Note: Descriptions are shown in the official language in which they were submitted.
CA 02421877 2003-03-11
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LIGAND/RECEPTOR SPECIFICITY EXCHANGERS THAT REDIRECT ANTIBODTES
TO RECEPTORS ON A PATHOGEN
FIELD OF THE INVENTION
S The present invention generally relates to compositions and methods for
preventing and
treating human diseases including, but not limited to, pathogens such as
bacteria, yeast, parasites,
fungus, viruses, and cancer. More specifically, embodiments described herein
concern the
manufacture and use of ligandlreceptor specificity exchangers, which redirect
existing antibodies
in a subject to receptors present on pathogens.
BACKGROUND OF THE INVENTION
Infection by pathogens, such as bacteria, yeast, parasites, fungus, and
viruses, and the onset
and spread of cancer present serious health concerns for all animals,
including humans, farm
livestock, and household pets. These health threats are exacerbated by the
rise of strains that are
resistant to vaccination andlor treatment. In the past, practitioners of
pharmacology have relied on
traditional methods of drug discovery to generate safe and efficacious
compounds for the treatment
of these diseases. Traditional drug discovery methods typically involve
blindly testing potential
drug candidate-molecules, often selected at random, in the hope that one might
prove to be an
effective treatment for some disease. With the advent of molecular biology,
however, the focus of
drug discovery has shifted to the identification of molecular targets
associated with the etiological
agent and the design of compounds that interact with these molecular targets.
One promising class of molecular targets are the receptors found on the
surface of bacteria,
yeast, parasites, fungus, viruses, and cancer cells, especially receptors that
allow for attachment to
a host cell or host protein (e.g., an extracelIular matrix protein). Research
in this area primarily
focuses on the identification of the receptor and its ligand and the discovery
of molecules that
interrupt the interaction of the ligand with the receptor and, thereby, block
adhesion to the host cell
or protein.
For example, many pathogenic bacteria (e.g., Staphylococcus aureus) produce
adhesion
receptors (e.g., CIfA, Efb, and FnBPA) that are capable of binding to a host's
extracellular matrix
proteins (e.g., fibrinogen, fibronectin, and laminin). (Flock, Mol. Med. Today
5:532-533 (1999)).
Investigators have shown that the adherence of some bacteria to host
extracellular matrix proteins
can be blocked by providing peptides that correspond to regions of the host
extracellular matrix
protein. (Pei et aL, Infection and Immunity 67(9):4525-4530 (I999)).
Similarly, many viruses
have receptors that interact with proteins present on the surface of host
cells. (See e.g., U.S. Pat.
Nos. 5,942,606 and 5,929,220). Investigators have shown that a fragment of the
T4 glycoprotein (a
host cell protein), can interact with gp120 of the human immunodeficiency
virus (HIV) and the T4
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peptide can be used to prevent or treat HIV infection. (See e.g., U.S. Pat.
No. 6,093,539).
Additionally, many types of cancer cells express receptors that interact with
host extracellular
matrix proteins and investigators have shown that molecules that block
integrin receptors can be
used to inhibit tissue attachment, metastasis, angiogenesis, and tumor growth.
(See e.g., U.S. Pat.
Nos.: 6,066,648; 6,087,330; 5,846,536; 5,766,591; and 5,627,263). Although
these inhibitory
peptides have promising therapeutic potential, there still remains a need for
new compositions and
methods to treat and prevent infection by pathogens and other diseases.
BRIEF SUMMARY OF THE INVENTION
The invention described herein concerns the manufacture, characterization, and
use of
novel agents that bind receptors on pathogens and redirect antibodies present
in a subject to the
pathogen. Embodiments include a ligand/receptor specificity exchanger having
at least one
specificity domain comprising a ligand for a receptor and at least one
antigenic domain joined to
said specificity domain, wherein said antigenic domain comprises an epitope of
a pathogen or
toxin.
Some embodiments of the ligand/receptor specificity exchanger have a
specificity domain
that comprises at least three consecutive amino acids of a peptide selected
from the group
consisting of an extracellular matrix protein, a ligand for a receptor on a
virus, and a ligand for a
receptor on a cancer cell. In some aspects of this embodiment, for example,
the peptide is an
extracellular matrix protein selected from the group consisting of fibrinogen,
collagen, vitronectin,
laminin, plasminogen, thrombospondin, and fibronectin. Preferably, the
extracellular matrix
protein comprises at least 3 amino acids of the alpha-chain of fibrinogen and
in the most preferred
embodiments the ligand comprises the sequence Arginine-Glycine-Aspartate
(RGD).
In other embodiments, the peptide described above is a ligand for a receptor
on a virus
selected from the group consisting of T4 glycoprotein and hepatitis B viral
envelope protein. In
still other aspects of this embodiment, the peptide is a ligand for a receptor
on a cancer cell
selected from the group consisting of a ligand for HER-2/neu and a ligand for
an integrin receptor.
Preferred embodiments have a specificity domain that comprises a sequence
provided by one of
SEQ. m. Nos. 1-42.
The ligand/receptor specificity exchangers described herein interact with a
receptor found
on a pathogen. In some embodiments, the receptor is a bacterial adhesion
receptor, for example, a
bacterial adhesion receptor selected from the group consisting of
extracellular fibrinogen binding
protein (Efb), collagen binding protein, vitronectin binding protein, laminin
binding protein,
plasminogen binding protein, thrombospondin binding protein, clumping factor A
(CIfA),
clumping factor B (CIfB), fibronectin binding protein, coagulase, and
extracellular adherence
protein.
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The ligand/receptor specificity exchangers described herein also interact with
a an
antibody present in a subject. In some embodiments, for example, the antigenic
domain comprises
at least three amino acids of a peptide selected from the group consisting of
a herpes simplex virus
protein, a hepatitis B virus protein, a TT virus protein, and a poliovirus
protein. In desirable
embodiments, the ligand/receptor specificity exchanger has an antigenic domain
that is a herpes
simplex virus protein comprising a sequence selected from the group consisting
of SEQ. ID. No.
53 and SEQ. ID. No. 54. In other desired embodiments, the antigenic domain is
a hepatitis B virus
protein comprising a sequence provided by one of SEQ. ID. No. 49, SEQ. ID. No.
50, SEQ. )m.
No. 52, and SEQ. >m. No. 59.
Some ligand/receptor specificity exchangers also have an antigenic domain that
is a TT
virus protein comprising a sequence provided by one of SEQ. )m. Nos. 43-47 and
SEQ. ID. Nos.
55-58. The ligand/receptor specificity exchangers can also have an antigenic
domain that is a polio
virus protein comprising a sequence selected from the group consisting of SEQ.
ID. No. 48 and
SEQ. ID. No. 51. Preferably, the ligand/receptor specificity exchanger has an
antigenic domain
that interacts with a high-titer antibody. In some embodiments, for example,
the antigenic domain
specifically binds to an antibody present in animal serum that has been
diluted to between
approximately 1:100 to 1:1000 or greater. The specif city exchangers of SEQ.
ID. Nos. 60-105 are
embodiments of the invention.
Aspects of the invention also concern methods of treating or preventing a
infection or
proliferation of a pathogen. One approach for example, involves a method for
treating and
preventing bacterial infection. This method is practiced by providing a
therapeutically effective
amount of a ligand/receptor specifcity exchanger to a subject, wherein said
ligand/receptor
specificity exchanger comprises a specificity domain that has a ligand that
interacts with a receptor
on a bacteria, and an antigenic domain that comprises an epitope for a
pathogen or toxin. A
method of treating or preventing viral infection is also an embodiment.
Accordingly, a method of
treating or preventing a viral infection is practiced by providing a
therapeutically effective amount
of a ligand/receptor specificity exchanger to a subject, wherein said
ligand/receptor specificity
exchanger comprises a specificity domain that has a ligand that interacts with
a receptor on a virus,
and an antigenic domain that comprises an epitope for a pathogen or toxin.
Similarly, a method of
treating or preventing cancer is an embodiment and this method can be
practiced by providing a
therapeutically effective amount of a ligand/receptor specificity exchanger to
a subject, wherein
said ligand/receptor specificity exchanger comprises a specificity domain that
has a ligand that
interacts with a receptor on a cancer cell, and an antigenic domain that
comprises an epitope for a
pathogen or toxin.
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DETAILED DESCRIPTION OF THE INVENTION
The following describes the manufacture, characterization, and use of novel
agents that
bind receptors on pathogens and redirect antibodies present in a subject to
the pathogen. The term
"antigen/antibody specificity exchanger" is known. in the art to refer to a
molecule that comprises
an amino acid sequence corresponding to an amino acid sequence of an antibody
(e.g., a
complementarity determining region) linked to an amino acid sequence to which
a certain antibody
binds (e.g., an epitope of a pathogen). (See e.g., Sallberg et al.,
Biochemical & Biophysical
Research Communications, 205:1386-90 (1994) and U.S. Pat. Nos. 5,869,232 and
6,040,137.)
Antigen/antibody specificity exchangers can redirect antibodies present in a
subject to a pathogen
and these exchanger agents have therapeutic and diagnostic use. (Id.).
The embodiments described herein concern a second generation of exchanger
agents
referred to as "ligand/receptor specificity exchangers". Unlike
antigen/antibody specificity
exchangers, ligand/receptor specificity exchangers do not comprise a sequence
found in an
antibody. Instead, ligand/receptor specificity exchangers comprise a first
domain that has a ligand
for a receptor and a second domain that has an epitope of a pathogen or a
toxin. Thus, for the
purposes of this disclosure, the term "ligandlreceptor specificity exchangers"
refers an exchanger
agent that comprises a "specificity domain" that has at least one ligand for a
receptor (a "ligand" is
not an antibody or portion thereof) joined to an "antigenic domain" that has
at least one epitope of
a pathogen or toxin (e.g., pertussis toxin or cholera toxin).
The ligand/receptor specificity exchangers can comprise more than a
specificity domain
and an antigenic domain. For example, some ligand/receptor specificity
exchangers comprise a
plurality of specifcity domains and/or antigenic domains. Ligand/receptor
specificity exchangers
having multiple specificity domains and/or antigenic domains are said to be
"multimerized"
because more than one specificity domain and/or antigenic domain are fused in
tandem. Other
embodiments concern ligand/receptor specificity exchangers that contain, in
addition to a
specificity domain and an antigenic domain, sequences that facilitate
purification (e.g., a poly
histidine tail), linkers (e.g., biotin and/or avidin or streptavidin or the
flexible arms of 8 phage (8-
linkers)), and sequences or modifications that either promote the stability of
the ligand/receptor
specificity exchanger (e.g., modifications that provide resistance to protease
digestion) or promote
the degradation of the ligand/receptor specificity exchanger (e.g., protease
cleavage sites).
Although the specificity and antigenic domains are preferably peptides, some
ligand/receptor
specificity exchangers have specificity and antigenic domains that are made of
modified or
derivatized peptides, peptidomimetics, or chemicals.
The diversity of ligand/receptor specificity exchangers is vast because the
embodiments
described herein can bind to many different receptors on many different
pathogens. Thus, the term
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"pathogen" is used herein in a general sense to refer to an etiological agent
of disease in animals
including, but not limited to, bacteria, parasites, fungus, mold, viruses, and
cancer cells. Similarly,
the term "receptor" is used in a general sense to refer to a molecule (usually
a peptide other than a
sequence found in an antibody, but can be a carbohydrate, lipid, or nucleic
acid) that interacts with
a "ligand" (usually a peptide other than a sequence found in an antibody, or a
carbohydrate, lipid,
nucleic acid or combination thereof). A "receptor", as used herein, does not
have to undergo signal
transduction and can be involved in a number of molecular interactions
including, but not limited
to, adhesion (e.g., integrins) and molecular signaling (e.g., growth factor
receptors). For example,
desired specificity domains comprise a ligand that has a peptide sequence that
is present in an
extracellular matrix protein (e.g., fibrinogen, collagen, vitronectin,
laminin, plasminogen,
thrombospondin, and fibronectin) and some specificity domains comprise a
Iigand that interacts
with a bacterial adhesion receptor (e.g., extracellular fibrinogen binding
protein (Efb), collagen
binding protein, vitronectin binding protein, laminin binding protein,
plasminogen binding protein,
thrombospondin binding protein, clumping factor A (CIfA), clumping factor B
(CIfB), fibronectin
binding protein, coagulase, and extracellular adherence protein).
In other embodiments, the specificity domain comprises a ligand that has a
peptide
sequence that interacts with a viral receptor (e.g., a fragment of T4
glycoprotein that binds gp120
or a fragment of the preS domain, which binds gp170 of the hepadnavirus
family). In still other
embodiments, the specificity domain comprises a ligand that interacts with a
receptor on a cancer
cell (e.g., HER-2/neu (C-erbB2)) or an integrin receptor such as a vitronectin
receptor, a Iaminin
receptor, a fibronectin receptor, a collagen receptor, a fibrinogen receptor,
an dq.~ 1 receptor, an
d6~ lreceptor, an H3~ lreceptor, an 'ds~ 1 receptor, and an b'v~3receptor.
Preferred embodiments,
however, have a specificity domain that comprises at least 8 amino acids of
the alpha-chain of
fibrinogen and/or the sequence Arginine-Glycine-Aspartic acid (RGD) and the
most preferred
embodiments have a specificity domain that comprises a sequence selected from
the group
consisting of SEQ. ID. Nos. 60-105.
Desired antigenic domains have an epitope that is recognized by an antibody
that already
exists in a subject. For example, many people are immunized against childhood
diseases
including, but not limited to, small pox, measles, mumps, rubella, and polio.
Thus, antibodies to
epitopes on these pathogens can be produced by an immunized person. Desirable
antigenic
domains have an epitope that is found on one of these etiological agents.
Some embodiments have antigenic domains that interact with an antibody that
has been
administered to the subject. For example, an antibody that interacts with an
antigenic domain on a
ligand/receptor specificity exchanger can be co-administered with the
ligand/receptor specificity
exchanger. Further, an antibody that interacts with a ligand/receptor
specificity exchanger may not
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normally exist in a subject but the subject has acquired the antibody by
introduction of a biologic
material (e.g., serum, blood, or tissue). For example, subjects that undergo
blood transfusion
acquire numerous antibodies, some of which can interact with an antigenic
domain of a
ligand/receptor specificity exchanger.
The most desirable antigenic domains comprise an epitope that is recognized by
a high
titer antibody. By "high titer antibody" is meant an antibody that has high
affinity for an antigen
(e.g., an epitope on an antigenic domain). For example, in a solid-phase
enzyme linked
immunosorbent assay (ELISA), a high titer antibody corresponds to an antibody
present in a serum
sample that remains positive in the assay after a dilution of the serum to
approximately the range
of 1:100-1:1000 in an appropriate dilution buffer, preferably, about 1:500.
The preferred antigenic
domains, however, have an epitope found on herpes simplex virus gG2 protein,
hepatitis B virus s
antigen (HBsAg), hepatitis B virus a antigen (HBeAg), hepatitis B virus c
antigen (HBcAg), TT
virus, and the poliovirus or combination thereof or comprise a sequence
selected from the group
consisting of SEQ. ID. Nos. 43-59.
The ligand/receptor specificity exchangers described herein can be made by
conventional
techniques in recombinant engineering and/or peptide chemistry. In some
embodiments, the
specificity and antigenic domains are made separately and are subsequently
joined together (e.g.,
through linkers or by association with a common carrier molecule). In other
embodiments, the
specificity domain and antigenic domain are made as part of the same molecule.
By one approach,
a ligand/receptor specificity exchanger having a specificity domain joined to
an antigenic domain
is made by a peptide synthesizer. By another approach, a nucleic acid encoding
the specificity
domain fused to an antigenic domain is cloned into an expression construct,
transfected to cells,
and the ligand/receptor specificity exchanger is purified or isolated from the
cells or cell
supernatent.
Once the ligand/receptor specificity exchanger is made, it can be screened to
determine its
ability to interact with the receptor on the pathogen and/or an antibody
specific for the antigenic
domain. Thus, the term "characterization assay" is used to refer to an
experiment or evaluation of
the ability of a ligand/receptor specificity exchanger to interact with a
receptor on a pathogen or
cancer cell or fragment thereof and/or an antibody specific for the antigenic
domain. Some
characterization assays, for example, evaluate the ability of a
ligand/receptor specificity exchanger
to bind to a support having a receptor of a pathogen or fragment thereof
disposed thereon or vice
versa. Other characterization assays assess the ability of a ligand/receptor
specificity exchanger to
bind to an antibody specific for the antigenic domain of the ligand/receptor
specificity exchanger.
Still other characterization assays evaluate the ability of the
ligand/receptor specificity exchanger
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to effect infection by the pathogen or cancer cell proliferation in cultured
cell lines or diseased
animals.
The ligand/receptor specificity exchangers described herein can be used as the
active
ingredients in pharmaceuticals for the treatment and prevention of pathogenic
infection, as well as
cancer, in animals including humans. The pharmaceutical embodiments can be
formulated in
many ways and may contain excipients, binders, emulsifiers, carriers, and
other auxiliary agents in
addition to the ligand/receptor specificity exchanger. Pharmaceuticals
comprising a
ligand/receptor specificity exchanger can be administered by several routes
including, but not
limited to, topical, transdermal, parenteral, gastrointestinal,
transbronchial, and transalveolar.
Ligand/receptor specificity exchangers can also be used as a coating for
medical equipment and
prosthetics to prevent infection or the spread of disease. The amount of
ligand/receptor specificity
exchanger provided in a pharmaceutical, therapeutic protocol, or applied to a
medical device varies
depending on the intended use, the patient, and the frequency of
administration.
Some of the methods disclosed concern the administration of a ligand/receptor
specificity
exchanger to a subject in need of treatment and/or prevention of bacterial
infection, fungal
infection, viral infection, and cancer. By one approach, a subject suffering
from bacterial infection
is provided a ligand/receptor specificity exchanger that comprises a
specificity domain, which
interacts with a bacterial receptor. Similarly, a subject suffering from a
viral infection can be
provided a ligand/receptor specificity exchanger that comprises a specificity
domain that interacts
with a viral receptor and a subject suffering from cancer is provided a
ligand/receptor specificity
exchanger that comprises a specificity domain that interacts with a receptor
on the cancer cells.
Once a receptor/specificity exchanger complex is formed, it is contemplated
that the pathogen or
cancer cell is cleared from the body by complement fixation and/or macrophage
degradation.
Methods of treatment and prevention of disease (e.g., bacterial, fungal, and
viral infection,
and cancer) are provided in which a subject suffering from disease or a
subject at risk for
contracting a disease is identified and then is provided a therapeutically
effective amount of a
ligand/receptor specificity exchanger that interacts with a receptor present
on the etiological agent.
Accordingly, subjects suffering from a bacterial infection, fungal infection,
viral infection, or
cancer are identified by conventional clinical and diagnostic evaluation and
are provided a
therapeutically effective amount of a ligand/receptor specificity exchanger
that interacts with the
particular pathogen or cancer cell. Although the ligand/receptor specificity
exchangers described
herein can be administered to all animals at risk of disease for prophylactic
purposes, it may be
desired to administer the ligand/receptor specificity exchangers only to those
individuals that are in
a high risk category (e.g., infants, the elderly, and those that come in close
contact with pathogens).
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As stated above, high risk individuals are identified by currently available
clinical and diagnostic
techniques.
The section below provides more description of various types of
ligand/receptor specificity
exchangers that interact with receptors on bacteria, parasites, fungus, mold,
viruses, and cancer
cells.
Ligahdl~ecepto~ specificity exchangers that ihte~act with s~eceptot s o~c a
patlrogeh
The ligand/receptor specificity exchangers that interact with receptors on a
pathogen have
a variety of chemical structures but, in a general sense, they are
characterized as having at least one
region that binds to the receptor (the specificity domain) and at least one
region that interacts with
an antibody that is specific for an epitope of a pathogen or toxin (the
antigenic domain). Preferred
ligand/receptor specificity exchangers are peptides but some embodiments
comprise derivatized or
modified peptides or a peptidomimetic structure. For example, a typical
peptide-based
ligand/receptor specificity exchanger can be modified to have substituents not
normally found on a
peptide or to have substituents that are normally found on a peptide but are
incorporated at regions
that are not normal. In this vein, a peptide-based ligand/receptor specificity
exchanger can be
acetylated, acylated, or aminated and the substituents that can be included on
the peptide so as to
modify it include, but are not limited to, H, alkyl, aryl, alkenyl, alkynl,
aromatic, ether, ester,
unsubstituted or substituted amine, amide, halogen or unsubstituted or
substituted sulfonyl or a 5 or
6 member aliphatic or aromatic ring. Thus, the term "ligand/receptor
specificity exchanger" is a
broad one that encompasses modified or unmodified peptide structures, as well
as peptidomimetics
and chemical structures.
There are many ways to make a peptidomimetic that resembles a peptide-based
ligand/receptor specificity exchanger. The naturally occurring amino acids
employed in the
biological production of peptides all have the L-configuration. Synthetic
peptides can be prepared
employing conventional synthetic methods, utilizing L-amino acids, D-amino
acids, or various
combinations of amino acids of the two different configurations. Synthetic
compounds that mimic
the conformation and desirable features of a peptide but that avoid the
undesirable features, e.g.,
flexibility (loss of conformation) and bond breakdown are known as a
"peptidomimetics". (See,
e.g., Spatola, A. F. Chemistry and Biochemistry of Amino Acids. Peptides, and
Proteins (Weistein,
B, Ed.), Vol. 7, pp. 267-357, Marcel Dekker, New York (I983), which describes
the use of the
methylenethio bioisostere [CH2 S] as an amide replacement in enkephalin
analogues; and Szelke et
al., In peptides: Structure and Function, Proceedings of the Eighth American
Peptide Symposium,
(Hruby and Rich, Eds.); pp. 579-582, Pierce Chemical Co., Rockford, Ill.
(1983), which describes
renin inhibitors having both the methyleneamino [CH2 NH] and hydroxyethylene
[CHOHCH2 ]
bioisosteres at the Leu-Val amide bond in the 6-13 octapeptide derived from
angiotensinogen).
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In general, the design and synthesis of a peptidomimetic that resembles a
ligand/receptor
specificity exchanger involves starting with the sequence of the
ligand/receptor specificity
exchanger and conformation data (e.g., geometry data, such as bond lengths and
angles) of a
desired ligand/receptor specificity exchanger (e.g., the most probable
simulated peptide), and using
S such data to determine the geometries that should be designed into the
peptidornimetic. Numerous
methods and techniques are known in the art for performing this step, any of
which could be used.
(See, e.g., Farmer, P. S., Drug Design, (Ariens, E. J. ed.), Vol. 10, pp. 119-
143 (Academic Press,
New York, London, Toronto, Sydney and San Francisco) (1980); Farmer, et al.,
in TIPS, 9/82, pp.
362-365; Verber et al., in TINS, 9/85, pp. 392-396; Kaltenbronn et al., in J.
Med. Chem. 33: 838-
845 (1990); and Spatola, A. F., in Chemistry and Biochemistry of Amino Acids.
Peptides, and
Proteins, Vol. 7, pp. 267-357, Chapter S, "Peptide Backbone Modifications: A
Structure-Activity
Analysis of Peptides Containing Amide Bond Surrogates. Conformational
Constraints, and
Relations" (B. Weisten, ed.; Marcell Dekker: New York, pub.) (1983); I~emp, D.
S.,
"Peptidomimetics and the Template Approach to Nucleation of ~-sheets and t-!-
helices in
1S Peptides," Tibech, Vol. 8, pp. 249-255 (1990)). Additional teachings can be
found in U.S. Patent
Nos. 5,288,707; 5,552,534; 5,811,515; 5,817,626; 5,817,879; 5,821,231; and
5,874,529. Once the
peptidomimetic is designed, it can be made using conventional techniques in
peptide chemistry
and/or organic chemistry.
Some embodiments comprise a plurality of specificity domains and/or a
plurality of
antigenic domains. One type of ligand/receptor specificity exchanger that has
a plurality of
specificity domains and/or antigenic domains is referred to as a "multimerized
ligand/receptor
specificity exchanger" because it has multiple specificity domains and/or
antigenic domains that
appear in tandem on the same molecule. For example, a multimerized specificity
domain may
have two or more ligands that interact with one type of receptor, two or more
ligands that interact
2S with different types of receptors on the pathogen, and two or more ligands
that interact with
different types of receptors on different pathogens.
Similarly, a multimerized antigenic domain can be constructed to have
multimers of the
same epitope of a pathogen or different epitopes of a pathogen, which can also
be multimerized.
That is, some multimerized antigenic domains are multivalent because the same
epitope is
repeated. In contrast, some multimerized antigenic domains have more than one
epitope present
on the same molecule in tandem but the epitopes are different. In this
respect, these antigenic
domains are multimerized but not multivalent. Further, some multimerized
antigenic domains are
constructed to have different epitopes but the different epitopes are
themselves multivalent because
each type of epitope is repeated.
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Some Iigand/receptor specificity exchangers comprise other elements in
addition to the
specificity domain and antigenic domain such as sequences that facilitate
purification, linkers that
provide greater flexibility and reduce steric hindrance, and sequences that
either provide greater
stability to the ligand/receptor specificity exchanger (e.g., resistance to
protease degradation) or
promote degradation (e.g., protease recognition sites). For example, the
ligand/receptor specificity
exchangers can comprise cleavable signal sequences that promote cytoplasmic
export of the
peptide and/or cleavable sequence tags that facilitate purification on
antibody columns, glutathione
columns, and metal columns.
Ligand/receptor specif city exchangers can comprise elements that promote
flexibility of
the molecule, reduce steric hindrance, or allow the ligand/receptor
specificity exchanger to be
attached to a support or other molecule. These elements are collectively
referred to as "linkers".
One type of linker that can be incorporated with a ligand/receptor specificity
exchanger, for
example, is avidin or streptavidin (or their ligand -- biotin). Through a
biotin-avidin/streptavidin
linkage, multiple ligand/receptor specificity exchangers can be joined
together (e.g., through a
I S support, such as a resin, or directly) or individual specificity domains
and antigenic domains can be
joined. Another example of a linker that can be included in a ligand/receptor
specificity exchanger
is referred to as a "8 linker" because it has a sequence that is found on 8
phage. Preferred 8
sequences are those that correspond to the flexible arms of the phage. These
sequences can be
included in a ligand/receptor specificity exchanger (e.g., between the
specificity domain and the
antigenic domain or between multimers of the specificity and/or antigenic
domains) so as to
provide greater flexibility and reduce steric hindrance.
Additionally, ligand/receptor specificity exchangers can include sequences
that either
confer resistance to protease degradation or promote protease degradation. By
incorporating
multiple cysteines in a ligand/receptor specificity exchanger, for example,
greater resistance to
protease degradation can be obtained. These embodiments of the ligand/receptor
specificity
exchanger are expected to remain in the body for extended periods, which may
be beneficial for
some therapeutic applications. In contrast, ligand/receptor specificity
exchangers can also include
sequences that promote rapid degradation so as to promote rapid clearance from
the body. Many
sequences that serve as recognition sites for serine, cysteine, and aspartic
proteases are known and
can be included in a ligand/receptor specificity exchanger.
The section below describes the specificity domains in greater detail.
Specificity donaairas
The types of specificity domains that can be used with a ligand/receptor
specificity
exchanger are diverse because a vast number of ligands are known to interact
with receptors on
bacteria, parasites, fungus, mold, viruses, and cancer cells. Many types of
bacteria, parasites,
CA 02421877 2003-03-11
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fungus, mold, viruses, and cancer cells, for example, interact with
extracellular matrix proteins.
Thus, desired specificity domains comprise at least one ligand that has a
peptide sequence that is
present in an extracellular matrix protein. That is, a specificity domain can
have a ligand that has a
peptide sequence found in, for example, fibrinogen, collagen, vitronectin,
laminin, plasminogen,
thrombospondin, and fibronectin.
Investigators have mapped the regions of extracellular matrix proteins that
interact with
several receptors. (See e.g., McDevvit et al., Eur. J. BiochenZ., 247:416-424
(1997); Flock,
Molecular Med. Today, 5:532 (1999); and Pei et al., Ifafect. and Immura.
67:4525 (1999)). Some
receptors bind to the same region of the extracellular matrix protein, some
have overlapping
binding domains, and some bind to different regions altogether. Preferably,
the ligands that make
up the specificity domain have an amino acid sequence that has been identified
as being involved
in adhesion to an extracellular matrix protein. It should be understood,
however, that random
fragments of known ligands for any receptor on a pathogen can be used to
generate ligand/receptor
specificity exchangers and these candidate ligand/receptor specificity
exchangers can be screened
in the characterization assays described infra to identify the molecules that
interact with the
receptors on the pathogen.
Some specificity domains have a ligand that interacts with a bacterial
adhesion receptor
including, but not limited to, extracellular fibrinogen binding protein (Efb),
collagen binding
protein, vitronectin binding protein, laminin binding protein, plasminogen
binding protein,
thrombospondin binding protein, clumping factor A (CIfA), clumping factor B
(CIfB), fibronectin
binding protein, coagulase, and extracellular adherence protein. Ligands that
have an amino acid
sequence corresponding to the C-terminal portion of the gamma-chain of
fibrinogen have been
shown to competitively inhibit binding of fibrinogen to CIfA, a Staphylococcus
aureus adhesion
receptor. (McDevvit et al., Eur. J. Biochena., 247:416-424 (1997)). Further,
Staplzylococcus
organisms produce many more adhesion receptors such as Efb, which binds to the
alpha chain
fibrinogen, CIfB, which interacts with both the b' and ~ chain of fibrinogen,
and Fbe, which binds
to the ~ chain of fibrinogen. (Pei et al., Infect. and Ir~zmun. 67:4525
(1999)). Accordingly,
preferred specificity domains comprise at least 3 amino acids of a sequence
present in a molecule
(e.g., fibrinogen) that can bind to a bacterial adhesion receptor.
Specificity domains can also comprise a ligand that interacts with a viral
receptor. Several
viral receptors and corresponding ligands are known and these ligands or
fragments thereof can be
incorporated into a ligand/receptor specificity exchanger. For example, Tong
et al., has identified
an Hepadnavirus receptor, a 170kd cell surface glycoprotein that interacts
with the pre-S domain of
the duck hepatitis B virus envelope protein (U.S. Pat No. 5,929,220) and
Maddon et al., has
determined that the T cell surface protein CD4 (or the soluble form termedT4)
interacts with gp120
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of HIV (U.S. Pat No. 6,093,539). Thus, specificity domains that interact with
a viral receptor can
comprise regions of the pre-S domain of the duck hepatitis B virus envelope
protein (e.g., amino
acid residues 80-102 or 80-104) or regions of the T cell surface protein CD4
(or the soluble form
termedT4) that interacts with gp120 of HIV (e.g., the extracellular domain of
CD4/T4 or fragments
thereof). Many more ligands for viral receptors exist and these molecules or
fragments thereof can
be used as a specificity domain.
Specificity domains can also comprise a ligand that interacts with a receptor
present on a
cancer cell. The proto-oncogene HER-2/neu (C-erbB2) encodes a surface growth
factor receptor
of the tyrosine kinase family, p185HER2. Twenty to thirty percent of breast
cancer patients over
express the gene encoding HER-2/neu (C-erbB2), via gene amplification. Thus,
ligand/receptor
specificity exchangers comprising a specificity domain that encodes a ligand
for HER-2/neu (C-
erbB2) are desirable embodiments. Many types of cancer cells also over express
or differentially
express integrin receptors. Many preferred embodiments comprise a specificity
domain that
interacts with an integrin receptor. Although integrins predominantly interact
with extracellular
matrix proteins, it is known that these receptors interact with other ligands
such as invasins, RGD-
containing peptides (i.e., Arginine-Glycine-Aspartate), and chemicals. (See
e.g., U.S. Pat. Nos.
6,090,944 and 6,090,388; and Brett et al., Eur Jlmnauraol, 23:1608 (1993)).
Ligands for integrin
receptors include, but are not limited to, molecules that interact with a
vitronectin receptor, a
laminin receptor, a fibronectin receptor, a collagen receptor, a fibrinogen
receptor, an t~/4~ 1
receptor, an H6~lreceptor, an b'3~lreceptor, an f/5~1 receptor, and an
~/v~3receptor. TABLE I
also lists several preferred specificity domains. The specificity domains
described above are
provided for illustrative purposes only and in no way should be construed to
limit the scope of
specificity domains that can be used with the embodiments described herein.
The next section describes antigenic domains in greater detail.
TABLE I
SPECIFICITY DOMAINS
YGEGQQHHLGGAKQAGDV (SEQ.
ID.
No.
1)
MSWSLHPRNLILYFYALLFL(SEQ. ID. No. 2)
ILYFYALLFLSTCVAYVAT (SEQ. ID. No. 3)
SSTCVAYVATRDNCCILDER(SEQ. ID. No. 4)
RDNCCILDERFGSYCPTTCG(SEQ. ID. No. 5)
FGSYCPTTCGIADFLSTYQT(SEQ. ID. No. 6)
IADFLSTYQTKVDKDLQSLE(SEQ. ID. No. 7)
KVDKDLQSLEDILHQVENKT(SEQ. ID. No. 8)
DILHQVENKTSEVKQLIKAI(SEQ. ID. No. 9)
SEVKQLIKAIQLTYNPDESS(SEQ. ID. No. 10)
QLTYNPDESSKPNMIDAATL(SEQ. ID. No. 11)
KPNMIDAATLKSRIMLEEIM(SEQ. ID. No. 12)
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KSRIMLEEIMKYEASILTHD (SEQ. ID. No.13)
KYEASILTHDSSIRYLQEIY (SEQ. ID. No.14)
SSIRYLQEIYNSNNQKIVNL (SEQ. ID. No.15)
NSNNQKIVNLKEKVAQLEAQ (SEQ. ID. No.16)
CQEPCKDTVQIHDITGKDCQ (SEQ. ID. No.17)
IHDITGKDCQDIANKGAKQS (SEQ. ID. No.18)
DIANKGAKQSGLYFIKPLKA (SEQ. ID. No.19)
GLYFIKPLKANQQFLVYCEI (SEQ. ID. No.20)
NQQFLVYCEIDGSGNGWTVF (SEQ. ID. No.21)
DGSGNGWTVFQKRLDGSVDF (SEQ. ID. No.22)
QKRLDGSVDFKKNWIQYKEG (SEQ. ID. No.23)
KKNWIQYKEGFGHLSPTGTT (SEQ. ID. No.24)
FGHLSPTGTTEFWLGNEKIH (SEQ. ID. No.25)
EFWLGNEKIHLISTQSAIPY (SEQ. ID. No.26)
LISTQSAIPYALRVELEDWN (SEQ. ID. No.27)
ALRVELEDWNGRTSTADYAM (SEQ. ID. No.28)
GRTSTADYAMFKVGPEADKY (SEQ. ID. No.29)
FKVGPEADKYRLTYAYFAGG (SEQ. ID. No.30)
RLTYAYFAGGDAGDAFDGFD (SEQ. ID. No.31)
DAGDAFDGFDFGDDPSDKFF (SEQ. ID. No.32)
FGDDPSDKFFTSHNGMQFST (SEQ. ID. No.33)
TSHNGMQFSTWDNDNDKFEG (SEQ. ID. No.34)
WDNDNDKFEGNCAEQDGSGW (SEQ. ID. No.35)
NCAEQDGSGWWMNKCHAGHL (SEQ. ID. No.36)
WMNKCHAGHLNGVYYQGGTY (SEQ. ID. No.37)
NGVYYQGGTYSKASTPNGYD (SEQ. ID. No.38)
SKASTPNGYDNGIIWATWKT (SEQ. ID. No.39)
NGIIWATWKTRWYSMKKTTM (SEQ. ID. No.40)
RWYSMKKTTMKIIPFNRLTI (SEQ. ID. No.41)
IKIIPFNRLTIGEGQQHHLGGAKQAGDV (SEQ. ID. No. 42)
Antigeyaic donaains
The diversity of antigenic domains that can be used in the ligand/receptor
specificity
exchangers is also quite large because a pathogen or toxin can present many
different epitopes.
S That is, the antigenic domains that can be incorporated into a
ligand/receptor specificity exchanger
include epitopes presented by bacteria, fungus, plants, mold, virus, cancer
cells, and toxins.
Desired antigenic domains comprise an epitope of a pathogen that already
exists in a subject by
virtue of naturally acquired immunity or vaccination. Epitopes of pathogens
that cause childhood
diseases, for example, can be used as antigenic domains.
Some embodiments have antigenic domains that interact with an antibody that
has been
administered to the subject. For example, an antibody that interacts with an
antigenic domain on a
specificity exchanger can be co-administered with the specificity exchanger.
Further, an antibody
that interacts with a ligand/receptor specificity exchanger may not normally
exist in a subject but
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WO 02/24887 PCT/IBO1/02327
the subject has acquired the antibody by introduction of a biologic material
(e.g., serum, blood, or
tissue). For example, subjects that undergo blood transfusion acquire numerous
antibodies, some
of which can interact with an antigenic domain of a ligand/receptor
specificity exchanger. Some
preferred antigenic domains for use in a ligand/receptor specificity exchanger
comprise viral
epitopes including, but not limited to, the herpes simplex virus, hepatitis B
virus, TT virus, and the
poliovirus.
In some embodiments, it is also preferred that the antigenic domains comprise
an epitope
of a pathogen or toxin that is recognized by a "high-titer antibody".
Approaches to determine
whether the epitope of a pathogen or toxin is recognizable by a high titer
antibody are provided
infra. Epitopes of a pathogen that can be included in an antigenic domain of a
ligand/receptor
specificity exchanger include epitopes on peptide sequences disclosed in
Swedish Pat No.
9901601-6; U.S. Pat. No. 5,869,232; Mol. Irnrnunol. 28: 719-726 (1991); and J.
Med. Virol.
33:248-252 (1991). TABLE II provides the amino acid sequence of several
preferred antigenic
domains.
The section following TABLE II, describes the preparation of ligand/receptor
specificity
exchangers in greater detail.
TABLE II:
ANTIGENIC DOMAINS
GLYSSIWLSPGRSYFET (SEQ.ID. No. 43)
YTDIKYNPFTDRGEGNM (SEQ.ID. No. 44)
DQNIHMNARLLIRSPFT (SEQ.ID. No. 45)
LIRSPFTDPQLLVHTDP (SEQ.ID. No. 46)
QKESLLFPPVKLLRRVP (SEQ.ID. No. 47)
PALTAVETGAT (SEQ. ID.
No.
48)
STLVPETT (SEQ. ID. No. 49)
TPPAYRPPNAPIL (SEQ. No. 50)
ID.
EIPALTAVE (SEQ. ID. 51)
No.
LEDPASRDLV (SEQ.
TD. No. 52)
HRGGPEEF (SEQ. ID. No. 53)
HRGGPEE (SEQ. ID. No.
54)
VLICGENTVSRNYATHS (SEQ.ID. No. 55)
KINTMPPFLDTELTAPS (SEQ.ID. No. 56)
PDEKSQREILLNKIASY (SEQ.ID. No. 57)
TATTTTYAYPGTNRPPV (SEQ.ID. No. 58)
STPLPETT (SEQ. ID. No. 59)
Methods of naaking ligandlreceptof~ specificity exchangers that interact with
receptors oh
bacteria, parasites, fungus, mold, viruses, arid cafacer cells
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WO 02/24887 PCT/IBO1/02327
In some embodiments, the specificity and antigenic domains are made separately
and are
subsequently joined together (e.g., through linkers or by association with a
common carrier
molecule) and in other embodiments, the specificity domain and antigenic
domain are made as part
of the same molecule. For example, any of the specificity domains listed in
TABLE I can be
joined to any of the antigenic domains of TABLE 1I. Although the specificity
and antigenic
domains could be made separately and joined together through a linker or
carrier molecule (e.g., a
complex comprising a biotinylated specif city domain, streptavidin, and a
biotinylated antigenic
domain), it is preferred that the ligand/receptor specificity exchanger is
made as a fusion protein.
Thus, preferred embodiments include fusion proteins comprising any of the
specificity domains
listed in TABLE I joined to any of the antigenic domains of TABLE II.
Ligand/receptor specificity exchangers can be generated in accordance with
conventional
methods of protein engineering, protein chemistry, organic chemistry, and
molecular biology.
Additionally, some commercial enterprises manufacture made-to-order peptides
and a
ligand/receptor specificity exchanger can be obtained by providing such a
company with the
sequence of a desired ligand/receptor specificity exchanger and employing
their service to
manufacture the agent according to particular specifications (e.g., Bachem AG,
Switzerland).
Preferably, the ligand/receptor specificity exchangers are prepared by
chemical synthesis methods
(such as solid phase peptide synthesis) using techniques known in the art,
such as those set forth by
Merrifield et al., J. AnZ. Cheni. Soc. 85:2149 (1964), Houghten et al., Proc.
Natl. Acad. Sci. USA,
82:51:32 (1985), Stewart and Young (Solid phasepeptide synthesis, Pierce Chem
Co., Rockford,
IL (1984), and Creighton, 1983, Proteins: Structures and Molecular Principles,
W. H. Freeman &
Co., N.Y.
By one approach, solid phase peptide synthesis is performed using an Applied
Biosystems
430A peptide synthesizer (Applied Biosystems, Foster City, CA). Each synthesis
uses a p
methylbenzylhydrylamine solid phase support resin (Peptide International,
Louisville, ICY)
yielding a carboxyl terminal amide when the peptides are cleaved off from the
solid support by
acid hydrolysis. Prior to use, the carboxyl terminal amide can be removed and
the ligand/receptor
specificity exchangers can be purified by high performance liquid
chromatography (e.g., reverse
phase high performance liquid chromatography (RP-HPLC) using a Peps-15 CI8
column
(Pharmacia, Uppsala, Sweden)) and sequenced on an Applied Biosystems 473A
peptide sequencer.
An alternative synthetic approach uses an automated peptide synthesizer (Syro,
Multisyntech,
Tubingen, Germany) and 9-fluorenylmethoxycarbonyl (fmoc) protected amino acids
(Milligen,
Bedford, MA).
While the ligand/receptor specificity exchangers can be chemically
synthesized, it can be
more efficient to produce these polypeptides by recombinant DNA technology
using techniques
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
well known in the art. Such methods can be used to construct expression
vectors containing
nucleotide sequences encoding a ligand/receptor specificity exchanger and
appropriate
transcriptional and translational control signals. These methods include, for
example, irr vi~o
recombinant DNA techniques, synthetic techniques, and in vivo genetic
recombination.
Alternatively, RNA capable of encoding a ligand/receptor specificity exchanger
can be chemically
synthesized using, for example, synthesizers. See, for example, the techniques
described in
Oli~onucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford.
A variety of host-expression vector systems can be utilized to express the
ligand/receptor
specificity exchangers. Where the ligand/receptor specificity exchanger is a
soluble molecule it
can be recovered from the culture, i.e., from the host cell in cases where the
peptide or polypeptide
is not secreted, and from the culture media in cases where the peptide or
polypeptide is secreted by
the cells. However, the expression systems also encompass engineered host
cells that express
membrane bound ligand/receptor specificity exchangers. Purification or
enrichment of the
ligand/receptor specificity exchangers from such expression systems can be
accomplished using
appropriate detergents and lipid micelles and methods well known to those
skilled in the art.
The expression systems that can be used include, but are not limited to,
microorganisms
such as bacteria (e.g., E. coli or B. subtilis) transformed with recombinant
bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing nucleotide sequences
encoding a
ligand/receptor specificity exchanger; yeast (e.g., Sacclaaronzyces, Pichia)
transformed with
recombinant yeast expression vectors containing nucleotide sequences encoding
Iigand/receptor
specificity exchangers; insect cell systems infected with recombinant virus
expression vectors
(e.g., Baculovirus) containing nucleic acids encoding the ligand/receptor
specificity exchangers; or
mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant
expression
constructs containing nucleic acids encoding ligand/receptor specificity
exchangers.
In bacterial systems, a number of expression vectors can be advantageously
selected
depending upon the use intended for the ligand/receptor specificity exchanger.
For example, when
a large quantity is desired (e.g., for the generation of pharmaceutical
compositions of
ligand/receptor specificity exchangers) vectors that direct the expression of
high levels of fusion
protein products that are readily purified can be desirable. Such vectors
include, but are not
limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J.,
2:1791 (1983), in which
the ligand/receptor specificity exchanger coding sequence can be ligated
individually into the
vector in frame with the lacZ coding region so that a fusion protein is
produced; pIN vectors
(Inouye & Inouye, Nueleic Acids Res., 13:3101-3109 (1985); Van Heeke &
Schuster, J. Biol.
Chem., 264:5503-5509 (1989)); and the like. pGEX vectors can also be used to
express foreign
polypeptides as fusion proteins with glutathione S-transferase (GST). In
general, such fusion
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WO 02/24887 PCT/IBO1/02327
proteins are soluble and can be purified from lysed cells by adsorption to
glutathione-agarose
beads followed by elution in the presence of free glutathione. The PGEX
vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the cloned
target gene product can be
released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV)
is used as
a vector to express foreign genes. The virus grows in Spodoptera fiugiperda
cells. The
ligandlreceptor specificity exchanger gene coding sequence can be cloned
individually into non-
essential regions (for example the polyhedrin gene) of the virus and placed
under control of an
AcNPV promoter (for example the polyhedrin promoter). Successful insertion of
ligand/receptor
specificity exchanger gene coding sequence will result in inactivation of the
polyhedrin gene and
production of non-occluded recombinant virus, (i.e., virus lacking the
proteinaceous coat coded for
by the polyhedrin gene). These recombinant viruses are then used to infect
Spodopte~a frugiperda
cells in which the inserted gene is expressed. (E.g., see Smith et al., J.
Virol. 46: 584 (1983); and
Smith, U.S. Pat. No. 4,215,051).
In mammalian host cells, a number of viral-based expression systems can be
utilized. In
cases where an adenovirus is used as an expression vector, a nucleic acid
sequence encoding a
ligandlreceptor specificity exchanger can be ligated to an adenovirus
transcription/translation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric gene can
then be inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-
essential region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is
viable and capable of expressing the ligand/receptor specificity exchanger
gene product in infected
hosts. (See e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659
(1984)). Specific
initiation signals can also be required for efficient translation of inserted
ligand/receptor specificity
exchanger nucleotide sequences (e.g., the ATG initiation codon and adjacent
sequences). In most
cases, an exogenous translational control signal, including, perhaps, the ATG
initiation codon,
should be provided. Furthermore, the initiation codon should be in phase with
the reading frame of
the desired coding sequence to ensure translation of the entire insert. These
exogenous
translational control signals and initiation codons can be of a variety of
origins, both natural and
synthetic. The efficiency of expression can also be enhanced by the inclusion
of appropriate
transcription enhancer elements, transcription terminators, etc. (See Bittner
et al., Methods in
Enzyrnol., 153:516-544 (1987)).
In addition, a host cell strain can be chosen that modulates the expression of
the inserted
sequences, or modifies and processes the gene product in the specific fashion
desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein
products can be
important for some embodiments. Different host cells have characteristic and
specific mechanisms
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for the post-translational processing and modification of proteins and gene
products. Appropriate
cell lines or host systems can be chosen to ensure the correct modification
and processing of the
foreign protein expressed. To this end, eukaryotic host cells that possess the
cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product
can be used. Such mammalian host cells include, but are not limited to, CHO,
VERO, BHK, HeLa,
COS, MDCK, 293, 3T3, and WI38.
For long-term, high-yield production of recombinant proteins, stable
expression is
preferred. For example, cell lines that stably express the ligandlreceptor
specificity exchangers
described above can be engineered. Rather than using expression vectors that
contain viral origins
of replication, host cells can be transformed with DNA controlled by
appropriate expression
control elements (e.g., promoter, enhancer sequences, transcription
terminators, polyadenylation
sites, etc.), and a selectable marker. Following the introduction of the
foreign DNA, engineered
cells are allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective
media. The selectable marker in the recombinant plasmid confers resistance to
the selection and
allows cells to stably integrate the plasmid into their chromosomes and grow
to form foci which in
turn are cloned and expanded into cell lines. This method is advantageously
used to engineer cell
lines which express a ligand/receptor specificity exchanger.
A number of selection systems can be used, including but not limited to the
herpes simplex
virus thymidine kinase (Wigler, et al., Cell 11:223 (1977)), hypoxanthine-
guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA
48:2026 ( 1962)),
and adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes
can be employed
in tk-, hgprt- or aprt- cells, respectively. Also,
antimetabolite resistance can be used
as the basis of selection for the following genes: dhfr, which confers
resistance to methotrexate
(Wigler, et al., Proc. Natl. Acad. Sci. USA 77:3567 (1980)); O'Hare, et al.,
Pf~oc. Natl. Acad. Sci.
USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, Proc.
Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the
aminoglycoside G-418
(Colberre-Garapin, et al., J. Mol. Biol. 150:1 (1981)); and hygro, which
confers resistance to
hygromycin (Santerre, et al., Gene 30:147 (1984)).
The following section describes the ligand/receptor specificity exchanger
characterization
assays in greater detail.
Ligandlreceptor specificity exchanger charaeter~izatiorz assays
Preferably, Iigand/receptor specificity exchangers are analyzed for their
ability to interact
with a receptor and/or the ability to interact with an antibody that may be
present in a subject. The
term "characterization assay" refers to an assay, experiment, or analysis made
on a ligand/receptor
specificity exchanger, which evaluates the ability of a ligand/receptor
specificity exchanger to
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interact with a receptor (e.g., a surface receptor present in bacteria, virus,
mold, or fungi) or an
antibody (e.g., an antibody that recognizes an epitope found on a pathogen),
or effect the
proliferation of a pathogen. Encompassed by the term "characterization assay"
are binding studies
(e.g., enzyme immunoassays (EIA), enzyme-linked immunoassays (ELISA),
competitive binding
assays, computer generated binding assays, support bound binding studies, and
one and two hybrid
systems), and infectivity studies (e.g., reduction of viral infection,
propagation, and attachment to a
host cell).
Preferred binding assays use multimeric agents. One form of multimeric agent
concerns a
composition comprising a ligand/receptor specificity exchanger, or fragments
thereof disposed on
a support. Another form of multimeric agent involves a composition comprising
a receptor or an
antibody specific for the antigenic domain of a ligand/receptor specificity
exchanger disposed on a
support. A "support" can be a carrier, a protein, a resin, a cell membrane, or
any macromolecular
structure used to join or immobilize such molecules. Solid supports include,
but are not limited to,
the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic
beads, nitrocellulose
strips, membranes, microparticles such as latex particles, animal cells,
Duracyte~, artificial cells,
and others. A ligand/receptor specificity exchanger can also be joined to
inorganic supports, such
as silicon oxide material (e.g. silica gel, zeolite, diatomaceous earth or
arninated glass) by, for
example, a covalent linkage through a hydroxy, carboxy, or amino group and a
reactive group on
the support.
In some multimeric agents, the macromolecular support has a hydrophobic
surface that
interacts with a portion of the ligand/receptor specificity exchanger,
receptor or ligand by a
hydrophobic non-covalent interaction. In some cases, the hydrophobic surface
of the support is a
polymer such as plastic or any other polymer in which hydrophobic groups have
been linked such
as polystyrene, polyethylene or polyvinyl. Additionally, a ligand/receptor
specificity exchanger,
receptor or an antibody specific for the antigenic domain of a ligand/receptor
specificity exchanger
can be covalently bound to supports including proteins and
oligo/polysaccarides (e.g. cellulose,
starch, glycogen, chitosane or aminated sepharose). In these later multimeric
agents, a reactive
group on the molecule, such as a hydroxy or an amino group, is used to join to
a reactive group on
the carrier so as to create the covalent bond. Additional multimeric agents
comprise a support that
has other reactive groups that are chemically activated so as to attach the
ligand/receptor
specificity exchanger, receptor, or antibody specific for the antigenic domain
of a ligand/receptor
speciEcity exchanger. For example, cyanogen bromide activated matrices, epoxy
activated
matrices, thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxy
succinimide
chlorformate linkages, or oxirane acrylic supports can be used. (Sigma).
Furthermore, in some
embodiments, a liposome or lipid bilayer (natural or synthetic) is
contemplated as a support and a
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ligand/receptor specificity exchanger, receptor, or an antibody specific for
the antigenic domain of
a ligand/receptor specificity exchanger can be attached to the membrane
surface or are
incorporated into the membrane by techniques in liposome engineering. By one
approach,
liposome multimeric supports comprise a ligand/receptor specificity exchanger,
receptor, or an
antibody specific for the antigenic domain of a ligand/receptor specificity
exchanger that is
exposed on the surface.
The insertion of linkers (e.g., "7~ linkers" engineered to resemble the
flexible regions of ~,
phage) of an appropriate length between the ligand/receptor specificity
exchanger, receptor, or
antibody specific for the antigenic domain of a ligand/receptor specificity
exchanger and the
support are also contemplated so as to encourage greater flexibility and
overcome any steric
hindrance that can be presented by the support. The determination of an
appropriate length of
linker that allows for optimal binding can be found by screening the attached
molecule with
varying linkers in the characterization assays detailed herein.
Several approaches to characterize ligand/receptor specificity exchangers
employ a
multimeric described above. For example, support-bound ligand/receptor
specificity exchanger
can be contacted with "free" adhesion receptors and an association can be
determined directly (e.g.,
by using labeled adhesion receptors) or indirectly (e.g., by using a labeled
ligand directed to an
adhesion receptor). Thus, candidate ligand/receptor specificity exchangers are
identified as bona
fide ligand/receptor specificity exchangers by virtue of the association of
the receptors with the
support-bound candidate ligandlreceptor specificity exchanger. Alternatively,
support-bound
adhesion receptors can be contacted with "free" ligand/receptor specificity
exchangers and the
amount of associated ligand/receptor specificity exchanger can be determined
directly (e.g., by
using labeled ligand/receptor specificity exchanger) or indirectly (e.g., by
using a labeled antibody
directed to the antigenic domain of the ligand/receptor specificity
exchanger). Similarly, by using
an antibody specific for the antigenic domain of a ligand/receptor specificity
exchanger disposed
on a support and labeled ligand/receptor specificity exchanger (or a secondary
detection reagent,
e.g., a labeled receptor or antibody to the ligand/receptor specificity
exchanger) the ability of the
antibody to bind to the antigenic domain of the ligand/receptor specificity
exchanger can be
determined.
Some characterization assays evaluate the ability of the ligand/receptor
specificity
exchanger to interact with the target receptor and the redirecting antibody
while other
characterization assays are designed to determine whether a ligand/receptor
specificity exchanger
can bind to both the target receptor and the redirecting antibody. In general,
the characterization
assays can be classified as: (I) in vitro characterization assays, (2)
cellular characterization assays,
and (3) in vivo characterization assays.
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A discussion of each type of characterization assay is provided in the
following sections.
In vitro characterization assays
There are many types of in vitro assays that can be used to determine whether
a
ligand/receptor specificity exchanger binds to a particular receptor and
whether an antibody found
in a subject can bind to the ligand/receptor specificity exchanger. Most
simply, the receptor is
bound to a support (e.g., a petri dish) and the association of the
ligand/receptor specificity
exchanger with the receptor is monitored directly or indirectly, as described
above. Similarly, a
primary antibody directed to the antigenic domain of a ligand/receptor
specificity exchanger (e.g.,
an antibody found in a subject) can be bound to a support and the association
of a ligand/receptor
specificity exchanger with the primary antibody can be determined directly
(e.g., using labeled
ligand/receptor specificity exchanger) or indirectly (e.g., using labeled
receptor or a labeled
secondary antibody that interacts with an epitope on the ligand/receptor
specificity exchanger that
does not compete with the epitope recognized by the primary antibody).
Another approach involves a sandwich-type assay, wherein the receptor is bound
to a
support, the ligand/receptor specificity exchanger is bound to the receptor,
and the primary
antibody is bound to the ligand/receptor specificity exchanger. If labeled
primary antibody is used,
the presence of a receptor/specificity exchanger/primary antibody complex can
be directly
determined. The presence of the receptor/specificity exchanger/primary
antibody complex can
also be determined indirectly by using, for example, a labeled secondary
antibody that reacts with
the primary antibody at an epitope that does not interfere with the binding of
the primary antibody
to the ligand/receptor specificity exchanger. In some cases, it may be desired
to use a labeled
tertiary antibody to react with an unlabeled secondary antibody, thus, forming
a
receptor/specificity exchanger/primary antibody/secondary antibody/labeled
tertiary antibody
complex.
The example below describes a characterization assay that was performed to
determine
whether a specificity domain derived from the C-terminal domain of fibrinogen
inhibits the
binding of clumping factor (Clf) to fibrinogen.
EXAMPLE 1
In this example, several peptides corresponding to the C-terminal domain of
fibrinogen
(Fib) were analyzed for their ability to block the binding of clumping factor
(Clf) to fibrinogen.
(See TABLE III). These peptides were manufactured using standard techniques in
peptide
synthesis using fmoc chemistry (Syro, MultiSynTech, Germany). Preferably, the
peptides are
purified by reverse-phase HPLC. A competition enzyme immunoassay was then
performed to
determine whether the peptides were able to block the interaction between Clf
and fibrinogen. The
results of these experments are shown in TABLE III. The smallest peptide from
fibrinogen found
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to inhibit the interaction between Clf and fibrinogen was HLGGAKQAGD (SEQ. ID
No. 124).
Substitution of the first two amino acids of this peptide with alanine and
lysine had a significant
effect on the ability of the peptide to block the interaction between Clf and
fibrinogen (e.g., the
peptide ALGGAKQAGD (SEQ. ID No.123) was unable to block the Clf/fibrinogen
interaction).
TABLE III
SEQ ID NO. (Fib) peptide Inhibition of~Fib/CIf1 interaction
106 LTIGEGQQHHLGGAKQAGDV +
107 GEGQQHHLGGAKQAGDV +
108 QQHHLGGAKQAGDV +
109 QHHLGGAKQAGDV +
110 HHLGGAKQAGDV +
111 HLGGAKQAGDV +
112 LGGAKQAGDV -
123 GGAKQAGDV -
114 GAKQAGDV -
115 QHHLGGAKQAGD +
116 QHHLGGAKQAG +
117 QHHLGGAKQA -
118 QHHLGGAKQ -
119 QHHLGGAK +/-
120 QHHLGGA -
121 HHLGGAKQAGDV +
122 HHLGGAKQAGD +
123 HHLGGAKQAG +
124 HLGGAKQAGDV +
125 HLGGAKQAGD +
126 ALGGAKQAG -
127 HAGGAKQAG +
128 HLAGAKQAG +
129 HLGAAKQAG +
130 HLGGGKQAG +
131 HLGGAAQAG +/-
132 HLGGAKAAG +
133 HLGGAKQGG +
134 HLGGAKQAA +
The example below describes a characterization assay that was performed to
determine
whether a ligand/receptor specificity exchanger interacts with bacteria having
the CIfA receptor.
EXAMPLE 2
Ligand/receptor specificity exchangers having specificity domains
(approximately 20
amino acids long) corresponding to various regions of the fibrinogen gamma-
chain sequence were
produced using standard techniques in peptide synthesis using finoc chemistry
(Syro,
MultiSynTech, Germany) and these ligand/receptor specificity exchangers were
analyzed fox their
ability to bind the CIfA receptor and an antibody specific for their antigenic
domains. The
sequences of these ligand/receptor specificity exchangers are listed in TABLE
IV and are provided
in the Sequence listing (SEQ. lD. Nos. 60-103). The ligand/receptor
specificity exchangers used
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in this analysis have an antigenic domain that presents an epitope of herpes
simplex virus gG2
protein, which is recognized by a monoclonal antibody for herpes simplex virus
gG2 proteins.
Serial dilutions of these ligand/receptor specificity exchangers were made in
phosphate buffered
saline (PBS) containing 2:g/ml goat serum. (Sigma Chemicals, St. Louis, MO)
and 0.5% Tween
20 (PBS-GT). The receptor CIfA was passively adsorbed at 10 pg/ml to 96 well
microtiter plates
in SOmM sodium carbonate buffer, pH 9.6, overnight at +4° C.
The diluted ligand/receptor specificity exchangers were then incubated on the
plates for 60
minutes. The ability of the ligand/receptor specificity exchanger to interact
with the receptor was
determined by applying a primary antibody to the plate and incubating for 60
minutes (a 1:1000
dilution of mAb for herpes simplex virus gG2 proteins). The bound primary mAb
was then
indicated by a rabbit anti-mouse IgG (Sigma) secondary antibody and a
peroxidase labeled goat
anti-rabbit IgG (Sigma) tertiary antibody. The plates were developed by
incubation with dinitro-
phenylene-diamine (Sigma) and the absorbance at 405 nm was analyzed.
Every ligand/receptor specificity exchanger provided in TABLE IV (SEQ. ID Nos.
60-
103) appreciably bound the immobilized CIfA and also allowed for the binding
of the mAb
specific for HSV gG2 protein. The method described above for determining the
affinity of a
ligand/receptor specificity exchanger for an adhesion receptor and a primary
antibody can be
performed for any candidate ligand/receptor specificity exchanger comprising
any specificity
domain and any antigenic domain provided that the appropriate sequences and
adhesion receptors
are used.
The example following TABLE IV describes another characterization assay that
was
performed to determine whether a ligand/receptor specificity exchanger
interacts with bacteria
having the CIfA receptor.
TABLE IV
LIGAND/RECEPTOR SPECIFICITY EXCHANGERS
YGEGQQHHLGGAKQAGDV HRGGPEEF(SEQ. ID.No. 60)
YGEGQQHHLGGAKQAGDVHRGGPEE (SEQ.
ID.
No.
61)
YGEGQQHHLGGAKQAGDVSTPLPETT (SEQ. ID.No. 62)
MSWSLHPRNLILYFYALLFLHRGGPEE(SEQ. ID.No. 63)
ILYFYALLFLSTCVAYVATHRGGPEE (SEQ. TD.No. 64)
SSTCVAYVATRDNCCILDERHRGGPEE(SEQ. ID.No. 65)
RDNCCILDERFGSYCPTTCGHRGGPEE(SEQ. ID.No. 66)
FGSYCPTTCGIADFLSTYQTHRGGPEE(SEQ. ID.No. 67)
IADFLSTYQTKVDKDLQSLEHRGGPEE(SEQ. ID.No. 68)
KVDKDLQSLEDILHQVENKTHRGGPEE(SEQ. ID.No. 69)
DILHQVENKTSEVKQLIKAIHRGGPEE(SEQ. ID.No. 70)
SEVKQLIKAIQLTYNPDESSHRGGPEE(SEQ. ID.No. 71)
QLTYNPDESSKPNMTDAATLHRGGPEE(SEQ. ID.No. 72)
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KPNMIDAATLKSRIMLEEIMHRGGPEE (SEQ. ID, No.73)
KSRIMLEEIMKYEASILTHDHRGGPEE (SEQ. ID, No.74)
KYEASILTHDSSIRYLQEIYHRGGPEE (SEQ. ID. No.75)
SSIRYLQEIYNSNNQKIVNLHRGGPEE (SEQ. ID. No.76)
NSNNQKIVNLKEKVAQLEAQHRGGPEE (SEQ. ID. No.77)
CQEPCKDTVQIHDITGKDCQHRGGPEE (SEQ. ID. No.78)
IHDITGKDCQDIANKGAKQSHRGGPEE (SEQ. ID. No.79)
DIANKGAKQSGLYFIKPLKAHRGGPEE (SEQ. ID. No.80)
GLYFIKPLKANQQFLVYCEIHRGGPEE (SEQ. ID. No.81)
NQQFLVYCEIDGSGNGWTVFHRGGPEE (SEQ. ID. No.82)
DGSGNGWTVFQKRLDGSVDFHRGGPEE (SEQ. ID. No.83)
QKRLDGSVDFKKNWIQYKEGHRGGPEE (SEQ. ID. No.84)
KKNWIQYKEGFGHLSPTGTTHRGGPEE (SEQ. ID. No.85)
FGHLSPTGTTEFWLGNEKIHHRGGPEE (SEQ. ID. No.86)
EFWLGNEKIHLISTQSAIPYHRGGPEE (SEQ. ID. No.$7)
LISTQSAIPYALRVELEDWNHRGGPEE (SEQ. ID. No.88)
ALRVELEDWNGRTSTADYAMHRGGPEE (SEQ. ID. No.89)
GRTSTADYAMFKVGPEADKYHRGGPEE (SEQ. ID. No.90)
FKVGPEADKYRLTYAYFAGGHRGGPEE (SEQ. ID. No.91)
RLTYAYFAGGDAGDAFDGFDHRGGPEE (SEQ. ID. No.92)
DAGDAFDGFDFGDDPSDKFFHRGGPEE (SEQ. ID. No.93)
FGDDPSDKFFTSHNGMQFSTHRGGPEE (SEQ. ID. No.94)
TSHNGMQFSTWDNDNDKFEGHRGGPEE (SEQ. ID. No.95)
WDNDNDKFEGNCAEQDGSGWHRGGPEE (SEQ. ID. No.96)
NCAEQDGSGWWMNKCHAGHLHRGGPEE (SEQ. ID. No.97)
WMNKCHAGHLNGVYYQGGTYHRGGPEE (SEQ. ID. No.98)
NGVYYQGGTYSKASTPNGYDHRGGPEE (SEQ. ID. No.99)
SKASTPNGYDNGIIWATWKTHRGGPEE (SEQ. ID. No.100)
NGIIWATWKTRWYSMKKTTMHRGGPEE (SEQ. ID. No.101)
RWYSMKKTTMKIIPFNRLTIHRGGPEE (SEQ. ID. No.102)
IKIIPFNRLTIGEGQQHHLGGAKQAGDVHRGGPEE (SEQ.ID.No. 103)
EXAMPLE 3
Ligand/receptor specificity exchangers having specificity domains that bind to
clumping
factor (Clf) and antigenic domains that correspond to an epitope derived from
the polio virus were
produced using standard techniques in peptide synthesis using fmoc chemistry
(Syro,
MultiSynTech, Germany). See TABLE V. These ligand/receptor specificity
exchangers were
analyzed for their ability to inhibit the interaction between CLF and
fibrinogen. In these
experiments, the ligand/specificity exchangers described in TABLE V were
manufactured and
various concentrations of these molecules were added to an enzyme competition
immunoassay
containing Clf and fibrinogen. The lowest inhibiting concentration, which is
the lowest peptide
concentration needed to inhibit the Clf/Fib interaction, was ascertained.
Accordingly, the lower the
concentration needed to inhibit the Fib/Clf interaction, the more effective
the inhibitor.
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Additionally, the lowest solid-phase bound peptide concentration, which is the
lowest tested
concentration of peptide recognized by anti-poliovirus antibodies in the
immunoassay, was
determined. Some of the peptides used (e.g., CPALTAVETGCTNPLAAHI-E,GGAKQAG
(SEQ
ID No. 135), ~GGAKQAG-AA-CPALTAVETGCTNPL (SEQ ID No. 137),
CPALTAVETGC-TNPLHHI.,GGAKQAG (SEQ ID No. 139), and HHI.,GGAKQAG-
CPALTAVETGCTNPL (SEQ TD No. 141)), designated by asterisks in TABLE V, were
cyclized
between the two artificially introduced cystiene residues. These experiments
revealed that
I~GGAKQAG-AA-CPALTAVETGCTNPL (SEQ ID No. 137) and I~LGGAKQAG-
CPALTAVETGCTNPL (SEQ ID No. 142) effectively inhibited the interaction of Clf
with
fibrinogen and retained functional poliovirus epitopes.
TABLE V
Lowest
Lowest epitope
inhibiting on soIid-
Conc. phase
ID Peptide sequence (~ /~ ml) ( ~/ml)
135 CPALTAVETGCTNPL-AA-HHI.,GGAKQAG* >625 1.6
136 CPALTAVETGCTNPL-AA-HHI~GGAKQAG 625 1.6
137 I-IHL,GGAKQAG-AA-CPALTAVETGCTNPL* 69 8
138 HHI.,GGAKQAG-AA-CPALTAVETGCTNPL 625 >200
139 CPALTAVETGC-TNPLHHI,GGAKQAG* 625 1.6
140 CPALTAVETGC-TNPLHHI,GGAKQAG 208 1.6
141 HHI.,GGAKQAG-CPALTAVETGCTNPL* 208 >200
142 HHI.,GGAKQAG-CPALTAVETGCTNPL 23 1.6
143 PALTAVETGATNPL-HHI.,GGAKQAG >625 1.6
144 HHIJGGAKQAG-PALTAVETGATNPL >625 >200
The next section describes several cellular-based characterization assays that
can be
performed to determine whether a ligand/receptor specificity exchanger has an
effect on the
proliferation of a pathogen.
Pathogen-based characterisation assays
In another type of characterization assay, a pathogen-based approach is used
to evaluate
the ability of a ligand/receptor specificity exchanger to interact with a
pathogen and an antibody
directed to the antigenic domain of the ligand/receptor specificity exchanger.
This analysis also
reveals the ability of the Iigand/receptor specificity exchanger to effect
proliferation of a pathogen
because, in the body of a subject, the interaction of the ligand/receptor
specificity exchanger with a
pathogen and an antibody directed to the antigenic domain of the
ligand/receptor specificity
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exchanger is followed by humoral and cellular responses that purge the
pathogen from the subject
(e.g., complement fixation and macrophage degradation). In general, the
pathogen-based
characterization assays involve providing ligand/receptor specificity
exchangers to cultured
pathogens and monitoring the association of the ligand/receptor specificity
exchanger with the
cells or virus. Several types of pathogen-based characterization assays can be
used and the
example below describes some of the preferred characterization assays in
greater detail.
EXAMPLE 4
One type of pathogen-based characterization assay involves binding of a
ligand/receptor
specificity exchanger to bacteria disposed on a support. Accordingly, bacteria
that produce Clfa
(e.g., Staphylococcus aureus, or Escherichia coli.) are grown in culture or on
a agar plate in a
suitable growth media (e.g., LB broth, blood broth, LB agar or blood agar).
The cells are then
transferred to a membrane (e.g., nitrocellulose or nylon) by either placing
the culture on the
membrane under vacuum (e.g., using a dot-blot manifold apparatus) or by
placing the membrane
on the colonies for a time sufficient to permit transfer. The cells that are
bound to the membrane
are then provided a serial dilution of a ligand/receptor specificity exchanger
(e.g., SOOng, 1:g, S:g,
10:g, 25:g, and SO:g of ligand/receptor specificity exchanger in a total
volume of 200:1 of PBS). In
one experiment, the ligand/receptor specificity exchangers listed in TABLE IV
or V are used. The
diluted ligand/receptor specificity exchangers are then incubated on the
membranes for 60 minutes.
Subsequently, the non-bound ligand/receptor specificity exchangers are removed
and the
membrane is washed with PBS (e.g., 3 washes with 2m1 of PBS per wash). Next, a
1:100 - 1:1000
dilution of a primary antibody that interacts with the antigenic domain of the
ligand/receptor
specificity exchanger (e.g., mAb for herpes simplex virus gG2 protein) is
provided and the binding
reaction is allowed to occur for 60 minutes. Again, the membrane is washed
with PBS (e.g., 3
washes with 2m1 of PBS per wash) to remove unbound primary antibody.
Appropriate controls
include the membrane itself, bacteria on the membrane without a
ligand/receptor specificity
exchanger, and bacteria on the membrane with Iigand/receptor specificity
exchanger but no
primary antibody.
To detect the amount of ligand/receptor specificity exchanger bound to the
bacteria on the
membrane, a secondary antibody (e.g., rabbit anti-mouse IgG (Sigma)) and a
tertiary antibody
(e.g., a peroxidase labeled goat anti-rabbit IgG (Sigma)) are used. Of course,
a labeled secondary
antibody that interacts with the primary antibody can be used as well. As
above, the secondary
antibody is contacted with the membrane for 60 minutes and the non-bound
secondary antibody is
washed from the membrane with PBS (e.g., 3 washes with 2m1 of PBS per wash).
Then, the
tertiary antibody is contacted with the membrane for 60 minutes and the non-
bound tertiary
antibody is washed from the membrane with PBS (e.g., 3 washes with 2m1 of PBS
per wash). The
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bound tertiary antibody can be detected by incubating the membrane with
dinitro-phenylene-
diamine (Sigma).
Another approach involves the use of an immobilized ligand/receptor
specificity
exchanger. Accordingly, primary antibody (e.g., mAb for herpes simplex virus
gG2 protein) is
bound to a petri dish. Once the primary antibody is bound, various dilutions
of a ligand/receptor
specificity exchanger (e.g., a ligand/receptor specificity exchanger provided
in TABLE IV or V)
are added to the coated dish. The ligand/receptor specificity exchanger is
allowed to associate with
the primary antibody for 60 minutes and the non-bound ligand/receptor
specificity exchanger is
washed away (e.g., three washes with 2m1 of PBS). Appropriate controls include
.petri dishes
without primary antibody or ligand/receptor specificity exchanger.
Subsequently, a turbid solution of bacteria (e.g., E. coli) are added to the
petri dishes and
the bacteria are allowed to interact with the immobilized ligandlreceptor
specificity exchanger for
60 minutes. The non-bound bacteria are then removed by washing with PBS (e.g.,
3 washes with
2m1 of PBS). Next, growth media (e.g., LB broth) is added to the petri dish
and the culture is
I5 incubated overnight. Alternatively, LB agar is added to the petri dish and
the culture is incubated
overnight. An interaction between the ligand/receptor specificity exchanger
and the bacteria can
be observed visually (e.g., turbid growth media, which can be quantified using
spectrophotometric
analysis or the appearance of colonies on the agar).
By modifying the approaches described above, one of skill in the art can
evaluate the
ability of a ligand/receptor specificity exchanger to interact with a virus.
For example, soluble
fragments of T4 glycoprotein have been shown to interact with a human
immunodeficiency virus
(HIV) envelope gIycoprotein. (See e.g., U.S. Pat No. 6,093,539).
Ligand/receptor specificity
exchangers having a specificity domain comprising a fragment of T4
glycoprotein that interacts
with HIV envelope glycoprotein (e.g., amino acids 1-419 of the T4 glycoprotein
sequence provided
in U.S. Pat No. 6,093,539 or a portion thereof) can be made by synthesizing a
fusion protein
having the specificity domain joined to an antigenic domain (e.g., an
antigenic domain Iisted in
TABLE II). Although peptide chemistry can be used to make the ligand/receptor
specificity
exchanger, it is preferred that an expression construct having the fragment of
T4 glycoprotein
joined to an antigenic domain is made and transfected into a suitable cell.
The expression and
purification strategies described in U.S. Pat No. 6,093,539 and above can also
be employed.
Once the Iigand/receptor specificity exchanger has been constructed a filter
binding assay
is performed. Accordingly, serial ten-fold dilutions of HIV inoculum are
applied to a membrane
(e.g. nitrocellulose or nylon) in a dot blot apparatus under constant vacuum.
Then serial ten fold
dilutions of the ligand/receptor specificity exchanger are applied to the
bound HIV particles. The
ligand/receptor specificity exchanger is contacted with the particles for 60
minutes before applying
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vacuum and washing With PBS (e.g., 3 washes with 2m1 of PBS per wash)). Once
the non-bound
ligand/receptor specificity exchanger is removed, ten fold serial dilutions of
the primary antibody,
which binds to the antigenic domain, are added to the samples and the binding
reaction is allowed
to occur for 60 minutes. Then a vacuum is applied and the non-bound primary
antibody is washed
with PBS (e.g., 3 washes with 2m1 of PBS per wash)). The detection of the
bound primary
antibody can be accomplished, as described above.
The ability of a ligand/receptor specificity exchanger to interact with a
virus can also be
evaluated in a sandwich-type assay. Accordingly, a primary antibody that
interacts with the
antigenic domain of the ligand/receptor specificity exchanger is immobilized
in micro titer wells
and serial dilutions of ligand/receptor specificity exchanger are added to the
primary antibody so as
to create a primary antibody/specificity exchanger complex, as described
above. Next, ten fold
serial dilutions of HIV inoculum are added and the binding reaction is allowed
to occur for 60
minutes. Non-bound HIV particles are removed by successive washes in PBS.
Detection of the
bound HIV particles can be accomplished using a radiolabeled anti-HIV antibody
(e.g., antibody
obtained from sera from a person suffering with HIV infection).
While the examples above describe pathogen-based assays using bacteria and a
virus,
modifications of these approaches can be made to study the interaction of
ligand/receptor
specificity exchangers with mammalian cells. For example, the ability of a
ligand/receptor
specificity exchanger to interact with an integrin receptor present on a
cancer cell can be
determined as follows. Melanoma cells that express an 'dv~3 receptor (e.g.,
M21 human
melanoma cells) bind fibrinogen and this interaction can be blocked by
administering an RGD
containing peptide ( See e.g., I~atada et al., J. Biol. Clzem. 272: 7720
(1997) and Felding-
Habermann et al., J. Biol. Cherra. 271:5892-5900 (1996)). Similarly, many
other types of cancer
cells express integrins that interact with RGD peptides. By one approach,
cancer cells that
expresses an RGD-responsive integrin (e.g., M21 human melanoma cells) are
cultured to
confluency. M21 cells can be grown in DMEM media with 10% fetal bovine serum,
20 mM
Hepes, and 1 mM pyruvate.
Preferably, the cells are stained with hydroethidine (Polysciences, Inc.,
Warrington, PA) at
20 pg/ml final concentration (2 x 106 cells/ml) for 30 min at 37°C and
then washed twice to
remove excess dye. Hydroethidine intercalates into the DNA resulting in a red
fluorescent labeling
of the cells and does not impair the cell's adhesive functions. The staining
provides a way to
quantify the binding of a ligand/receptor specificity exchanger to the cells.
That is, the total
number of hydroethidine stained cells can be compared to the number of cells
bound to a
fluorescently labeled primary antibody/specificity exchanger complex so as to
determine the
binding efficiency.
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Accordingly, the stained cells are incubated with various dilutions of a
ligand/receptor
specificity exchanger comprising a RGD sequence (e.g., GRGDSPHRGGPEE (SEQ. ID
No. 104)
or WSRGDWHRGGPEE (SEQ. ID No. 105)). After a 60 minute incubation, the non-
bound
ligand/receptor specificity exchanger is removed by several washes in DMEM
media with 10%
fetal bovine serum, 20 rnM Hepes, and 1 mM pyruvate (e.g., 3 washes of Sml of
media). Next, a
1:100 - 1:1000 dilution of a primary antibody that interacts with the
antigenic domain of the
ligand/receptor specificity exchanger (e.g., mAb for herpes simplex virus gG2
protein) is provided
and the binding reaction is allowed to occur for 60 minutes. Subsequently,
several washes in
media are performed to remove any non-bound primary antibody. Appropriate
controls include
stained cells without ligand/receptor specificity exchanger or stained cells
without primary
antibody.
Following binding of the primary antibody, a goat anti-mouse FITC labeled
antibody
(1:100 dilution) (Sigma) is added and binding is allowed to occur for 60
minutes. Again, several
media washes are made to remove any non-bound secondary antibody. Analysis is
made by flow
cytometry with filter settings at 543/590 nm for hydroethidine and 495/525 nm
for fluorescin. One
will observe an appreciable binding of primary antibody to the ligand/receptor
specificity
exchanger/cell complex, which will demonstrate that the ligand/receptor
specificity exchanger will
have an effect on the cell.
The example below describes a characterization assay that verified that RGD-
containing
ligand/receptor specificity exchangers effectively bind to mammalian cells and
redirect antibodies
to these cells.
EXAMPLE 5
The peptide RGDSAATPPAYR (SEQ ID NO. 145) was manufactured using standard
techniques in peptide synthesis using fmoc chemistry (Syro, MultiSynTech,
Germany). This
peptide has a specificity domain that binds integrin receptors, a spacer (the
AA), and an antigenic
domain that has an epitope recognized by the monoclonal antibody 57/8, an
epitope present on the
hepatitis B virus a antigen (HBeAg).
Murine myeloma cells (SP2/0 cells) were washed in serum free media and were
then
incubated with the RGDSAATPPAYR (SEQ m NO. 145) peptide or a control peptide
derived
from hepatitis C virus (HCV) NS3 domain at a concentration of SOpg/ml. The
cells were then
washed and the amount surface bound peptide was detected by labeling the cells
with the the 57/8
antibody. Surface bound antibody was indicated by an FITC labelled anti-mouse
IgG conjugate
diluted 1/500 and the level of surface staining was determined by fluorescent
microscopy.
This experiment revealed that cells incubated with the control peptide did not
show
staining. In contrast, cells incubated with the RGDSAATPPAYR (SEQ ID NO. 145)
peptide
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showed significant surface staining consistent with the location of surface
expressed integrins.
Accordingly, RGD-containing ligand/receptor specificity exchangers effectively
bind integrin
producing mammalian cells and these molecules can be used to redirect and
target antibodies to
tumor cells.
The next section describes characterization assays that are performed in
animals.
In vivo characterization assays
Characterization assays also include experiments that evaluate ligand/receptor
specificity
exchangers in vivo. There are many animal models that are suitable for
evaluating the ability of a
Iigand/receptor specificity exchanger to inhibit pathogenic infection. Mice
are preferred because
they are easy to maintain and are susceptible to bacterial infection, viral
infection, and cancer.
Chimpanzees are also preferred because of their close genetic relationship to
humans.
An approach to evaluate the efficacy of a ligand/receptor specificity
exchanger in mice is
provided in the next example.
EXAMPLE 6
To test the ability of a ligand/receptor specificity exchanger to treat a
bacterial infection
the following characterization assay can be performed. Several female CF-1
outbred mice (Charles
Rivers Laboratories) of approximately 8 weeks of age and 25 gram body mass are
vaccinated with
the antigenic domains of the ligand/receptor specificity exchangers to be
tested. Preferably, the
antigenic domains are coupled to a carrier and are administered with an
adjuvant. For example, the
antigenic domains can be fused to keyhole limpet hemocyanin or bovine serum
albumin, which act
as both a carrier and adjuvant or an adjuvant such as Freund's adjuvant,
aluminum hydroxide, or
lysolecithin can be used. Once a high titer of antibody to the antigenic
domains can be verified by,
for example, irnmunodiffusion or EIA, the immunized mice are inoculated
intraperitoneally with
overnight cultures of Staphylococcus aureus NTCC 10649. The inoculums are
adjusted to yield
approximately 100 x LDSO or log 6.6 for S. aureus.
Serial dilutions of Iigand/receptor specificity exchangers (e.g., the
ligand/receptor
specificity exchangers provide in TABLE I~ are formulated in sterile water for
injection and are
administered by the subcutaneous (SC) or oral (PO) route at one and five hours
post infection.
Concurrently with each trial, the challenge LD50 is validated by inoculation
of untreated mice with
log dilutions of the bacterial inoculum. Preferably, a five log dilution range
of the bacterial
challenges is inoculated into five groups of ten mice each (ten mice per log
dilution). A mortality
rate of 100% will be produced in all groups of untreated mice at the 100 X
LD50 challenge
inoculum. Mice are monitored daily for mortality for seven days. The mean
effective dose to
protect 50% of the mice (EDSp) can be calculated from cumulative mortality by
logarithmic-probit
analysis of a plotted curve of survival versus dosage as described in
A~atirnicrob. Agents
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Chemotlzer. 31: 1768-1774 and Proc. Soc. Exp. Biol. Med. 1994, 57, 261-264. As
one of skill in
the art will appreciate, similar approaches can be used to test the ability of
ligand/receptor
specificity exchangers to inhibit viral infection and cancer.
The ligand/receptor specificity exchangers described herein can be formulated
in
pharmaceuticals and administered to subjects in need of an agent that inhibits
the proliferation of a
pathogen. The section below describes several pharmaceuticals comprising
ligand/receptor
specificity exchangers that interact with a receptor on a pathogen.
Pharmaceuticals comprising a ligandlreceptor specificity exchanger that
interacts with a
receptor on a pathogen
The ligand/receptor specificity exchangers described herein are suitable for
incorporation
into pharmaceuticals for administration to subjects in need of a compound that
treats or prevents
infection by a pathogen. These pharmacologically active compounds can be
processed in
accordance with conventional methods of galenic pharmacy to produce medicinal
agents for
administration to mammals including humans. The active ingredients can be
incorporated into a
pharmaceutical product with and without modification. Further, the manufacture
of
pharmaceuticals or therapeutic agents that deliver the pharmacologically
active compounds of this
invention by several routes are aspects of the present invention. For example,
and not by way of
limitation, DNA, RNA, and viral vectors having sequences encoding a
ligandlreceptor specificity
exchanger that interacts with a receptor on a pathogen are used with
embodiments of the invention.
Nucleic acids encoding a ligand/receptor specificity exchanger can be
administered alone or in
combination with other active ingredients.
The compounds can be employed in admixture with conventional excipients, i.e.,
pharmaceutically acceptable organic or inorganic carrier substances suitable
for parenteral, enteral
(e.g., oral) or topical application that do not deleteriously react with the
pharmacologically active
ingredients described herein. Suitable pharmaceutically acceptable carriers
include, but are not
limited to, water, salt solutions, alcohols, gum arabic, vegetable oils,
benzyl alcohols, polyetylene
glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium
stearate, talc, silicic
acid, viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, pentaerythritol
fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Many
more vehicles that
can be used are described in Rernrnihgtora's Pharmaceutical Sciences, 15th
Edition, Easton:Mack
Publishing Company, pages 1405-1412 and 1461-1487(1975) and The National
Fornaulary XIV,
14th Edition, Washington, American Pharmaceutical Association (1975). The
pharmaceutical
preparations can be sterilized and if desired mixed with auxiliary agents,
e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure,
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buffers, coloring, flavoring and/or aromatic substances and the like so long
as the auxiliary agents
does not deleteriously react with the ligand/receptor specificity exchangers.
The effective dose and method of administration of a particular pharmaceutical
having a
ligand/receptor specificity exchanger can vary based on the individual needs
of the patient and the
treatment or preventative measure sought. Therapeutic efficacy and toxicity of
such compounds
can be determined by standard pharmaceutical procedures in cell cultures or
experimental animals,
e.g., ED50 (the dose therapeutically effective in 50% of the population). Fox
example, the
effective dose of a ligand/receptor specificity exchanger can be evaluated
using the
characterization assays described above. The data obtained from these assays
is then used in
formulating a range of dosage for use with other organisms, including humans.
The dosage of such
compounds lies preferably within a range of circulating concentrations that
include the ED50 with
no toxicity. The dosage varies within this range depending upon type of
ligand/receptor specificity
exchanger, the dosage form employed, sensitivity of the organism, and the
route of administration.
Normal dosage amounts of a ligand/receptor specificity exchanger can vary from
approximately 1 to 100,000 micrograms, up to a total dose of about 10 grams,
depending upon the
route of administration. Desirable dosages include about 250:g-lmg, about SOmg-
200mg, and
about 250mg-SOOmg.
In some embodiments, the dose of a ligand/receptor specificity exchanger
preferably
produces a tissue or blood concentration or both from approximately 0.1 :M to
SOOmM. Desirable
doses produce a tissue or blood concentration or both of about 1 to 800 p,M.
Preferable doses
produce a tissue or blood concentration of greater than about 10 pM to about
SOO:M. Although
doses that produce a tissue concentration of greater than 800:M are not
preferred, they can be used.
A constant infusion of a ligand/receptor specificity exchanger can also be
provided so as to
maintain a stable concentration in the tissues as measured by blood levels.
The exact dosage is chosen by the individual physician in view of the patient
to be treated.
Dosage and administration are adjusted to provide sufficient levels of the
active moiety or to
maintain the desired effect. Additional factors that can be taken into account
include the severity
of the disease, age of the organism being treated, and weight or size of the
organism; diet, time and
frequency of administration, drug combination(s), reaction sensitivities, and
tolerance/response to
therapy. Short acting pharmaceutical compositions are administered daily or
more frequently
whereas long acting pharmaceutical compositions are administered every 2 or
more days, once a
week, or once every two weeks or even less frequently.
Routes of administration of the pharmaceuticals include, but are not limited
to, topical,
transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar.
Transdermal
administration is accomplished by application of a cream, rinse, gel, etc.
capable of allowing the
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ligand/receptor specificity exchangers to penetrate the skin. Parenteral
routes of administration
include, but are not limited to, electrical or direct injection such as direct
injection into a central
venous line, intravenous, intramuscular, intraperitoneal, intradermal, or
subcutaneous injection.
Gastrointestinal routes of administration include, but are not limited to,
ingestion and rectal.
Transbronchial and transalveolar routes of administration include, but are not
limited to,
inhalation, either via the mouth or intranasally.
Compositions having the ligand/receptor specificity exchangers described
herein that are
suitable for transdermal or topical administration include, but are not
limited to, pharmaceutically
acceptable suspensions, oils, creams, and ointments applied directly to the
skin or incorporated into
a protective carrier such as a transdermal device ("transdermal patch").
Examples of suitable
creams, ointments, etc. can be found, for instance, in the Physician's Desk
Reference. Examples of
suitable transdermal devices are described, for instance, in U.S. Patent No.
4,818,540 issued April
4, 1989 to Chinen, et al.
Compositions having pharmacologically active compounds that are suitable for
parenteral
administration include, but are not limited to, pharmaceutically acceptable
sterile isotonic
solutions. Such solutions include, but are not limited to, saline and
phosphate buffered saline for
injection into a central venous line, intravenous, intramuscular,
intraperitoneal, intradermal, or
subcutaneous injection.
Compositions having pharmacologically active compounds that are suitable for
transbronchial and transalveolar administration include, but are not limited
to, various types of
aerosols for inhalation. Devices suitable for transbronchial and transalveolar
administration of
these are also embodiments. Such devices include, but are not limited to,
atomizers and
vaporizers. Many forms of currently available atomizers and vaporizers can be
readily adapted to
deliver compositions having the ligand/receptor specificity exchangers
described herein.
Compositions having pharmacologically active compounds that are suitable for
gastrointestinal administration include, but not limited to, pharmaceutically
acceptable powders,
pills or liquids for ingestion and suppositories for rectal administration.
Due to the ease of use,
gastrointestinal administration, particularly oral, is a preferred embodiment.
Once the
pharmaceutical comprising the ligand/receptor specificity exchanger has been
obtained, it can be
administered to an organism in need to treat or prevent pathogenic infection.
Aspects of the invention also include a coating for medical equipment such as
prosthetics,
implants, and instruments. Coatings suitable for use on medical devices can be
provided by a gel
or powder containing the ligand/receptor specificity exchanger or by a
polymeric coating into
which a ligand/receptor specificity exchanger is suspended. Suitable polymeric
materials for
coatings of devices are those that are physiologically acceptable and through
which a
33
CA 02421877 2003-03-11
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therapeutically effective amount of the ligand/receptor specificity exchanger
can diffuse. Suitable
polymers include, but are not limited to, polyurethane, polymethacrylate,
polyamide, polyester,
polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyvinyl-
chloride, cellulose
acetate, silicone elastomers, collagen, silk, etc. Such coatings are
described, for instance, in U.S.
Patent No. 4,612,337.
The section below describes methods of treating and preventing disease using
the
ligand/receptor specificity exchangers described herein.
Ti~eatnaent and prevention of disease using a ligandlreceptor specificity
exchanger
Pharmaceuticals comprising a ligand/receptor specificity exchanger can be
administered to
a subject in need to treat and/or prevent infection by a pathogen that has a
receptor. Such subjects
in need can include individuals at risk of contacting a pathogen or
individuals who are already
infected by a pathogen. These individuals can be identified by standard
clinical or diagnostic
techniques.
By one approach, for example, a subject suffering from a bacterial infection
is identified as
a subject in need of an agent that inhibits proliferation of a pathogen. This
subject is then provided
a therapeutically effective amount of ligand/receptor specificity exchanger.
The Iigand/receptor
specificity exchanger used in this method comprises a specificity domain that
interacts with a
receptor present on the bacteria (e.g., extracellular fibrinogen binding
protein (Efb), collagen
binding protein, vitronectin binding protein, laminin binding protein,
plasminogen binding protein,
thrombospondin binding protein, clumping factor A (CIfA), clumping factor B
(CIfB), fibronectin
binding protein, coagulase, and extracellular adherence protein). The
ligand/receptor specificity
exchanger also comprises an antigenic domain that has an epitope of a pathogen
or toxin,
preferably, an epitope recognized by high titer antibodies present in the
subject in need. It may
also be desired to screen the subject in need for the presence of high titer
antibodies that recognize
the antigenic domain prior to providing the subject the ligand/receptor
specificity exchanger. This
screening can be accomplished by EIA or ELISA using immobilized antigenic
domain or
ligand/receptor specificity exchanger, as described above.
Similarly a subject in need of an agent that inhibits viral infection can be
administered a
ligand/receptor specificity exchanger that recognizes a receptor present on
the particular etiologic
agent. Accordingly, a subject in need of an agent that inhibits viral
infection is identified by
standard clinical or diagnostic procedures. Next, the subject in need is
provided a therapeutically
effective amount of a ligand/receptor specificity exchanger that interacts
with a receptor present on
the type of virus infecting the individual. As above, it may be desired to
determine whether the
subject has a sufficient titer of antibody to interact with the antigenic
domain of the ligand/receptor
specificity exchanger prior to administering the ligand/receptor specificity
exchanger.
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In the same vein, a subject in need of an agent that inhibits the
proliferation of cancer can
be administered a ligand/receptor specificity exchanger that interacts with a
receptor present on the
cancer cell. For example, a subject in need of an agent that inhibits
proliferation of cancer is
identified by standard clinical or diagnostic procedures; then the subject in
need is provided a
therapeutically effective amount of a Iigand/receptor specificity exchanger
that interacts with a
receptor present on the cancer cells infecting the subject. As noted above, it
may be desired to
determine whether the subject has a sufficient titer of antibody to interact
with the antigenic
domain of the ligand/receptor specificity exchanger prior to administering the
ligand/receptor
specificity exchanger.
Ligandlreceptor specificity exchangers described herein can also be
administered to
subjects as a prophylactic to prevent the onset of disease. Virtually anyone
can be administered a
ligand/receptor specificity exchanger described herein for prophylactic
purposes, (e.g., to prevent a
bacterial infection, viral infection, or cancer). It is desired, however, that
subjects at a high risk of
contracting a particular disease are identified and provided a ligand/receptor
specificity exchanger.
Subjects at high risk of contracting a disease include individuals with a
family history of disease ,
the elderly or the young, or individuals that come in frequent contact with a
pathogen (e.g., health
care practitioners). Accordingly, subjects at risk of becoming infected by a
pathogen that has a
receptor are identified and then are provided a prophylactically effective
amount of ligand/receptor
specificity exchanger.
One prophylactic application for the a ligand/receptor specificity exchangers
described
herein concerns coating or cross-Linking the Iigand/receptor specificity
exchanger to a medical
device or implant. Implantable medical devices tend to serve as foci for
infection by a number of
bacterial species. Such device-associated infections are promoted by the
tendency of these
organisms to adhere to and colonize the surface of the device. Consequently,
there is a
considerable need to develop surfaces that are less prone to promote the
adverse biological
reactions that typically accompany the implantation of a medical device.
By one approach, the medical device is coated in a solution of containing a
ligand/receptor
specificity exchanger. Prior to implantation, medical devices (e.g., a
prosthetic valve) can be
stored in a solution of ligand/receptor specificity exchangers, for example.
Medical devices can
also be coated in a powder or gel having a ligand/receptor specificity
exchanger. For example,
gloves, condoms, and intrauterine devices can be coated in a powder or gel
that contains a
specificity exchanger that interacts with a bacterial or viral receptor. Once
implanted in the body,
these ligand/receptor specificity exchangers provide a prophylactic barrier to
infection by a
pathogen.
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In some embodiments, the ligand/receptor specificity exchanger is immobilized
to the
medical device. As described above, the medical device is a support to which a
ligand/receptor
specificity exchanger can be attached. Immobilization may occur by hydrophobic
interaction
between the ligand/receptor specificity exchanger and the medical device but a
preferable way to
immobilize a ligand/receptor specificity exchanger to a medical device
involves covalent
attachment. For example, medical devices can be manufactured with a reactive
group that interacts
with a reactive group present on the specificity exchanger.
By one approach, a periodate is combined with a ligand/receptor specificity
exchanger
comprising a 2-aminoalcohol moiety to form an aldehyde-functional exchanger in
an aqueous
solution having a pH between about 4 and about 9 and a temperature between
about 0 and about 50
degrees Celsius. Next, the aldehyde-functional exchanger is combined with the
biomaterial surface
of a medical device that comprises a primary amine moiety to immobilize the
ligand/receptor
specificity exchanger on the support surface through an imine moiety. Then,
the imine moiety is
reacted with a reducing agent to form an immobilized ligand/receptor
specificity exchanger on the
biomaterial surface through a secondary amine linkage. Other approaches for
cross-linking
molecules to medical devices, (such as described in U.S. Pat. No. 6017741);
can be modified to
immobilize the ligand/receptor specificity exchanger described herein.
Although the invention has been described with reference to embodiments and
examples, it
should be understood that various modifications can be made without departing
from the spirit of
the invention. Accordingly, the invention is limited only by the following
claims.
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SEQUENCE LISTING
<110> TRIPEP AB
SALLBERG, Matti
FLOCK, Jan-Ingmar
<120> LIGAND/RECEPTOR SPECIFICITY EXCHANGERS
THAT REDIRECT ANTIBODIES TO RECEPTORS ON A PATHOGEN
<130> TRIPEP.022VPC
<150> US 09/664,025
<151> 2000-09-19
<160> 145
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 1
Tyr Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln Ala Gly
1 5 10 15
Asp Val
<210> 2
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 2
Met Ser Trp Ser Leu His Pro Arg Asn Leu Ile Leu Tyr Phe Tyr Ala
1 5 10 l5
Leu Leu Phe Leu
<210> 3
<211> 19
<212> PRT
<213> Artificial Sequence
- 1 -
CA 02421877 2003-03-11
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<220>
<223> Specificity domain peptide
<400> 3
Ile Leu Tyr Phe Tyr Ala Leu Leu Phe Leu Ser Thr Cys Val Ala Tyr
1 5 10 15
Val Ala Thr
<210> 4
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 4
Ser Ser Thr Cys Val Ala Tyr Val Ala Thr Arg Asp Asn Cys Cys Ile
1 5 10 15
Leu Asp Glu Arg
<210> 5
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 5
Arg Asp Asn Cys Cys Ile Leu Asp Glu Arg Phe Gly Ser Tyr Cys Pro
1 5 10 15
Thr Thr Cys Gly
<210> 6
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 6
Phe Gly Ser Tyr Cys Pro Thr Thr Cys Gly Ile Ala Asp Phe Leu Ser
1 5 10 15
Thr Tyr Gln Thr
- 2 -
CA 02421877 2003-03-11
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<210> 7
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 7
Ile Ala Asp Phe Leu Ser Thr Tyr Gln Thr Lys Val Asp Lys Asp Leu
1 5 10 15
Gln Ser Leu Glu
<210> 8
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 8
Lys Val Asp Lys Asp Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val
1 5 10 15
Glu Asn Lys Thr
<210> 9
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 9
Asp Ile Leu His Gln Val Glu Asn Lys Thr Ser Glu Val Lys Gln Leu
1 5 10 15
Ile Lys Ala Ile
<210> 10
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 10
Ser Glu Val Lys Gln Leu Ile Lys Ala Ile Gln Leu Thr Tyr Asn Pro
- 3 -
CA 02421877 2003-03-11
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1 5 10 15
Asp Glu Ser Ser
<210> 11
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 11
Gln Leu Thr Tyr Asn Pro Asp Glu Ser Ser Lys Pro Asn Met Ile Asp
1 5 10 15
Ala Ala Thr Leu
<210> 12
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 12
Lys Pro Asn Met Ile Asp Ala Ala Thr Leu Lys Ser Arg Ile Met Leu
1 5 10 15
Glu Glu Ile Met
<210> 13
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 13
Lys Ser Arg Ile Met Leu Glu Glu Ile Met Lys Tyr Glu Ala Ser Ile
1 5 10 15
Leu Thr His Asp
<210> 14
<211> 20
<212> PRT
<213> Artificial Sequence
- 4 -
CA 02421877 2003-03-11
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<220>
<223> Specificity domain peptide
<400> 14
Lys Tyr Glu Ala Ser Ile Leu Thr His Asp Ser Ser Ile Arg Tyr Leu
1 5 10 15
Gln Glu Ile Tyr
<210> 15
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 15
Sex Ser Ile Arg Tyr Leu Gln Glu Ile Tyr Asn Ser Asn Asn Gln Lys
1 5 10 15
Ile Val Asn Leu
<210> 16
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 16
Asn Ser Asn Asn Gln Lys Ile Val Asn Leu Lys Glu Lys Val Ala Gln
1 5 10 15
Leu Glu Ala Gln
<210> 17
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 17
Cys Gln Glu Pro Cys Lys Asp Thr Val Gln Ile His Asp Ile Thr Gly
1 5 10 15
Lys Asp Cys Gln
- 5 -
CA 02421877 2003-03-11
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<210> 18
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 18
Ile His Asp Ile Thr Gly Lys Asp Cys Gln Asp Ile Ala Asn Lys Gly
1 5 10 15
Ala Lys Gln Ser
<210> 19
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 19
Asp Ile Ala Asn Lys Gly Ala Lys Gln Ser Gly Leu Tyr Phe Ile Lys
1 5 10 15
Pro Leu Lys Ala
<210> 20
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 20
Gly Leu Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val
1 5 10 15
Tyr Cys Glu Ile
<210> 21
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 21
Asn Gln Gln Phe Leu Val Tyr Cys Glu Ile Asp Gly Ser Gly Asn Gly
- 6 -
CA 02421877 2003-03-11
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1 5 10 15
Trp Thr Val Phe
<210> 22
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 22
Asp Gly Ser Gly Asn Gly Trp Thr Val Phe Gln Lys Arg Leu Asp Gly
1 5 10 15
Ser Val Asp Phe
<210> 23
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 23
Gln Lys Arg Leu Asp Gly Ser Val Asp Phe Lys Lys Asn Trp Ile Gln
1 5 10 15
Tyr Lys Glu Gly
<210> 24
<211> 20
<212> PRT
<2l3> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 24
Lys Lys Asn Trp Ile Gln Tyr Lys Glu Gly Phe Gly His Leu Ser Pro
1 5 10 15
Thr Gly Thr Thr
<210> 25
<211> 20
<212> PRT
<213> Artificial Sequence
CA 02421877 2003-03-11
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<220>
<223> Specificity domain peptide
<400> 25
Phe Gly His Leu Ser Pro Thr Gly Thr Thr Glu Phe Trp Leu Gly Asn
1 5 10 15
Glu Lys Ile His
<210> 26
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 26
Glu Phe Trp Leu Gly Asn Glu Lys Ile His Leu Ile Ser Thr Gln Ser
1 5 10 15
Ala Ile Pro Tyr
<210> 27
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 27
Leu Ile Ser Thr Gln Ser Ala Ile Pro Tyr Ala Leu Arg Val Glu Leu
1 5 10 15
Glu Asp Trp Asn
<210> 28
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
~400> 28
Ala Leu Arg Val Glu Leu Glu Asp Trp Asn Gly Arg Thr Ser Thr Ala
1 5 10 15
Asp Tyr Ala Met
_ g _
CA 02421877 2003-03-11
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<210> 29
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 29
Gly Arg Thr Ser Thr Ala Asp Tyr Ala Met Phe Lys Val Gly Pro Glu
1 5 10 15
Ala Asp Lys Tyr
<210> 30
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 30
Phe Lys Val Gly Pro Glu Ala Asp Lys Tyr Arg Leu Thr Tyr Ala Tyr
1 5 10 15
Phe Ala Gly Gly
<210> 31
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400>. 31
Arg Leu Thr Tyr Ala Tyr Phe Ala Gly Gly Asp Ala Gly Asp Ala Phe
1 5 10 15
Asp Gly Phe Asp
<210> 32
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 32
Asp Ala Gly Asp Ala Phe Asp Gly Phe Asp Phe Gly Asp Asp Pro Ser
- 9 -
CA 02421877 2003-03-11
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1 5 10 15
Asp Lys Phe Phe
<210> 33
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 33
Phe Gly Asp Asp Pro Ser Asp Lys Phe Phe Thr Ser His Asn Gly Met
1 5 10 15
Gln Phe Ser Thr
<210> 34
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 34
Thr Ser His Asn Gly Met Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp
1 5 20 15
Lys Phe Glu Gly
<210> 35
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 35
Trp Asp Asn Asp Asn Asp Lys Phe Glu Gly Asn Cys Ala Glu Gln Asp
1 5 10 15
Gly Ser Gly Trp
<210> 36
<21l> 20
<212> PRT
<213> Artificial Sequence
- 10 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<220>
<223> Specificity domain peptide
<400> 36
Asn Cys Ala Glu Gln Asp Gly Ser Gly Trp Trp Met Asn Lys Cys His
1 5 10 I5
Ala Gly His Leu
<210> 37
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 37
Trp Met Asn Lys Cys His Ala Gly His Leu Asn Gly Val Tyr Tyr Gln
1 5 10 15
Gly Gly Thr Tyr
<210> 38
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 38
Asn Gly Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala Ser Thr Pro
1 5 10 15
Asn Gly Tyr Asp
<210> 39
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 39
Ser Lys Ala Ser Thr Pro Asn Gly Tyr Asp Asn Gly Ile Ile Trp Ala
1 5 10 15
Thr Trp Lys Thr
- 11 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<210> 40
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 40
Asn Gly Ile Ile Trp Ala Thr Trp Lys Thr Arg Trp Tyr Ser Met Lys
1 5 10 15
Lys Thr Thr Met
<210> 41
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 41
Arg Trp Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Tle Pro Phe Asn
1 5 10 15
Arg Leu Thr Ile
<210> 42
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Specificity domain peptide
<400> 42
Lys Ile Ile Pro Phe Asn Arg Leu Thr Ile Gly Glu Gly Gln Gln His
1 5 10 15
His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val
20 25
<210> 43
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 43
Gly Leu Tyr Ser Ser Ile Trp Leu Ser Pro Gly Arg Ser Tyr Phe Glu
- 12 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
I 5 10 15
Thr
<210> 44
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 44
Tyr Thr Asp Ile Lys Tyr Asn Pro Phe Thr Asp Arg Gly Glu Gly Asn
1 5 10 15
Met
<210> 45
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 45
Asp Gln Asn Ile His Met Asn Ala Arg Leu Leu Ile Arg Ser Pro Phe
1 5 10 15
Thr
<210> 46
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 46
Leu Ile Arg Ser Pro Phe Thr Asp Pro Gln Leu Leu Val His Thr Asp
1 5 10 15
Pro
<210> 47
<211> 17
<212> PRT
<213> Artificial Sequence
- 13 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<220>
<223> Antigenic domain peptide
<400> 47
Gln Lys Glu Ser Leu Leu Phe Pro Pro Val Lys Leu Leu Arg Arg Val
1 5 10 15
Pro
<210> 48
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 48
Pro Ala Leu Thr Ala Val Glu Thr Gly Ala Thr
1 5 10
<210> 49
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 49
Ser Thr Leu Val Pro Glu Thr Thr
1 5
<210> 50
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 50
Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu
1 5 10
<210> 51
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
- 14 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<223> Antigenic domain peptide
<400> 51
Glu Ile Pro Ala Leu Thr Ala Val Glu
1 5
<2l0> 52
<2l1> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 52
Leu Glu Asp Pro Ala Ser Arg Asp Leu Val
1 5 10
<210> 53
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 53
His Arg Gly Gly Pro Glu Glu Phe
1 5
<220> 54
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 54
His Arg Gly Gly Pro Glu Glu
Z 5
<210> 55
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 55
- 15 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
Val Leu Ile Cys Gly Glu Asn Thr Val Ser Arg Asn Tyr Ala Thr His
1 5 10 15
Ser
<210> 56
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 56
Lys Ile Asn Thr Met Pro Pro Phe Leu Asp Thr Glu Leu Thr Ala Pro
1 5 10 15
Ser
<210> 57
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigenic domain peptide
<400> 57
Pro Asp Glu Lys Ser Gln Arg Glu Ile Leu Leu Asn Lys Ile Ala Ser
1 5 10 15
Tyr
<210> 58
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> .Antigenic domain peptide
<400> 58
Thr Ala Thr Thr Thr Thr Tyr Ala Tyr Pro Gly Thr Asn Arg Pro Pro
1 5 10 15
Val
<210> 59
<211> 8
<212> PRT
<213> Artificial Sequence
- 16 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<220>
<223> Antigenic domain peptide
<400> 59
Ser Thr Pro Leu Pro Glu Thr Thr
1 5
<220> 60
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 60
Tyr Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln Ala Gly
1 5 10 15
Asp Val His Arg Gly Gly Pro Glu Glu Phe
20 25
<210> 61
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 61
Tyr Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys. Gln Ala Gly
1 5 10 15
Asp Val His Arg Gly Gly Pro Glu Glu
20 25
<210> 62
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 62
Tyr Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln Ala Gly
1 5 10 15
Asp Val Ser Thr Pro Leu Pro Glu Thr Thr
20 25
<210> 63
- 17 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 63
Met Ser Trp Ser Leu His Pro Arg Asn Leu Ile Leu Tyr Phe Tyr Ala
1 5 10 Z5
Leu Leu Phe Leu His Arg Gly Gly Pro Glu Glu
20 25
<210> 64
<211> 26
<212> PRT
<2I3> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 64
Ile Leu Tyr Phe Tyr Ala Leu Leu Phe Leu Ser Thr Cys Val Ala Tyr
1 5 10 15
Val AIa Thr His Arg Gly Gly Pro Glu Glu
20 25
<210> 65
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 65
Ser Ser Thr Cys Val Ala Tyr Val Ala Thr Arg Asp Asn Cys Cys Ile
1 5 10 15
Leu Asp Glu Arg His Arg Gly Gly Pro Glu Glu
20 25
<210> 66
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 66
Arg Asp Asn Cys Cys Ile Leu Asp Glu Arg Phe Gly Ser Tyr Cys Pro
1 5 10 15
- 18 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
Thr Thr Cys Gly His Arg Gly Gly Pro Glu Glu
20 25
<210> 67
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 67
Phe Gly Ser Tyr Cys Pro Thr Thr Cys Gly Ile Ala Asp Phe Leu Ser
1 5 10 15
Thr Tyr Gln Thr His Arg Gly Gly Pro Glu Glu
20 25
<210> 68
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 68
Ile Ala Asp Phe Leu Ser Thr Tyr Gln Thr Lys Val Asp Lys Asp Leu
1 5 10 15
Gln Ser Leu Glu His Arg Gly Gly Pro Glu Glu
20 25
<210> 69
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 69
Lys Val Asp Lys Asp Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val
1 5 10 15
Glu Asn Lys Thr His Arg Gly Gly Pro Glu Glu
20 25
<210> 70
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
- 19 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<223> Ligand/Receptor specificity exchanger peptide
<400> 70
Asp Ile Leu His Gln Val Glu Asn Lys Thr Ser Glu Val Lys Gln Leu
1 5 10 15
Ile Lys Ala Ile His Arg Gly Gly Pro Glu Glu
20 25
<210> 71
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 71
Ser Glu Val Lys Gln Leu Ile Lys Ala Ile Gln Leu Thr Tyr Asn Pro
1 5 10 15
Asp Glu Ser Ser His Arg Gly Gly Pro Glu Glu
20 25
<210> 72
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 72
Gln Leu Thr Tyr Asn Pro Asp Glu Ser Ser Lys Pro Asn Met Ile Asp
1 5 10 15
Ala Ala Thr Leu His Arg Gly Gly Pro Glu Glu
20 25
<210> 73
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 73
Lys Pro Asn Met Ile Asp Ala Ala Thr Leu Lys Ser Arg Ile Met Leu
1 5 10 15
Glu Glu Ile Met His Arg Gly Gly Pro Glu Glu
20 25
<210> 74
- 20 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 74
Lys Ser Arg Ile Met Leu Glu Glu Ile Met Lys Tyr Glu Ala Ser Ile
1 5 10 15
Leu Thr His Asp His Arg Gly Gly Pro Glu Glu
20 25
<210> 75
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 75
Lys Tyr Glu Ala Ser Ile Leu Thr His Asp Ser Ser Ile Arg Tyr Leu
1 5 10 15
Gln Glu Ile Tyr His Arg Gly Gly Pro Glu Glu
20 25
<210> 76
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 76
Ser Ser Ile Arg Tyr Leu Gln Glu Ile Tyr Asn Ser Asn Asn Gln Lys
1 5 10 15
Ile Val Asn Leu His Arg Gly Gly Pro Glu Glu
20 25
<210> 77
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 77
Asn Ser Asn Asn Gln Lys Ile Val Asn Leu Lys Glu Lys Val Ala Gln
1 5 10 15
- 21 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
Leu Glu Ala Gln His Arg Gly Gly Pro Glu Glu
20 25
<210> 78
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 78
Cys Gln Glu Pro Cys Lys Asp Thr Val Gln Ile His Asp Ile Thr Gly
1 5 10 15
Lys Asp Cys Gln His Arg Gly Gly Pro Glu Glu
20 25
<210> 79
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 79
Ile His Asp Ile Thr Gly Lys Asp Cys Gln Asp Ile Ala Asn Lys Gly
1 5 10 15
Ala Lys Gln Ser His Arg Gly Gly Pro Glu Glu
20 25
<210> 80
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 80
Asp Tle Ala Asn Lys Gly Ala Lys Gln Ser Gly Leu Tyr Phe Ile Lys
1 5 10 15
Pro Leu Lys Ala His Arg Gly Gly Pro Glu Glu
20 25
<210> 81
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
- 22
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<223> Ligand/Receptor specificity exchanger peptide
<400> 81
Gly Leu Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val
1 5 10 15
Tyr Cys Glu Tle His Arg Gly Gly Pro Glu Glu
20 25
<210> 82
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 82
Asn Gln Gln Phe Leu Val Tyr Cys Glu Ile Asp Gly Ser Gly Asn Gly
1 5 10 15
Trp Thr Val Phe His Arg Gly Gly Pro Glu Glu
20 25
<210> 83
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 83
Asp Gly Ser Gly Asn Gly Trp Thr Val Phe Gln Lys Arg Leu Asp Gly
1 5 10 15
Ser Val Asp Phe His Arg Gly Gly Pro Glu Glu
20 25
<210> 84
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 84
Gln Lys Arg Leu Asp Gly Ser Val Asp Phe Lys Lys Asn Trp Ile Gln
1 5 l0 15
Tyr Lys Glu Gly His Arg Gly Gly Pro Glu Glu
20 25
<210> 85
- 23 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 85
Lys Lys Asn Trp Ile Gln Tyr Lys Glu Gly Phe Gly His Leu Ser Pro
1 5 10 15
Thr Gly Thr Thr His Arg Gly Gly Pro Glu Glu
20 25
<210> 86
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 86
Phe Gly His Leu Ser Pro Thr Gly Thr Thr Glu Phe Trp Leu Gly Asn
1 5 10 15
Glu Lys Ile His His Arg Gly Gly Pro Glu Glu
20 25
<210> 87
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 87
Glu Phe Trp Leu Gly Asn Glu Lys Ile His Leu Ile Ser Thr Gln Ser
1 5 10 15
Ala Ile Pro Tyr His Arg Gly Gly Pro Glu Glu
20 25
<210> 88
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 88
Leu Ile Ser Thr Gln Ser Ala Ile Pro Tyr Ala Leu Arg Val Glu Leu
1 5 10 15
- 24 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
Glu Asp Trp Asn His Arg Gly Gly Pro Glu Glu
20 25
<210> 89
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 89
Ala Leu Arg Val Glu Leu Glu Asp Trp Asn Gly Arg Thr Ser Thr Ala
1 5 20 15
Asp Tyr Ala Met His Arg Gly Gly Pro Glu Glu
20 25
<210> 90
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 90
Gly Arg Thr Ser Thr Ala Asp Tyr Ala Met Phe Lys Val Gly Pro Glu
1 5 10 l5
Ala Asp Lys Tyr His Arg Gly Gly Pro Glu Glu
20 25
<210> 91
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 91
Phe Lys Val Gly Pro Glu Ala Asp Lys Tyr Arg Leu Thr Tyr Ala Tyr
1 5 10 15
Phe Ala Gly Gly His Arg Gly Gly Pro Glu Glu
20 25
<210> 92
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
- 25 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<223> Ligand/Receptor specificity exchanger peptide
<400> 92
Arg Leu Thr Tyr Ala Tyr Phe Ala Gly GIy Asp Ala Gly Asp Ala Phe
1 5 10 15
Asp Gly Phe Asp His Arg Gly Gly Pro Glu Glu
20 25
<210> 93
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 93
Asp Ala Gly Asp Ala Phe Asp Gly Phe Asp Phe Gly Asp Asp Pro Ser
1 5 10 15
Asp Lys Phe Phe His Arg Gly Gly Pro Glu Glu
20 25
<210> 94
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 94
Phe GIy Asp Asp Pro Ser Asp Lys Phe Phe Thr Ser His Asn Gly Met
1 5 10 15
Gln Phe Ser Thr His Arg Gly Gly Pro Glu Glu
20 25
<210> 95
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 95
Thr Ser His Asn Gly Met Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp
1 5 10 15
Lys Phe Glu Gly His Arg Gly Gly Pro Glu Glu
20 25
<210> 96
- 26 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 96
Trp Asp Asn Asp Asn Asp Lys Phe Glu Gly Asn Cys Ala Glu Gln Asp
1 5 10 15
Gly Ser Gly Trp His Arg Gly Gly Pro Glu Glu
20 25
<210> 97
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 97
Asn Cys Ala Glu Gln Asp Gly Ser Gly Trp Trp Met Asn Lys Cys His
1 5 10 15
Ala Gly His Leu His Arg Gly Gly Pro Glu Glu
20 25
<210> 98
<211> 27
<212> FRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 98
Trp Met Asn Lys Cys His Ala Gly His Leu Asn Gly Val Tyr Tyr Gln
1 5 10 15
Gly Gly Thr Tyr His Arg Gly Gly Pro Glu Glu
20 25
<210> 99
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 99
Asn Gly Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala Ser Thr Pro
1 5 10 15
- 27 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
Asn Gly Tyr Asp His Arg Gly Gly Pro Glu Glu
20 25
<210> 100
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 100
Ser Lys Ala Ser Thr Pro Asn Gly Tyr Asp Asn Gly Ile Ile Trp Ala
1 5 10 15
Thr Trp Lys Thr His Arg Gly Gly Pro Glu Glu
20 25
<210> 101
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/Receptor specificity exchanger peptide
<400> 101
Asn Gly Ile Ile Trp Ala Thr Trp Lys Thr Arg Trp Tyr Ser Met Lys
1 5 10 15
Lys Thr Thr Met His Arg Gly Gly Pro Glu Glu
20 25
<210> 102
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> LigandJReceptor specificity exchanger peptide
<400> 102
Arg Trp Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro Phe Asn
1 5 10 15
Arg Leu Thr Ile His Arg Gly Gly Pro Glu Glu
20 25
<210> 103
<2l1> 34
<212> PRT
<213> Artificial Sequence
<220>
_ 28 _
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<223> Ligand/Receptor specificity exchanger peptide
<400> 103
Lys Ile Ile Pro Phe Asn Arg Leu Thr Ile Gly Glu Gly Gln Gln His
1 5 10 Z5
His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val His Arg Gly Gly Pro
20 25 30
Glu Glu
<210> 104
<211> 13
<212> PRT
<213> IArtificial Sequence
<220>
<223> Integrin specific ligand/receptor specificity
exchanger peptide
<400> 104
Gly Arg Gly Asp Ser Pro His Arg Gly Gly Pro Glu Glu
1 5 10
<210> 105
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Integrin specific ligand/receptor specificity
exchanger peptide
<400> 105
Trp Ser Arg Gly Asp Trp His Arg Gly Gly Pro Glu Glu
1 5 10
<210> 106
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 106
Leu Thr Ile Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln
1 5 10 15
Ala Gly Asp Val
<210> 107
- 29 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 107
Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln Ala Gly Asp
1 5 10 15
Val
<210> 108
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 108
Gln Gln His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val
1 5 10
<210> 109
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 109
Gln His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val
1 5 10
<210> 110
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 110
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val
1 5 l0
<210> 111
<211> 11
- 30 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 111
His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val
1 5 10
<210> 112
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 112
Leu Gly Gly Ala Lys Gln Ala Gly Asp Val
1 5 10
<210> 113
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 113
Gly Gly Ala Lys Gln Ala Gly Asp Val
1 5
<210> 114
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 114
Gly Ala Lys Gln Ala Gly Asp Val
1 5
<210> 115
<211> 12
<212> PRT
<213> Artificial Sequence
- 31 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<220>
<223> Fibrinogen Peptide
<400> 115
Gln His His Leu Gly Gly Ala Lys Gln Ala Gly Asp
1 5 10
<210> 116
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 116
Gln His His Leu Gly Gly Ala Lys Gln Ala Gly
1 5 10
<210> 117
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 117
Gln His His Leu Gly Gly Ala Lys Gln Ala
1 5 10
<210> 118
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 118
Gln His His Leu Gly Gly Ala Lys Gln
1 5
<210> 119
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
- 32 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<400> 119
Gln His His Leu Gly Gly Ala Lys
1 5
<210> 120
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 120
Gln His His Leu Gly Gly Ala
1 5
<210> 121
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 121
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val
1 5 10
<210> 122
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 122
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp
1 5 10
~210> 123
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 123
His His Leu Gly Gly Ala Lys Gln Ala G1y
1 5 to
- 33 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<2I0> I24
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 124
His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val
1 5 10
<210> 125
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 125
His Leu Gly Gly Ala Lys Gln Ala Gly Asp
1 5 10
<210> 126
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 126
Ala Leu Gly Gly Ala Lys Gln Ala Gly
1 5
<210> 127
<2l1> 9
<212> PRT
<2l3> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 127
His Ala Gly Gly Ala Lys Gln Ala Gly
1 5
<210> 128
- 34 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 128
His Leu Ala Gly Ala Lys Gln Ala Gly
1 5
<210> 129
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 129
His Leu Gly Ala Ala Lys Gln Ala Gly
1 5
<210> 130
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 130
His Leu Gly Gly Gly Lys Gln Ala Gly
1 5
<210> 131
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 131
His Leu Gly Gly Ala Ala Gln Ala Gly
1 5
<210> 132
<211> 9
<212> PRT
<213> Artificial Sequence
- 35 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<220>
<223> Fibrinogen Peptide
<400> 132
His Leu Gly Gly Ala Lys Ala Ala Gly
1 5
<210> 133
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 133
His Leu GIy GIy Ala Lys Gln Gly Gly
1 5
<210> 134
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Fibrinogen Peptide
<400> 134
His Leu Gly Gly Ala Lys Gln Ala Ala
1 5
<210> 135
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/receptor specificity exchanger peptide;
cyclized between cystiene residues
<400> 135
Cys Pro Ala Leu Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu Ala
1 5 ~ 10 15
Ala His His Leu Gly Gly Ala Lys Gln Ala Gly
20 25
<210> 136
<211> 27
<212> PRT
<213> Artificial Sequence
- 36 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<220>
<223> Ligand/receptor specificity exchanger,peptide
<400> 136
Cys Pro Ala Leu Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu Ala
1 5 10 15
Ala His His Leu Gly Gly Ala Lys Gln Ala Gly
20 25
<210> 137
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/receptor specificity exchanger peptide;
cyclized between cystiene residues
<400> 137
His His Leu Gly Gly Ala Lys Gln Ala Gly Ala Ala Cys Pro Ala Leu
1 5 10 15
Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu
20 25
<210> 138
<2l1> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/receptor specificity exchanger peptide
<400> 138
His His Leu Gly Gly Ala Lys Gln Ala Gly Ala Ala Cys Pro Ala Leu
1 5 10 15
Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu
20 25
<210> 139
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/receptor specificity exchanger peptide;
cyclized between cystiene residues
<400> 139
Cys Pro Ala Leu Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu His
1 5 10 15
His Leu Gly Gly Ala Lys Gln Ala Gly
- 37 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
20 25
<210> 140
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/receptor specificity exchanger peptide
<400> 140
Cys Pro Ala Leu Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu His
1 5 10 15
His Leu Gly Gly Ala Lys Gln Ala Gly
20 25
<210> 141
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/receptor specificity exchanger peptide;
cyclized between cystiene residues
<400> 141
His His Leu Gly Gly Ala Lys Gln Ala Gly Cys Pro Ala Leu Thr Ala
1 5 10 15
Val Glu Thr Gly Cys Thr Asn Pro Leu
20 25
<210> 142
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/receptor specificity exchanger peptide
<400> 142
His His Leu Gly Gly Ala Lys Gln Ala Gly Cys Pro Ala Leu Thr Ala
1 5 10 15
Val Glu Thr Gly Cys Thr Asn Pro Leu
20 25
<210> 143
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
- 38 -
CA 02421877 2003-03-11
WO 02/24887 PCT/IBO1/02327
<223> Ligand/receptor specificity exchanger peptide
<400> 143
Pro Ala Leu Thr Ala Val Glu Thr Gly Ala Thr Asn Pro Leu His His
1 5 10 15
Leu Gly Gly Ala Lys Gln Ala Gly
<210> 144
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Ligand/receptor specificity exchanger peptide
<400> 144
His His Leu Gly Gly Ala Lys Gln Ala Gly Pro Ala Leu Thr Ala Val
1 5 10 15
Glu Thr Gly Ala Thr Asn Pro Leu
<210> 145
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> Integrin specificity peptide
<400> 145
Arg Gly Asp Ser Ala Ala Thr Pro Pro Ala Tyr Arg
1 5 10
- 39 -
