.
toll; or be retained on the outer surface of a cell or viral membrane. The
phrase "recovering the protein", as well as similar phrases, refers to
collecting
the whole fermentation medium containing the protein and need not imply
additional steps of separation or purification. Proteins of the present
invention can be purified using a variety of standard protein purification
techniques, such as, but not limited to, affinity f~hromatography, ion
exchange
chromatography, filtration; electrophoresis, hydrophobic interaction
chromatography, gel filtration chromatography, reverse phase
chromatography, concanavalin A chromatography, chrornatofocusing and
differential solubilization. Proteins of the present invention are preferably
retrieved in "substantially pure" form. As used herein, "substantially pure"
refers to a purity that allows for the effecitive use of the protein as a
therapeutic composition or diagnostic. A .therapeutic composition for
animals, for example, should exhibit few impurities.
In addition, a recombinant HA mimic can be separated from other
cellular proteins by use of an irnmunoa,ffinity column made with
monoclonal or polycional antibodies specific for the HA mimic, or
polypeptide fragments of the HA mimic.
The present invention also includes isolated (i.e., removed from their
. natural milieu) antibodies that selectively bind to an HA mimic protein of
the present invention or a mimetope thereof (i.e., anti-HA mimic antibodies).
As used herein, the term "selectively binds to'" the HA mimic protein refers
to the ability of antibodies of the present invention to preferentially bind
to
specified proteins and rnimetopes thereof of the present invention. Binding
can be measured using a variety of methods standard in the art including
enzyme immunoassays (e.g., ELISA); imrnunoblot assays, etc.; see, for
example, Sambrook et al., ibid. An anti-HA mimic antibody preferably
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selectively binds to A HA mimic in such a way as to reduce the activity of
that
protein. These antibodies may be admixed or conjugated with additional
materials, such as cytotic agents or other antibody fragments.
Isolated antibodies of the present invention can include antibodies in a
bodily fluid (such as, but not limited to, serum;l, or antibodies that have
been
purified to varying degrees. Antibodies of l~he present invention can be
polyclonai or monoclonal. Functional equivalents of such antibodies, such as
antibody fragments and genetically-engineered antibodies {including single
chain antibodies or chirneric antibodies that can bind to more than one
epitope) are also included in the present invention.
A preferred method to produce antibodies of the present invention
includes (a) administering to an animal an e~Efective amount of a protein,
peptide or mimetope thereof of the present invention to produce the
antibodies and {b) recovering the antibodies. I:n another method, antibodies
of the present invention are produced recombinantly using techniques as
heretofore disclosed to produce HA mimic proteins of the present invention.
Compositions of the present invention can be administered to any
animal having at least one HA mimic-target enzyme that can be inhibited by
a therapeutic compound of the present invention or by a protein expressed by
a nucleic acid molecule contained in a therapeutic composition. Preferred
animals to treat are humans, although other mammals, such as cattle, pigs,
sheep, horses, cats, dogs, and other pets, work and/or economic food animals
are also within the scope of the present invention.
Therapeutic compositions of the present invention can be formulated
in an excipient that the animal to be treated can tolerate. Examples of such
excipients include water, saline, Ringer's solutiion, dextrose solution,
Hank's
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solution, and other aqueous physiologically balanced salt solutions.
Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or
triglycerides may also be used. Other useful formulations include
suspensions containing viscosity enhancing agents, such as sodium
carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain
minor amounts of additives, such as substances that enhance isotonicity and
chemical stability. Examples of buffers include phosphate buffer, bicarbonate
buffer and Tris buffer, while examples of preservatives include thimerosal,
cresols, forrnalin and benzyl alcohol. Standard formulations can either be
liquid injectables or solids which can be taken up in a suitable liquid as a
suspension or solution for injection. Thus, in a non-liquid formulation, the
excipient can comprise dextrose, human serurrt albumin, preservatives, etc.,
to which sterile water or saline can be added prior to administration.
i5 Administration of the present compounds can be by a variety of routes
known to those skilled in the art including, but not limited to, subcutaneous,
intradermal, intravenous, intranasal, oral, transdermal, intramuscular routes
and other parenteral routes.
In one embodiment of the present: invention, a therapeutic
composition can include an adjuvant. A preferred adjuvant is a member of
the group of non-steroidal antiinflammatory drugs, such as ibuprofen.
In another embodiment of the present invention, a therapeutic
composition can include a carrier. Carriers include compounds that increase
the half-life of a therapeutic composition in i:he treated animal. Suitable
carriers include, but are not limited to, polymeric controlled release
vehicles,
biodegradable implants, liposomes, bacteria, viruses, other cells, oils,
esters,
and glycols.
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Another embodiment of the present invention is a controlled release
formulation that is capable of slowly releasing a composition of the present
invention into an animal. As used herein, a controlled release formulation
comprises a composition of the present invention in a controlled release
vehicle. Suitable controlled release vehicles include, but are not limited to,
biocompatible polymers, other polymeric matrices, capsules, microcapsules,
microparticles, bolus preparations, osmotic pumps, diffusion devices,
liposomes, lipospheres, and transdermal delivery systems. Other controlled
release formulations of the present invention include liquids that, upon
administration to an animal, form a solid or a gel in situ. Preferred
controlled release formulations are biodegradabi~e {i.e., bioerodible).
A preferred controlled release formulation of the present invention is
capable of releasing a composition of the present invention into the blood of
1S an animal at a constant rate sufficient to attain therapeutic dose levels
of the
composition to reduce HA-mediated biological responses in the animal. The
therapeutic composition is preferably released ~wer a period of time ranging
from about 1 day to about 12 months, and include release over a 2, 3, 4 , 5,
6, 7
day through a 30 day time period.
Acceptable protocols to administer therapeutic compositions of the
present invention in an effective manner include individual dose size,
number of doses, frequency of dose administration, and mode of
administration. Determination of such protocols can be accomplished by
those skilled in the art. A suitable single dose is a dose that is capable of
protecting {i.e., preventing or treating) an animal from disease when
administered one or more times over a suitable time period. The need for
additional administrations of a therapeutic composition can be determined by
one of skill in the art in accordance with the given condition of a patient.
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According to one embodiment, a nucleic acid molecule of the present
invention can be administered to an animal in a~ fashion to enable expression
of that nucleic acid molecule into an HA mimic protein in the animal.
Nucleic acid molecules can be delivered to an animal in a variety of methods
including, but not limited to, (a) administering a naked (i.e., not packaged
in a
viral coat or cellular membrane} nucleic acid molecule (e.g., as naked DNA
molecules, such as is taught, for example in ~olff et al., 1990, Science 247,
I4b5-1468) or (b) administering a nucleic acid molecule packaged as a
recombinant virus or as a recombinant cell (i.e., the nucleic acid molecule is
i0 delivered by a viral or cellular vehicle).
A naked nucleic acid molecule of the present invention includes a
nucleic acid molecule of the present invention and preferably includes a
recombinant molecule of the present invention that preferably is replication,
or otherwise amplification, competent. A naked nucleic acid of the present
invention can comprise one or more nucleic acid molecules of the present
invention in the form of, for example, a bicistronic recombinant molecule
having, for example one or more internal ribosome entry sites. Preferred
naked nucleic acid molecules include at Least a portion of a viral genome
(i.e.,
a viral vector). Preferred viral vectors include those based on alphaviruses,
poxviruses, adenoviruses, herpesviruses, pico~°naviruses; and
retroviruses,
with those based on alphaviruses (such as Sindbis or Sernliki virus), species-
specific herpesviruses and species-specific poxviruses being particularly
preferred. Any suitable transcription control sequence can be used, including
those disclosed as suitable for protein production. Particularly preferred
transcription control sequence include cytomegalovirus intermediate early
(preferably in conjunction with lntron-A), Rous Sarcoma Virus long terminal
repeat, and tissue-specific transcription control sequences, as well as
transcription control sequences endogenous to viral vectors if viral vectors
are used. The incorporation of "strong" poly(A) sequences are also preferred.
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Naked nucleic acid molecules of the present invention can be
administered by a variety of methods. Suitable delivery methods include, for
example, intramuscular injection, subcutaneous injection, intradermal
injection, intradermal scarification, particle bombardment, oral application,
and nasal application, with intramuscular injection, intradermal injection,
intradermal scarification and particle bombardment being preferred. A
preferred single dose of a naked DNA molecule ranges from about 1
nanogram (ng) to about 1 milligram (mg), depending on the route of
administration and/or method of delivery, as can be determined by those
skilled in the art. Examples of administration methods are disclosed; for
example, in U.S. Patent No. 5,204,253, by Brunei, et al., issued April 20,
1993,
PCT Publication No. W0 95/19799, published July 27, 1995, by McCabe, and
PCT Publication No. WO 95/05853, published March 2, 1995, by Carson, et al.
Naked DNA molecules c~f the present invention can be contained in an
aqueous excipient (e.g., phosphate buffered saline) and/or with a carrier
{e.g.,
lipid-based vehicles), or it can be bound to microparticles (e.g., gold
particles).
A recombinant virus of the present invention includes a recombinant
molecule of the present invention that is packaged in a viral coat and that
can
be expressed in an animal after administration. Preferably, the recombinant
molecule is packaging-deficient and/or encodes an attenuated virus. A
number of recombinant viruses can be used, including,, but not limited to,
those based on alphaviruses, poxviruses, adenoviruses, herpesviruses,
picornaviruses and retroviruses.
When administered to an animal, a recombinant virus of the present
invention infects cells within the recipient animal and directs the production
of a protein molecule that is capable of affecting HA-mediated biological
responses in the animal. For example, a recombinant virus comprising an
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HA mimic nucleic acid molecule of the present invention is administered
according to a protocol that results in the animal producing an amount of
protein sufficient to affect HA-mediated biological responses. Administration
protocols are similar to those described. herein for protein-based
compositions, with subcutaneous, intramuscular, intranasal and oral
administration routes being preferred.
Pharmaceutically useful compositions comprising -an HA mimic DNA
or an HA mimic protein, may be formulated according to known methods
such as by the admixture of a pharmaceutically acceptable carrier, or by
modification with additional chemical moieties so as to form a chemical
derivative. Examples of such carriers, modifications and methods of
formulation may be found in Remington's Pharmaceutical Sciences. To form
a pharmaceutically acceptable composition suitable for effective
administration, such compositions will contain an effective amount of the
protein or DNA.
The present invention also has the objective of providing suitable
topical, oral, systemic and parenteral formulations of the pharmaceutical
compounds herein provided. The formulaticms can be administered in a
wide variety of therapeutic dosage forms :in conventional vehicles for
administration. For example, the compounds can be formulated for oral
administration in the form of tablets, capsules (each including timed release
and sustained release formulations), pills, powders, granules, elixirs,
tinctures, solutions, susper<sions, syrups and emulsions, or by injection.
Likewise, they may also be administered intravenously (both bolus and
infusion), during angioplasty/catheterization, intraperitoneally,
subcutaneously, topically with or without occlusion, or intramuscularly, all
using forms well known to those of ordinary skill in the pharmaceutical arts.
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An HA mimic of the present invention can be combined with a buffer
in which the HA mimic molecule is solubilized, and/or with a carrier.
Suitable buffers and carriers are known to those skilled in the art. Examples
of suitable buffers include any buffer in which an HA mimic can function to
inhibit its target enzyme(s), such as, but not limited to, phosphate buffered
saline, water, saline, phosphate buffer, bicarbonate buffer, HEPES buffer (N-2-
hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered saline), TES buffer
(Tris-EDTA buffered saline), Tris buffer and TAE buffer (Tris-acetate-EDTA).
Examples of carriers include, but are not limited to, polymeric matrices,
toxoids, and serum albumins, such as bovine serum albumin.
In the methods of the present inveni:ion, the compounds herein
described in detail can form the active ingredient, and are typically
administered in admixture with suitable pharmaceutical diluents, excipients
ar carriers (collectively referred to herein as "carrier" materials) suitably
selected with respect to the intended form of administration, that is, oral
tablets, capsules, elixirs, syrups arid the like, and consistent with
conventional
pharmaceutical practices.
2p For instance, for oral administration in the form of a tablet or capsule,
the active drug component can be combined with an oral, non-toxic
pharmaceutically acceptable inert carrier such as ethanol, glycerol, water,
and
the like. Moreover, .when desired or necessary, suitable binders, lubricants,
disintegrating agents and coloring agents can also be incorporated into the
mixture. Suitable binders include, -without Iimiitation, starch, gelatin,
natural
sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic
gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose,
polyethylene glycol, waxes and the like. Lubricants used in these dosage
forms include, without limitation, sodium oleate, sodium stearate,
magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and
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the like. Disintegrators include, without limitation, starch, methyl
cellulose,
agar, bentonite, xanthan gum and the like.
For liquid forms the active drug component can be combined in
suitably flavored suspending or dispersing agents such as the synthetic and
natural gums, for example, tragacanth, acacia, methylcellulose and the like.
Other dispersing agents which maybe employed include glycerin and the like.
For parenterai administration, sterile suspensions and solutions are desired.
Isotonic preparations which generally contain suitable preservatives are
employed when intravenous administration is desired.
Topical preparations containing the active drug component can be
admixed with a variety of carrier materials wE~ll known in the art, such as,
e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils,
mineral
oil, PPG2 myristyl propionate, and the like, to :Form, e.g., alcoholic
solutions,
topical cleansers, cleansing creams, skin gels, skin lotions formulations. The
compounds of the present invention can also be administered in the form of
liposome delivery systems, such as small unilamellar vesicles, large
unilam.ellar vesicles and multilamellar vesicles. Liposomes can be formed
from a variety of phospholipids, such as cholesterol, stearylamine or
phosphatidylcholines.
The compounds of the present invention may also be coupled with
soluble polymers as targetable drug carriers. Such polymers can include
polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-
amidephenol, polyhydroxy-ethylaspartamidephenol, or
polyethyleneoxidepolylysine substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be coupled to a
class of biodegradable polymers useful in achieving controlled release of a
drug, for example, polylactic acid, polyepsilo:n caprolactone, polyhydroxy
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butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,
polycyanoacrylates and cross-linked or amphipathic block copolymers of
hydrogels.
Therefore, methods are also provided herein, which utilize the
compounds, formulations, compositions and protocols described above. In
particular, there are provided methods to antagonize or inhibit HA-target
enzymes, comprising administering an HA mimic of the present invention.
Preferred methods utilize the preferred and most preferred HA mimics.
The present invention also provides methods to treat and/or reduce
the risk of HA-related diseases in a patient in need of such treatment,
comprising administering the presently-disclosed HA mimics.
Lastly, the present invention also provides methods for producing the
present HA mimics in bodily fluid, comprising: producing a transgenic
animal that expresses in bodily fluid a transgene which encodes an HA mimic
of the present invention, wherein the HA mimic is secreted into the bodily
fluid produced by the transgenic animal; collecting bodily fluid from the
transgenic animal, which bodily fluid contains the HA mimic; and isolating
the HA mimic from the callected bodily fluid. Preferred are methods wherein
the bodily fluid is selected from the group consisting of: milk or urine.
Those
methods wherein the bodily fluid is milk and the animal is selected from the
group consisting of: goat; sheep; and cow are more preferred. Most preferred
are methods for producing an HA mimic in goat milk, comprising: producing
a transgenic goat that expresses in mammary tissue a transgene which
encodes an HA mimic of the present invention, wherein the HA mimic is
secreted into the milk produced by the transgenic goat; collecting milk from
the transgenic goat which milk contains the HA. mimic; and isolating the HA
mimic from the collected milk. This aspect of the invention can be
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accomplished according to US Patent Serial Number 5,843,705, which patent is
hereby incorporated by reference in its entirety.
The present invention also provides methods to identify the ability of a
test compound to interfere with the present HA mimic/target enzyme
interaction, comprising: contacting the test compound with a protein of the
present invention; and determining whether the test compound and said
protein interact.
The following examples illustrate the present invention without,
however, limiting it. It is to be noted that the Examples include a number of
molecular biology, microbiology, immunology and biochemistry techniques
considered to be known to those skilled in the art. Disclosure of such
techniques can be found, for example, in Sambrook et aL, ibid., and related
references.
Examples
Example 2 Screening of Phage Library
A completely random phage library expressing; a 15-amino acid peptide was
used to screen phage that can bind to the HA binding domains. The principle
of the screening process relies on selectively eluting phage that bind to the
HA binding domains. Three constructs expressing the HA binding domains
of RHAMM (Receptor of HA Mediated Motility;) were prepared. The first two
GST-P1 (70 amino acids) and GST-P6 (68 annino acids are glutathione-S-
transferase fusion constructs. The third is a thioredoxin fusion construct (62
amino acids 312c). The HA binding domain portions of these polypeptides
axe identical except for several amino acids on the N or C terminus. A
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portion of the phage screens were performed ~~ith 312c as the target peptide
while all binding assays used GST-P1. All constructs have a thrombin
cleavage site, which was key to the selective elution method used.
To screen phage which selectively bound to t:he HA binding domains, the
following method was used. Purified GST-I'6 or 312c (0.lmg/mL) were
immobilized on a 30 mm polystyrene petri dish in 50 mM Tris, pH 8Ø The
phage library was applied in binding buffer (Tris buffered saline pH 7.5,
0.05%
Tween, 0.1% polyvinylpyrolidone) (TBST-PVP). Nonspecific phage were
washed off,and the phage bound to the HA binding domains were recovered
either by cleaving the HA binding domain from the fusion protein using
thrombin (first and second rounds of screenin;g), or eluting with HA (third
round of screening), as summarized in FIG. 1. Finally, the peptide encoding
DNAs present in the phages were sequenced. 'lCable 1 shows the numbers of
phages recovered during the second round of screening.
Table 1
.
Screen - cfU/mL
Step 312c GST-P6 Control
Input 1x1010 1x1010 1x1010
First Wash 7 x 104 6 x 104 6 x I04
Last Wash 8 10 3
40~g/ml HA elution 1160 1320 -
I mg/mL HA elution 2S0 160 -
Thrombin cleavage 128 16$ 20
From the results shown in Table 1, it was concluded that most or all
nonspecific phage were washed off. After adding HA, some specific phage
were replaced by HA. Therefore, the phage from the HA elution were
CA 02346742 2001-O1-08
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considered to have specifically bound to the H.A binding domain. The amino
acid sequences of eleven peptides (HA1 -HA11; :SEQ ID NO: 1 through SE(~ ID
NO: 11) were determined. Peptides corresponding to HA2-HA5 were
synthesized with an amino terminal biotin. The ability of these peptides to
bind competitively to GST-Pl was tested. First, GST-Pl was immobilized onto
a 96 well plate as described above. After rigorous washing with binding
buffer, the biotinylated peptides were appl!.ied. Each experiment was
performed in quadruplicate. The concentration of peptide applied was
titrated such that binding curves could be calculated. The plate was agitated
at
room temperature for two hours and then .washed with binding buffer.
Streptavidin alkaline phosphatase (Pharmacia Biotec) was applied in binding
buffer at a 1:5000 dilution. This was followed toy washing and application of
commercially available BCIP/NBT substrate (.3igma). Results are shown in
FIG. 2, which shows specific, compatible binding of HA2 and HA3 with GST-
Pl L.
Example 2 Screening of One-bead, One-peptide Library
Two bead libraries were constructed in this lab with eight amino acid peptides
covalently attached to 100~,M Tentagel S (Rapp Polymere GmbH). This
method ensures that only one species of peptide is attached to one bead,
generally 50nmoles of peptide/bead. One of the bead libraries was designed to
be an anionic biased library of 8-mers was designed and prepared on the basis
of alternating amino acids with carboxylic acid chains that mimic the 6-
carboxylic acids of glucuronic acid (XNXNXNx:N; X= any amino acid except
Cys, Arg, Lys; N= negatively charged side chains,, D-Asp, L-Asp, D-Glu, L-
Glu).
Both libraries were screened against GST-P1. the method is described as
follows:
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About 2 ml of beads (approx. 105 beads), were prepared in a 5-ml fritted
column by sequentially washing and equilibrating in binding buffer. Pure
GST-PI was added to a final concentration of 0.5 ng/ml protein. The beads
were incubated with shaking at room temperature for two hours. After
vigorous washing in binding buffer, anti-GST-P:l antibody was added (1:2000),
the beads were incubated and washed, This was followed by a secondary
antibody anti-IgG conjugated to alkaline phosphatase (1:3000). The beads were
then washed and placed in a large petri dish. The dish was drained and
alkaline phosphatase substrate was added. The substrate was prepared by
IO suspending 50 mg of 5-bromo-4-chloro-3-indoiyl-phosphate (BCIP) in 1 ml of
DMF and adding 33 ~,l of this mixture to 10 ml of reaction buffer {TBS pH 8.5
+ 1mM MgCl2) Positive beads in reaction buffer would begin to turn
blue/purple after 15 minutes. Positive beads were removed using a pipettor
and treated with 6 M guanidium hydrochloride, followed by a series of
IS washes in DMF. This screening process was repeated twice, after which
approximately 300 candidate beads remained. A third screen was similar to
the process mentioned above except that the beads were preincubated with
excess natural Iigand, in this case 1 mg/rnL of partially degraded HA. The
beads then collected were those that did not turn blue/purple, indicating that
20 they bound to the same place as the natural ligand. At least 7 candidates
were
discovered through this screening process that have unique, but related
amino acid sequences. The first such positive bead sequences as the primary
structure Phe-Asp-Phe.Asp-Ser-Glu-Tyr-Glu (SEQ ID NO: 12)...
Example 3 Peptide Binding Assays
To verify that the peptides obtained from phage screening indeed bound to
the HA binding domains of RHAMM, a solid phase 96-well plate assay was
employed. 50 ~,L GST-Pl (0.25 mg/rnL) in glutathione elution buffer was
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immobilized in wells of a polystyrene 96-well plate {Greiner). The plate is
then blocked with 250 ~cL TBS-T-PVP-40 bovine serum albumin (BSA) (20
mM Tris, pH 7.5, 130 mM NaCl, 0:1% polyvinylpyrolidone-40, 1% BSA) for 2
hr to overnight with shaking. The plate is washed with TBS-T-PVP-40 (no
BSA) and if blocking is required, 'the plate is incubated with excess HA or
chondroitin sulfate (CS) for 1 hr prior to the addition of biotinylated
Iigand.
The biotinyiated ligand is incubated for 25 min (determined from an
initial timecourse). The plate is washed 3 X, and blocked with TBS-T-PVP-40-
BSA for 1 hr. Streptavidin HRP is added at a dilution of 0.5 ~.g /mL in TBS-
T-PVP-40. HA is added to the streptavidin solution, since it is known that
streptavidin HRP binds nonspecifically to HA and CS, which inherently
causes misleading results in the competition experiments. After 40 min
incubation, the plate is washed, and the presence of biotinylated peptide is
determined using TMB. Colorimetric development is read at 590 nm (Perkin
Elmer HTS 7000). All measurements are performed in quadruplicate. The
figure on the following page outlines this method.
For the purposes of generating binding data, the color generated from
streptavidin HRP is calibrated and used to estimate the total biotinylated
molecules immobilized on the plate. Eadie Hofstee analyses were to calculate
binding constants.
Example 4 Pepspots assay
Since the biased library contains both D or L isomers of the alternating
charged residues it was necessary to decipher which set of natural or
unnatural amino acids provided the best binding to GST-RHAMM-P1. For
each peptide obtained from sequencing there are 16 possible combinations
(44). This peptide library was constructed as an array of spots on cellulose
membrane (Pepspots membranes, Jerini Biotool;s) where each spot contains a
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single species of peptide (3-5nmoles/spot). This membrane contained a total
of 68 spots representing 6-18, 6-19, 6-20, 6-2i and 4 control spots taken from
a
negative bead.
To detect binding we incubated the blocked membrane with GST-RHAMM-
PI. After washing, the bound protein was semi--dry transferred from the
cellulose membrane onto a PVDF membrane. 7i he PVDF membrane was
blocked and incubated with anti-GST, followed by anti-goat antibody-
horseradish peroxidase conjugated. To detect bound material, a
chemilumiscence detection kit was used followed by exposure to film. The
developed film was digitized and quantified to .evaluate the strength of GST-
RHAMM-P1 binding to each well. To verify the results the experiment was
repeated several times at various GST-RHAMM-P1 concentrations.
Example 5 Preferred Binding motifs
Depicted below is a summary of the results found from phage and bead
screening experiments. The X stands for unknown amino acids that arose
from sequencing difficulties.
Random phage libraries
HAl WPVSLTVCSAVWCPL (SEQ ID NO 1)
HA2 GVCNADFCWLPAVVV (SEQ ID NO 2}
HA3 SASPSASKLSLMSTV (SEQ ID NO 3)
HA4 IPPILPAYTLLGHPR (SEQ ID NO 4)
HA5 YSVYLSVAHNFVLPS (SEQ ID NO 5)
HA6 HWCLPLLACDTFARA (SEQ ID NO 6)
3Q
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Biased bead peptides
6-I8 MDYEPEQE (SEQ ID NO ~
6-19 YDSEYESE {SEQ ID NO 8)
6-20 FDFDSEYE (SEQ ID NO 9)
6-21 EDQEAXEX (SEQ ID NO 10)
6-22 EDAENXDX (SEQ ID 1V0 11)
Random bead peptides
R-1 SGRPYKPP (SEQ ID NO 12)
R-2 YXSSNKPG (SEQ ID NO I3)
R-3 EGEWPVYP {SEQ ID NO 14)
R-4 WNYTEAKG {SEQ iD NO 15)
Figure 1 shows the pertinent results from such an experiment.
The phage results show a motif: ILPA (SEQ ID NO 16) or WLPA (SEQ ID NO
17) with the potential for a hydrophobic amino <~cid to further govern
binding. The presence of prolines is enforced by the random bead libraries
where motifs as WPVYP (SEQ ID NO I8) and YKPP (SEQ iD NO 19) are seen.
It is believed that a four amino acid sequence in the proper conformation is
sufficient to regulate binding to the HA binding domains of RHAMM. The
biased library showed that hydrophobic amino acids are important in
predisposing a peptide to specific binding: YDSE',YESE (SEQ ID NO 20). This
information was capitalized on by the Pepspots assay exemplified in Example
4.
The results can be summarized in the following way: In all cases unnatural
amino acids facilitated binding. In all but one case the binding of completely
natural amino acids provided no binding. Further, we note that a C-terminal
D-glutamate acid provides optimal binding and the presence of D-glutamate
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or D-aspartate as the sixth ensures high binding. From this information we
have constructed a model for fihe best binding peptides in which the pattern
of amino acids follows this sequence: X-A/a-X-.A/a-X-a-X-a. (X} can be any
amino acid and is often hydrophobic, {A) is L-glutamate or L-aspartate and (a}
is D-glutamate or D-aspartate.
Example 6 Preferred examples of TSG-6 binding peptides
Tumor necrosis factor (TNF) stimulated gene-6 (TSG-6} is an HA binding
protein whose carbohydrate binding domain is a member of the /ink module
consensus family of which CD44 is also a member(Bajorath et al., 1998).
TSG-6 is present in higher than normal concentrations at sites of joint
and cartilage inflammation (Maier et al., 1996) and is thought to
mediate some of the pathological conditions of arthritis. The structure
1S of the HA binding domain was solved using NMR and is the only structure
presently available of an HA binding domain (I'DB ascension # 1TSG)
(Kohda et al., 1996}.
Using TSG-6 as a target protein we screened our random library against
this protein using the same experimental methods described previously.
We performed this experiment at two different pH solutions: pH 5.7 and
7.5. This is because we wanted to compare neui:ral pH conditions to that
of conditions where maximal HA binding occurs (Parkar et al., 1998}. The
following sequences were found:
pH 5.7
GYYFNVAM
WAYNFLVM
TQSLNNHM
WWPFINAY
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WWKADMVG
W WPFINAY
MALQLPYY
IIYEEFFV
S ISINNRWY
VTPPVYFT
pH 7.5
QIRNGWFW
SW WFGPLA
GDWEQILT
PAGFGWNL
NMRFNIEN
QMTFFDGV
While these sequences are different from those found in the screening
experiment using GST-Pl as the target protein, i:here are similarities.
There is a motif of double hydrophobic amino acids in these peptides.
Although the present invention has been fully described herein, it is to
be noted that various changes and modifications are apparent to those skilled
in the art. Such changes and modifications are to be understood as included
-- within the scope of the present invention as defined by the appended
claims.
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