Note: Descriptions are shown in the official language in which they were submitted.
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PdIAl1/nVLAT IAN CHl DELETED NQMETIBODIES,
COMPOSITIONS, METHODS AND USES
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates to mammalian CH1-deleted mimetibodies, specified
portions and
variants specific for bologically active proteins, fragment or ligands, CH1-
deleted mimetibody
encoding and complementary nucleic acids, host cells, and methods of making
and using thereof,
including therapeutic formulations, administration and devices.
RELATED ART
Recombinant proteins are an emerging class of therapeutic agents. Such
recombinant
therapeutics have engendered advances in protein formulation and chemical
modification. Such
modifications can potentially enhance the therapeutic utility of therapeutic
proteins, such as by
increaseing half lives (e.g., by blocking their exposure to proteolytic
enzymes), enhancing biological
activity, or reducing unwanted side effects. One such modification is the use
of immunoglobulin
fragments fused to receptor proteins, such as enteracept. Therapeutic proteins
have also been
constructed using the Fc domain to attempt to provide a longer half life or to
incorporate functions
such as Fc receptor binding, protein A binding, and complement fixation.
Accordingly, there is a need to provide improved and/or modified versions of
therapeutic
2 0 proteins, which overcome one more of these and other problems known in the
art.
SUMMARY OF THE INVENTION
The present invention provides isolated human CHl-deleted mimetibodies,
including modified
immunoglobulins, cleavage products and other specified portions and variants
thereof, as well as CHl-
2 5 deleted mimetibody compositions, encoding or complementary nucleic acids,
vectors, host cells,
compositions, formulations, devices, transgenic animals, transgenic plants,
and methods of making and
using thereof, as described and/or enabled herein, in combination with what is
known in the art.
The present invention also provides at least one isolated CH1-deleted
mimetibody or specified
portion or variant as described herein and/or as known in the art. The CH1
deleted mimetibody can
3 0 optionally comprise at least one CH3 region directly linked with at least
one CH2 region directly
linked with at least one hinge region or fragment thereof directly linleed
with at least one partial V
region, directly linked with an optional linker sequence, directly linked to
at least one therapeutic
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peptide, optionally further directly linked with at least a portion of at
least one variable antibody
sequence. In a preferred embodiment a pair of a CH3-CH2-hinge-partial J
sequence-linker-therapeutic
peptide with an option N-terminal antibody sequence, the pair optionally
linked by association or
covalent linkage, such as, but not limited to, a Cys-Cys disulfide bond. In
one embodiment, a CHl
deleted mimetibody comprises formula (I):
(V 1 (n)-Pep(n)-Flex(n)-V2(n)-pHinge(n)-CH2(n)-CH3 (n))(m),
where V 1 is at least one portion of an N-terminus of an immunoglobulin
variable region, Pep is at least
one bioactive peptide, Flex is polypeptide that provides structural flexablity
by allowing the
mimietibody to have alternative orientations and binding properties, V2 is at
least one portion of a C-
terminus of an immunoglobulin variable region, pHinge is at least a portion of
an immunoglobulin
variable hinge region, CH2 is at least a portion of an immunoglobulin CH2
constant region, CH3 is at
least a portion of an immunoglobulin CH3 constant region, n and m can be an
integer between 1 and
10, mimicing different types of immunoglobulin molecules, e.g., but not
limited to IgGl, IgG2, IgG3,
IgG4, IgA, IgM, IgD, IgE, and the like, or combination thereof.
Thus, a CHl-deleted mirnetibody of the present invention mimics at least a
portion of an
antibody or immnuoglobulin structure or function with its inherent properties
and functions, while
providing a therapeutic peptide and its inherent or acquired in vitro, in vivo
or in situ properties or
activities. The various portions of the antibody and therapeutic peptide
portions of at least one CHl-
deleted mimetibody of the present invention can vary as described herein in
combinatoin with what is
2 0 known in the art.
At least one CH1-deleted mimetibody or specified portion or variant of the
invention mimics
the binding of the Pep portion of the mimetibody to at least one ligand, or
has at least one biological
activity of, at least one protein, subunit, fragment, portion or any
combination thereof.
The present invention also provides at least one isolated CHl-deleted
mimetibody or specified
2 5 portion or variant as described herein and/or as known in the art, wherein
the CH1-deleted mimetibody
or specified portion or variant has at least one activity, such as, but not
limited to known biological
activities of at least one bioactive peptide or polypeptide corresponding to
the Pep portion of formula I.
A CHl-deleted mimetibody can thus be screened for a corresponding activity
according to known
methods, such as at least one neutralizing activity towards a protein or
fragment thereof.
3 0 In one aspect, the present invention provides at least one isolated
mammalian CH1-deleted
mimetibody, comprising at least one Pep(n) region comprising at least a
bilogically active portion of at
least one of SEQ ID NOS:l-979, or optionally with one or more substitutions,
deletions or insertions as
described herein and/or as known in the art.
In another aspect, the present invention provides at least one isolated
mammalian CHl-deleted
3 5 mimetibody, wherein the CHl-deleted mimetibody specifically binds at least
one epitope comprising at
2
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least 1-3, to the entire amino acid sequence of at least one ligand or binding
region which ligand binds
to at least a portion of at least one of SEQ ID NOS: 1-1109, or optionally
with one or more
substitutions, deletions or insertions as described herein or as known in the
art.
The at least one CHl-deleted mimetibody can optionally further comprise at
least one
characteristic selected from (i) bind at least one protein with an affinity of
at least 10'9 M, at least 10'10
M, at least 10'11 M, or at least 10'12 M; and/or (ii) substantially neutralize
at least one activity of at least
one protein or portion thereof.
The present invention provides, in one aspect, isolated nucleic acid molecules
comprising,
complementary, having significant identity or hybridizing to, a polynucleotide
encoding specific
mimetibodies or specified portions or variants thereof, comprising at least
one specified sequence,
domain, portion or variant thereof. The present invention further provides
recombinant vectors
comprising at least one of said isolated CHl-deleted mimetibody nucleic acid
molecules, host cells
containing such nucleic acids and/or recombinant vectors, as well as methods
of making and/or using
such CH1-deleted mimetibody nucleic acids, vectors and/or host cells.
Also provided is an isolated nucleic acid encoding at least one isolated
mammalian CHl-
deleted mimetibody; an isolated nucleic acid vector comprising the isolated
nucleic acid, and/or a
prokaryotic or eukaryotic host cell comprising the isolated nucleic acid. The
host cell can optionally
be at least one selected from COS-1, COS-7, HEI~2,93, BHK21, CHO, BSC-1, Hep
G2, 653, SP2/0, 293,
HeLa, myeloma, or lymphoma cells, or any derivative, immortalized or
transformed cell thereof. Also
2 0 provided is a method for producing at least one CHl-deleted mimetibody,
comprising translating the
CHl-deleted mimetibody encoding nucleic acid under conditions in vitro, in
vivo or in situ, such that
the CHl-deleted mimetibody is expressed in detectable or recoverable amounts.
The present invention also provides at least one composition comprising (a) an
isolated CHl-
deleted mimetibody or specified portion or variant encoding nucleic acid
and/or CH1-deleted
2 5 mimetibody as described herein; and (b) a suitable carrier or diluent. The
carrier or diluent can
optionally be pharmaceutically acceptable, according to known methods. The
composition can
optionally further comprise at least one further compound, protein or
composition.
Also provided is a composition comprising at least one isolated mammalian CHl-
deleted
mimetibody and at least one pharmaceutically acceptable carrier or diluent.
The composition can
3 0 optionally further comprise an effective amount of at least one compound
or protein selected from at
least one of a detectable label or reporter, an anti-infective drug, a
cardiovascular (CV) system drug, a
central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a
respiratory tract drug,
a gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or
electrolyte balance, a
hematologic drug, an antineoplactic, an immunomodulation drug, an ophthalmic,
otie or nasal drug, a
3
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topical drug, a nutritional drug, a TNF antagonist, an antirheumatic, a muscle
relaxant, a narcotic, a
non-steroid anti-inflammatory drug (LATHE), an analgesic, an anesthetic, a
sedative, a local anethetic, a
neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod,
an anabolic steroid, an
erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a
growth hormone, a
hormone replacement drug, a radiopharmaceutical, an antidepressant, an
antipsychotic, a stimulant, an
asthma medication, a beta agonist, an inhaled steroid, an epinephrine or
analog, a cytokine, or a
cytokine antagonist.
The present invention further provides at least one anti-idiotype antibody to
at least one CHl-
deleted mimetibody of the present invention. The anti-idiotype antibody
includes any protein or
peptide containing molecule that comprises at Least a portion of an
immunoglobulin molecule, such as
but not limited to at least one complimetarity determing region (CDR) of a
heavy or light chain or a
ligand binding portion thereof, a heavy chain or light chain variable region,
a heavy chain or light chain
constant region, a framework region, or any portion thereof, that can be
incorporated into a CHl-
deleted mimetibody of the present invention. A CH1-deleted mimetibody of the
invention can include
or be derived from any mammal, such as but not limited to a human, a mouse, a
rabbit, a rat, a rodent, a
primate, and the like.
The present invention further provides an anti-idiotype antibody or fragment
that specifically
binds at least one CH1 deleted mimetibody of the present invention.
The present invention provides, in one aspect, isolated nucleic acid molecules
comprising,
2 0 complementary, or hybridizing to, a polynucleotide encoding at least one
CH1-deleted mimetibody
anti-idiotype antibody, comprising at least one specified sequence, domain,
portion or variant thereof.
The present invention further provides recombinant vectors comprising said CHl-
deleted mimetibody
anti-idiotype antibody encoding nucleic acid molecules, host cells containing
such nucleic acids and/or
recombinant vectors, as well as methods of making and/or using such anti-
idiotype antiobody nucleic
2 5 acids, vectors and/or host cells.
The present invention also provides at least one method for expressing at
least one CH1-
deleted mimetibody or specified portion or variant, or CH1-deleted mimetibody
anti-idiotype antibody,
in a host cell, comprising culturing a host cell as described herein and/or as
known in the art under
conditions wherein at least one CHl-deleted rnimetibody or specified portion
or variant, or anti-
3 0 idiotype antibody is expressed in detectable and/or recoverable amounts.
The present invention further provides at least one CHl-deleted mimetibody,
specified portion
or variant in a method or composition, when administered in a therapeutically
effective amount, for
modulation, for treating or reducing the symptoms of at least one of a bone
and joint disorder,
cardiovascular disoder, a dental or oral disorder, a dermatologic disorder, an
ear, nose or throat
3 5 disorder, an endocrine or metabolic disorder, a gastrointestinal disorder,
a gynecologic disorder, a
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hepatic or biliary disorder, a an obstetric disorder, a hematologic disorder,
an immunologic or allergic
disorder, an infectious disease, a museuloskeletal disorder, a oncologic
disorder, a neurologic disorder,
a nutritrional disorder, an opthalmologic disorder, a pediatric disorder, a
poisoning disorder, a
psychiatric disorder, a renal disorder, a pulmonary disorder, or any other
known disorder. (See., e.g.,
The Merck Manual, 17th ed. , Merck Research Laboratories, Merck and Co.,
Whitehouse Station, NJ
(1999), entirely incoporated herein by reference), as needed in many different
conditions, such as but
not limited to, prior to, subsequent to, or during a related disease or
treatment condition, as known in
the art.
The present invention further provides at least one CHl-deleted mimetibody,
specified portion
or variant in a method or composition, when administered in a therapeutically
effective amount, for
modulation, for treating or reducing the symptoms of, at least one immune,
cardiovascular, infectious,
malignant, and/or neurologic disease in a cell, tissue, organ, animal or
patient and/or, as needed in
many different conditions, such as but not limited to, prior to, subsequent
to, or during a related disease
or treatment condition, as known in the art and/or as described herein.
The present invention also provides at least one composition, device and/or
method of delivery
of a therapeutically or prophylactically effective amount of at least one CHl-
deleted mimetibody or
specified portion or variant, according to the present invention.
The present invention also provides at least one composition comprising (a) an
isolated CHl-
deleted mimetibody encoding nucleic acid and/or CH1-deleted mimetibody as
described herein; and (b)
2 0 a suitable carrier or diluent. The carrier or diluent can optionally be
pharmaceutically acceptable,
according to known carriers or diluents. The composition can optionally
further comprise at least one
further compound, protein or composition.
The present invention further provides at least one CHl-deleted mimetibody
method or
composition, for administering a therapeutically effective amount to modulate
or treat at least one
2 5 protein related condition in a cell, tissue, organ, animal or patient
andlor, prior to, subsequent to, or
during a related condition, as known in the art and/or as described herein.
The present invention also provides at least one composition, device and/or
method of delivery
of a therapeutically or prophylactically effective amount of at least one CH1-
deleted mimetibody,
according to the present invention.
3 0 The present invention further provides at least one CH1-deleted mimetibody
method or
composition, for diagnosing at least one protein related condition in a cell,
tissue, organ, animal or
patient and/or, prior to, subsequent to, or during a related condition, as
known in the art and/or as
described herein.
The present invention also provides at least one composition, device and/or
method of delivery
3 5 for diagnosing of at least one CHl-deleted mimetibody, according to the
present invention.
5
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Also provided is a method for diagnosing or treating a disease condition in a
cell, tissue, organ
or animal, comprising
(a) contacting or administering a composition comprising an effective amount
of at least one
isolated mammalian CHl-deleted mimetibody of the invention with, or to, the
cell, tissue, organ or
animal. The method can optionally further comprise using an effective amount
of 0.001-50
mg/kilogram of the cells, tissue, organ or animal. The method can optionally
further comprise using the
contacting or the administrating by at least one mode selected from
parenteral, subcutaneous,
intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular,
intracartilaginous, intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,
intrapelvic, intrapericardiac,
intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal,
intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal,
sublingual, intranasal, or transdermal. The method can optionally further
comprise administering, prior,
concurrently or after the (a) contacting or administering, at least one
composition comprising an
effective amount of at least one compound or protein selected from at least
one of a detectable label or
reporter, an anti-infective drug, a cardiovascular (CV) system drug, a central
nervous system (CNS)
drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, a
gastrointestinal (GI) tract
drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic
drug, an antineoplactic, an
immunomodulation drug, an ophthalmic, otic or nasal drug, a topical drug, a
nutritional drug, a TNF
2 0 antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid
anti-inflammatory drug
(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a
neuromuscular blocker, an
antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an
erythropoietin, an
immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a
hormone replacement
drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,
an asthma medication, a
2 5 beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine, or
a cytokine antagonist.
Also provided is a medical device, comprising at least one isolated mammalian
CHl-deleted
mimetibody of the invention, wherein the device is suitable to contacting or
administerting the at least
one CH1-deleted mimetibody by at least one mode selected from parenteral,
subcutaneous,
intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular,
3 0 intracartilaginous, intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,
intrapelvic, intrapericardiac,
intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal,
intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal,
sublingual, intranasal, or transdermal.
3 5 Also provided is an article of manufacture for human pharmaceutical or
diagnostic use,
6
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comprising packaging material and a container comprising a solution or a
lyophilized form of at least
one isolated mammalian CHl-deleted mimetibody of the present invention. The
article of manufacture
can optionally comprise having the container as a component of a parenteral,
subcutaneous,
intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular,
intracartilaginous, intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,
intrapelvic, intrapericardiac,
intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal,
intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal,
sublingual, intranasal, or transdermal delivery device or system.
Also provided is a method for producing at least one isolated mammalian CHl-
deleted
mimetibody of the present invention, comprising providing a host cell or
transgenic animal or
transgenic plant or plant cell capable of expressing in recoverable amounts
the CH1-deleted
mimetibody. Further provided in the present invention is at least one CHl-
deleted mimetibody
produced by the above method.
The present invention further provides any invention described herein.
DESCRIPTION OF THE INVENTION
The present invention provides isolated, recombinant and/or synthetic
mimetibodies or
specified portions or variants, as well as compositions and encoding nucleic
acid molecules comprising
at least one polynucleotide encoding at least one CHl-deleted mimetibody. Such
mimetibodies or
2 0 specified portions or variants of the present invention comprise specific
CHl-deleted mimetibody
sequences, domains, fragments and specified variants thereof. The present
invention also provides
methods of making and using said nucleic acids and mimetibodies or specified
portions or variants,
including therapeutic compositions, methods and devices.
The present invention also provides at least one isolated CH1-deleted
mimetibody or specified
2 5 portion or variant as described herein and/or as known in the art. The CHI
deleted mimetibody can
optionally comprise at least one CH3 region directly linked with at least one
CH2 region directly
linked with at least one hinge region or fragment thereof directly linked with
at least one partial V
region, directly linked with an optional linker sequence, directly linked to
at least one therapeutic
peptide, optionally further directly linked with at least a portion of at
least one variable antibody
3 0 sequence.
In a preferred embodiment a CHl deleted mimetibody comprises formula (I):
(V 1 (n)-Pep(n)-Flex(n)-V2(n)-pHinge(n)-CH2(n)-CH3 (n))(m),
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where V 1 is at least one portion of an N-terminus of an immunoglobulin
variable region, Pep is at least
one bioactive peptide, Flex is polypeptide that provides structural flexablity
by allowing the
mimietibody to have alternative orientations and binding properties, V2 is at
least one portion of a C-
terminus of an immunoglobulin variable region, pHinge is at least a portion of
an immunoglobulin
variable hinge region, CH2 is at least a portion of an immunoglobulin CH2
constant region, CH3 is at
least a portion of an immunoglobulin CH3 constant region, n and m can be an
integer between 1 and
10, mimicing different types of immunoglobulin molecules, e.g., but not
limited to IgGl, IgG2, IgG3,
IgG4, IgA, IgM, IgD, IgE, and the like, or combination thereof. The monomer
where m=1 can be linked
to other monomers by association or covalent linkage, such as, but not limited
to, a Cys-Cys disulfide
bond. Thus, a CHl-deleted mimetibody of the present invention mimics an
antibody structure with its
inherent properties and functions, while providing a therapeutic peptide and
its inherent or acquired in
vitro, in vivo or in situ properties or activities. The various portions of
the antibody and therapeutic
peptide portions of at least one CH1-deleted mimetibody of the present
invention can vary as described
herein in combinatoin with what is known in the art.
As used herein, a "CHl-deleted mimetibody," "CHl-deleted mimetibody portion,"
or "CH1-
deleted mimetibody fragment" and/or "CH1-deleted mimetibody variant" and the
like mimics, has or
simulates at least one ligand binding or at least one biological activity of
at least one protein, such as
but not limited to at least one biologically active portion of at least one of
SEQ ID NOS:1-979, in vitro,
ih situ and/or preferably in vivo. For example, a suitable CH1-deleted
mimetibody, specified portion or
2 0 variant of the present invention can bind at least one protein ligand and
includes at least one protein
ligand, receptor, soluble receptor, and the like. A suitable CHl-deleted
mimetibody, specified portion,
or variant can also modulate, increase, modify, activate, at least one protein
receptor signaling or other
measurable or detectable activity.
Mimetibodies useful in the methods and compositions of the present invention
are
2 5 characterized by suitable affinity binding to protein ligands or receptors
and optionally and preferably
having low toxicity. In particular, a CH1-deleted mimetibody, where the
individual components, such
as the portion of variable region, constant region (without a CHl portion) and
framework, or any
portion thereof (e.g., a portion of the J, D or V rgions of the variable heavy
or light chain; the hinge
region, the constant heavy chain or light chain, and the like) individually
and/or collectively optionally
3 0 and preferably possess low immunogenicity, is useful in the present
invention. The mimetibodies that
can be used in the invention are optionally characterized by their ability to
treat patients for extended
periods with good to excellent alleviation of symptoms and low toxicity. Low
immunogenicity and/or
high affinity, as well as other undefined properties, may contribute to the
therapeutic results achieved.
"Low immunogenicity" is defined herein as raising significant HAMA, HACA or
HAHA responses in
35 less than about 75%, or preferably less than about 50, 45, 40, 3S, 30, 35,
20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2,
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and/or 1 % of the patients treated and/or raising low titres in the patient
treated (less than about 300,
preferably less than about 100 measured with a double antigen enzyme
immunoassay) (see, e.g., Elliott
et al., Lancet 344:1125-1127 (1994)).
Utility
The isolated nucleic acids of the present invention can be used for production
of at least one
CHl-deleted mimetibody, fragment or specified variant thereof, which can be
used to effect in an cell,
tissue, organ or animal (including mammals and humans), to modulate, treat,
alleviate, help prevent the
incidence of, or reduce the symptoms of, at least one protein related
condition, selected from, but not
limited to, at least one of an immune disorder or disease, a cardiovascular
disorder or disease, an
2 0 infectious, malignant, and/or neurologic disorder or disease, an anemia;
an immune/autoimmune;
and/or an cancerous/infecteous, as well as other known or specified protein
related conditions.
Such a method can comprise administering an effective amount of a composition
or a
pharmaceutical composition comprising at least one CH1-deleted mimetibody or
specified portion or
variant to a cell, tissue, organ, animal or patient in need of such
modulation, treatment, alleviation,
prevention, or reduction in symptoms, effects or mechanisms. The effective
amount can comprise an
amount of about 0.0001 to 500 mg/kg per single or multiple administration, or
to achieve a serum
concentration of 0.0001-5000 pg/ml serum concentration per single or multiple
adminstration, or any
effective range or value therein, as done and determined using known methods,
as described herein or
known in the relevant arts.
2 0 Citations
All publications or patents cited herein are entirely incorporated herein by
reference as they
show the state of the art at the time of the present invention and/or to
provide description and
enablement of the present invention. Publications refer to any scientific or
patent publications, or any
other information available in any media format, including all recorded,
electronic or printed formats.
2 5 The following references are entirely incorporated herein by reference:
Ausubel, et al., ed., Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2002);
Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, 2°d Edition, Cold Spring
Harbor, NY (1989); Harlow and
Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989);
Colligan, et al., eds., Current
Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2002); Colligan et
al., Current Protocols
3 0 in Protein Science, John Wiley & Sons, NY, NY, (1997-2002).
Mimetibodies of the Present Invention
The CH1 deleted mimetibody can comprise at least one CH3 region directly
linked with at
least one CH2 region directly linked with at least one hinge region or
fragment thereof directly linked
with at least one partial V region, directly linked with an optional linker
sequence, directly linked to at
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least one therapeutic peptide, optionally further directly linked with at
least a portion of at least one
variable antibody sequence. In a preferred embodiment of a pair of a CH3-CH2-
hinge-partial J
sequence-linker-therapeutic peptide with an option N-terminal antibody
sequence, the pair can be
linked by association or covalent linkage, such as, but not limited to, a Cys-
Cys disulfide bond. Thus,
a CHl-deleted mimetibody of the present invention mimics an antibody structure
with its inherent
properties and functions, while providing a therapeutic peptide and its
inherent or acquired in vitro, in
vivo or in situ properties or activities. The various portions of the antibody
and therapeutic peptide
portions of at least one CHl-deleted mimetibody of the present invention can
vary as described herein
in combinatoin with what is known in the art.
In particular, mimetibodies comprise at least one ligand binding region (LBR)
that corresponds
to at least one portion of at least one complementarity determining region
(CDR, e.g., CDRl, CDRZ or
CDR3 of HC or LC variable region) of at least one antibody or fragment or
portion thereof where at
least one ligand protein is inserted into or replaces at least a portion of at
least one CDR of the
antibody or portion thereof. Such mimetibodies of the present invention thus
provide at least one
suitable property as compared to known proteins, such as, but not limited to,
at least one of increased
half life, increased activity, more specific activity, increased avidity,
increased or decreased off rate, a
selected or more suitable subset of activities, less immungenicity, increased
quality or duration of at
least one desired therapeutic effect, less side effects, and the like.
Such fragments can be produced by enzymatic cleavage, synthetic or recombinant
techniques,
2 0 as known in the art and/or as described herein. For example, papain or
pepsin cleavage can generate
CH1-deleted mimetibody Fab or F(af)2 fragments, respectively. Other proteases
with the requisite
substrate specificity can also be used to generate Fab or F(ab')2 fragments or
portions thereof.
Mimetibodies can also be produced in a variety of truncated forms using
antibody genes in which one
or more stop codons have been introduced upstream of the natural stop site.
For example, a chimeric
2 5 gene encoding a F(af)Z heavy chain portion can be designed to include DNA
sequences encoding the
CHl domain and/or hinge region of the heavy chain. The various portions of
mimetibodies can be
joined together chemically by conventional techniques, or can be prepared as a
contiguous protein
using genetic engineering techniques. For example, a nucleic acid encoding the
variable and constant
regions of a human antibody chain can be expressed to produce a contiguous
protein for use in
3 0 mimetibodies of the present invention. See, e.g., Ladner et al., U.S.
Patent No. 4,946,778 and Bird,
R.E, et al., Science, 242: 423-426 (1988), regarding single chain antibodies.
As used herein, the term "human mirnetibody" refers to an antibody in which
substantially
every part of the protein (e.g., LBR, framework, CL, CH domains (e.g., CHI,
CH2, CH3), hinge, (VL, VH))
is expected to be substantially non-immunogenic, with only minor sequence
changes or variations.
3 5 Such changes or variations optionally and preferably retain or reduce the
immunogenicity in humans
to
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
relative to non-modified human antibodies, or mimetibodies of the prsent
invention. Thus, a human
antibody and corresponding CH1-deleted mimetibody of the present invention is
distinct from a
chimeric or humanized antibody. It is pointed out that a human antibody and
CHI-deleted mimetibody
can be produced by a non-human animal or cell that is capable of expressing
functionally rearranged
human immunoglobulin (e.g., heavy chain and/or light chain) genes, and for a
CHI-deleted
mimetibody, wherein at least one Ig CDR is replaced by an LBR of at least one
ligand protein or
fragment.
Human mimetibodies that are specific for at least one protein ligand or
receptor thereof can be
designed against an appropriate ligand, such as isolated and/or protein
receptor or ligand, or a portion
thereof (including synthetic molecules, such as synthetic peptides).
Preparation of such mimetibodies
are performed using known techniques to identify and characterize ligand
binding regions or sequences
of at least one protein or portion thereof.
In a preferred embodiment a CHl deleted mimetibody comprises formula (I):
(V 1 (n)-Pep(n)-Flex(n)-V2(n)-pHinge(n)-CH2(n)-CH3 (n))(m),
where V 1 is at least one portion of an N-terminus of an immunoglobulin
variable region, Pep is
at least one bioactive peptide, Flex is polypeptide that provides structural
flexablity by allowing the
mimietibody to have alternative orientations and binding properties, V2 is at
least one portion of a C-
terminus of an immunoglobulin variable region, pHinge is at least a portion of
an immunoglobulin
variable hinge region, CH2 is at least a portion of an immunoglobulin CH2
constant region, CH3 is at
2 0 least a portion of an immunoglobulin CH3 constant region, n and m can be
an integer between 1 and
10, mimicing different types of immunoglobulin molecules, e.g., but not
limited to IgGl, IgG2, IgG3,
IgG4, IgA, IgM, IgD, IgE, and the like, or combination thereof. The monomer
where m=1 can be linked
to other monomers by association or covalent linkage, such as, but not limited
to, a Cys-Cys disulfide
bond.
In a preferred embodiment, at least one CH1-deleted mimetibody or specified
portion or
variant of the present invention is produced by at least one cell line, mixed
cell line, immortalized cell
or clonal population of immortalized and/or cultured cells. Immortalized
protein producing cells can
be produced using suitable methods. Preferably, the at least one CHl-deleted
mimetibody or specified
portion or variant is generated by providing nucleic acid or vectors
comprising DNA derived or having
3 0 a substantially similar sequence to, at least one human immunoglobulin
locus that is functionally
rearranged, or which can undergo functional rearrangement, and which further
comprises a mimetibody
structure as described herein, e.g., but not limited to Formula (I), wherein
known portions of :C- and N-
termiinal variable regions can be used for V l and V2, hinge regions for
pHinge, CH2 for CH2 and
CH3 for CH3, as known in the art.
m
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
The term "functionally rearranged," as used herein refers to a segment of
nucleic acid from an
immunoglobulin locus that has undergone V(D)J recombination, thereby producing
an immunoglobulin
gene that encodes an immunoglobulin chain (e.g., heavy chain, light chain), or
any portion thereof. A
functionally rearranged immunoglobulin gene can be directly or indirectly
identified using suitable
methods, such as, for example, nucleotide sequencing, hybridization (e.g.,
Southern blotting, Northern
blotting) using probes that can anneal to coding joints between gene segments
or enzymatic
amplification of immunoglobulin genes (e.g., polymerase chain reaction) with
primers that can anneal
to coding joints between gene segments. Whether a cell produces an CHl-deleted
mimetibody or
portion or variant comprising a particular variable region or a variable
region comprising a particular
sequence (e.g., at least one Pep sequence can also be determined using
suitable methods.
Mimetibodies, specified portions and variants of the present invention can
also be prepared
using at least one CHl-deleted mimetibody or specified portion or variant
encoding nucleic acid to
provide transgenic animals or mammals, such as goats, cows, horses, sheep, and
the like, that produce
such mimetibodies or specified portions or variants in their milk. Such
animals can be provided using
known methods as applied for antibody encoding sequences. See, e.g., but not
limited to, ITS patent
nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362;
5,304,489, and the like, each
of which is entirely incorporated herein by reference.
Mimetibodies, specified portions and variants of the present invention can
additionally be
prepared using at least one CH1-deleted mimetibody or specified portion or
variant encoding nucleic
2 0 acid to provide transgenic plants and cultured plant cells (e.g., but not
limited to tobacco and maize)
that produce such mimetibodies, specified portions or variants in the plant
parts or in cells cultured
therefrom. As a non-limiting example, transgenic tobacco leaves expressing
recombinant proteins have
been successfully used to provide large amounts of recombinant proteins, e.g.,
using an inducible
promoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118
(1999) and references
2 5 cited therein. Also, transgenic maize have been used to express mammalian
proteins at commercial
production levels, with biological activities equivalent to those produced in
other recombinant systems
or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol.
464:127-147 (1999) and
references cited therein. Antibodies have also been produced in large amounts
from transgenic plant
seeds including antibody fragments, such as single chain mimetibodies
(scFv's), including tobacco
3 0 seeds and potato tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101-
109 (1998) and references
cited therein. Thus, mimetibodies, specified portions and variants of the
present invention can also be
produced using transgenic plants, according to know methods. See also, e.g.,
Fischer et al., Biotechnol.
Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al., Trends Biotechnol. 13:522-7
(1995); Ma et al., Plant
Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans. 22:940-944
(1994); and references
3 5 cited therein. The above references are entirely incorporated herein by
reference.
12
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
The mimetibodies of the invention can bind human protein ligands with a wide
range of
affinities (KD). In a preferred embodiment, at least one human CH1-deleted
mimetibody of the present
invention can optionally bind at least one protein ligand with high affinity.
For example, at least one
CHl-deleted mimetibody of the present invention can bind at least one protein
ligand with a KD equal
S to or less than about 10-9 M or, more preferably, with a KD equal to or less
than about 0.1-9.9 (or any
range or value therein) X 10-1° M, 10-11, 10-12 , 10-13 or any range or
value therein.
The amity or avidity of a CHl-deleted mimetibody for at least one protein
ligand can be
determined experimentally using any suitable method, e.g., as used for
determing antibody-antigen
binding affinity or avidity. (See, for example, Berzofsky, et al., "Antibody-
Antigen Interactions," In
Fundame~tallnzmunology, Paul, W. E., Ed., Raven Press: New York, NY (1984);
Kuby, Janis
Immunology, W. H. Freeman and Company: New York, NY (1992); and methods
described herein).
The measured affinity of a particular CHl-deleted mimetibody-ligand
interaction can vary if measured
under different conditions (e.g., salt concentration, pH). Thus, measurements
of affinity and other
ligand-binding parameters (e.g., KD, Ka, Kd) are preferably made with
standardized solutions of CHl-
deleted mimetibody and ligand, and a standardized buffer, such as the buffer
described herein.
Nucleic Acid Molecules
Using the information provided herein, such as the nucleotide sequences
encoding at least 90-
100% of the contiguous amino acids of at least one of SEQID NOS:1-1009 as well
as at least one
portion of an antibody, wherein the above sequences are inserted as the Pep
sequence of Formula (I) to
2 0 provide a CHl-deleted mimetibody of the present invention, further
comprising specified fragments,
variants or consensus sequences thereof, or a deposited vector comprising at
least one of these
sequences, a nucleic acid molecule of the present invention encoding at least
one GH1-deleted
mimetibody or specified portion or variant can be obtained using methods
described herein or as
known in the art.
2 5 Nucleic acid molecules of the present invention can be in the form of RNA,
such as mRNA,
hnRNA, tRNA or any other form, or in the form of DNA, including, but not
limited to, cDNA and
genomic DNA obtained by cloning or produced synthetically, or any combination
thereof. The DNA
can be triple-stranded, double-stranded or single-stranded, or any combination
thereof. Any portion of
at least one strand of the DNA or RNA can be the coding strand, also known as
the sense strand, or it
3 0 can be the non-coding strand, also referred to as the anti-sense strand.
Isolated nucleic acid molecules of the present invention can include nucleic
acid molecules
comprising an open reading frame (ORF), optionally with one or more introns,
nucleic acid molecules
comprising the coding sequence for a CHl-deleted mimetibody or specified
portion or variant; and
nucleic acid molecules which comprise a nucleotide sequence substantially
different from those
3 5 described above but which, due to the degeneracy of the genetic code,
still encode at least one CH1-
Z3
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
deleted mimetibody as described herein and/or as known in the art. Of course,
the genetic code is well
known in the art. Thus, it would be routine for one skilled in the art to
generate such degenerate
nucleic acid variants that code for specific CHI-deleted mimetibody or
specified portion or variants of
the present invention. See, e.g., Ausubel, et al., supra, and such nucleic
acid variants are included in
the present invention.
As indicated herein, nucleic acid molecules of the present invention which
comprise a nucleic
acid encoding a CHl-deleted mimetibody or specified portion or variant can
include, but are not
limited to, those encoding the amino acid sequence of a CH1-deleted mimetibody
fragment, by itself;
the coding sequence for the entire CH1-deleted mirnetibody or a portion
thereof; the coding sequence
for a CHI-deleted mimetibody, fragment or portion, as well as additional
sequences, such as the coding
sequence of at least one signal leader or fusion peptide, intron, non-coding
5' and 3' sequences, such as
the transcribed, non-translated sequences that play a role in transcription,
mRNA processing, including
splicing and polyadenylation signals (for example - ribosome binding and
stability of mRNA); an
additional coding sequence that codes for additional amino acids, such as
those that provide additional
functionalities. Thus, the sequence encoding a CHl-deleted mimetibody or
specified portion or variant
can be fused to a marker sequence, such as a sequence encoding a peptide that
facilitates purification
of the fused CHI-deleted mimetibody or specified portion or variant comprising
a CHl-deleted
mimetibody fragment or portion.
Polynucleotides Which Selectively Hybridize to a Polynucleotide as Described
Herein
The present invention provides isolated nucleic acids that hybridize under
selective hybridization
conditions to a polynucleotide disclosed herein, or others disclosed herein,
including specified variants or
portions thereof. Thus, the polynucleotides of this embodiment can be used for
isolating, detecting,
and/or quantifying nucleic acids comprising such polynucleotides.
2 5 Low or moderate stringency hybridization conditions are typically, but not
exclusively, employed
with sequences having a reduced sequence identity relative to complementary
sequences. Moderate and
high stringency conditions can optionally be employed for sequences of greater
identity. Low stringency
conditions allow selective hybridization of sequences having about 40-99%
sequence identity and can be
employed to identify orthologous or paralogous sequences.
3 0 Optionally, polynucleotides of this invention will encode at least a
portion of a CHI-deleted
mimetibody or specified portion or variant encoded by the polynucleotides
described herein. The
polynucleotides of this invention embrace nucleic acid sequences that can be
employed for selective
hybridization to a polynucleotide encoding a CHI-deleted mimetibody or
specified portion or variant of
the present invention. See, e.g., Ausubel, supra; Colligan, supra, each
entirely incorporated herein by
3 5 reference.
l4
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Construction of Nucleic Acids
The isolated nucleic acids of the present invention can be made using (a)
recombinant. methods,
(b) synthetic techniques, (c) purification techniques, or combinations
thereof, as well-known in the art.
The nucleic acids can conveniently comprise sequences in addition to a
polynucleotide of the
present invention. For example, a mufti-cloning site comprising one or more
endonuclease restriction
sites can be inserted into the nucleic acid to aid in isolation of the
polynucleotide. Also, translatable
sequences can be inserted to aid in the isolation of the translated
polynucleotide of the present invention.
For example, a hexa-histidine marker sequence provides a convenient means to
purify the proteins of the
present invention. The nucleic acid of the present invention - excluding the
coding sequence - is
optionally a vector, adapter, or linleer for cloning andlor expression of a
polynucleotide of the present
invention.
Additional sequences can be added to such cloning and/or expression sequences
to optimize their
function in cloning and/or expression, to aid in isolation of the
polynucleotide, or to improve the
introduction of the polynucleotide into a cell. Use of cloning vectors,
expression vectors, adapters, and
linkers is well known in the art. See, e.g., Ausubel, supra; or Sambrook,
supra.
Recombinant Methods for Constructing Nucleic Acids
The isolated nucleic acid compositions of this invention, such as RNA, cDNA,
genomic DNA, or
any combination thereof, can be obtained from biological sources using any
number of cloning
methodologies known to those of skill in the art. In some embodiments,
oligonucleotide probes that
2 0 selectively hybridize, under suitable stringency conditions, to the
polynucleotides of the present invention
are used to identify the desired sequence in a cDNA or genomic DNA library.
The isolation of RNA, and
construction of cDNA and genomic libraries, is well known to those of ordinary
skill in the art. (See, e.g.,
Ausubel, supra; or Sambrook, supra).
Synthetic Methods for Constructing Nucleic Acids
2 5 The isolated nucleic acids of the present invention can also be prepared
by direct chemical
synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical
synthesis generally produces a
single-stranded oligonucleotide, which can be converted into double-stranded
DNA by hybridization with
a complementary sequence, or by polymerization with a DNA polymerise using the
single strand as a
template. One of skill in the art will recognize that while chemical synthesis
of DNA can be limited to
3 0 sequences of about 100 or more bases, longer sequences can be obtained by
the ligation of shorter
sequences.
Recombinant Expression Cassettes
The present invention further provides recombinant expression cassettes
comprising a nucleic
acid of the present invention. A nucleic acid sequence of the present
invention, for example a cDNA or a
3 5 genomic sequence encoding a CHl-deleted mimetibody or specified portion or
variant of the present
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
invention, can be used to construct a recombinant expression cassette that can
be introduced into at least
one desired host cell. A recombinant expression cassette will typically
comprise a polynucleotide of the
present invention operably linked to transcriptional initiation regulatory
sequences that will direct the
transcription of the polynucleotide in the intended host cell. Both
heterologous and non-heterologous
(i.e., endogenous) promoters can be employed to direct expression of the
nucleic acids of the present
invention.
In some embodiments, isolated nucleic acids that serve as promoter, enhancer,
or other elements
can be introduced in the appropriate position (upstream, downstream or in
intron) of a non-heterologous
form of a polynucleotide of the present invention so as to up or down regulate
expression of a
polynucleotide of the present invention. For example, endogenous promoters can
be altered in vivo or i~
vitro by mutation, deletion and/or substitution, as known in the art. A
polynucleotide of the present
invention can be expressed in either sense or anti-sense orientation as
desired. It will be appreciated that
control of gene expression in either sense or anti-sense orientation can have
a direct impact on the
observable characteristics. Another method of suppression is sense
suppression. Introduction of nucleic
acid configured in the sense orientation has been shown to be an effective
means by which to block the
transcription of target genes.
Vectors And Host Cells
The present invention also relates to vectors that include isolated nucleic
acid molecules of the
present invention, host cells that are genetically engineered with the
recombinant vectors, and the
2 0 production of at least one CHl-deleted mimetibody or specified portion or
variant by recombinant
techniques, as is well known in the art. See, e.g., Sambrook, et al., supra;
Ausubel, et al., supra, each
entirely incorporated herein by reference.
The polynucleotides can optionally be joined to a vector containing a
selectable marker for
propagation in a host. Generally, a plasmid vector is introduced into a cell
using suitable known
2 5 methods, such as electroporation and the like, other known methods include
the use of the vector as a
precipitate, such as a calcium phosphate precipitate, or in a complex with a
charged lipid. If the vector
is a virus, it can be packaged in vitro using an appropriate packaging cell
line and then transduced into
host cells.
The DNA insert should be operatively linked to an appropriate promoter. The
expression
3 0 constructs will further contain sites optionally for at least one of
transcription initiation, termination
and, in the transcribed region, a ribosome binding site for translation. The
coding portion of the mature
transcripts expressed by the constructs will preferably include a translation
initiating at the beginning
and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at
the end of the mRNA
to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell
expression.
16
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Expression vectors will preferably but optionally include at least one
selectable marker. Such
markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate
reductase (DHFR, US
Pat.Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,
ampicillin, neomycin
(G418), mycophenolic acid, or glutamine synthetase (GS, US Pat.Nos. 5,122,464;
5,770,359;
5,827,739) resistance for eukaryotic cell culture, and tetracycline or
ampicillin resistance genes for
culturing in E. coli and other bacteria or prokaryotics (the above patents are
entirely incorporated
hereby by reference). Appropriate culture mediums and conditions for the above-
described host cells
are known in the art. Suitable vectors will be readily apparent to the skilled
artisan. Introduction of a
vector construct into a host cell can be effected by calcium phosphate
transfection, DEAF-dextran
mediated transfection, cationic lipid-mediated transfection, electroporation,
transduction, infection or
other known methods. Such methods are described in the art, such as Sambrook,
supra, Chapters 1-4
and I6-I8; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
At least one CHl-deleted mimetibody or specified portion or variant of the
present invention
can be expressed in a modified form, such as a fusion protein, and can include
not only secretion
signals, but also additional heterologous functional regions. For instance, a
region of additional amino
acids, particularly charged amino acids, can be added to the N-terminus of a
CH1-deleted mimetibody
or specified portion or variant to improve stability and persistence in the
host cell, during purification,
or during subsequent handling and storage. Also, peptide moieties can be added
to a CHI-deleted
mimetibody or specified portion or variant of the present invention to
facilitate purification. Such
2 0 regions can be removed prior to final preparation of a CHl-deleted
mimetibody or at least one
fragment thereof. Such methods are described in many standard laboratory
manuals, such as
Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters
16, 17 and I8.
Those of ordinary skill in the art are knowledgeable in the numerous
expression systems available
for expression of a nucleic acid encoding a protein of the present invention.
2 5 Illustrative of cell cultures useful for the production of the
mimetibodies, specified portions or
variants thereof, are mammalian cells. Mammalian cell systems often will be in
the form of monolayers of
cells although mammalian cell suspensions or bioreactors can also be used. A
number of suitable host
cell lines capable of expressing intact glycosylated proteins have been
developed in the art, and include
the COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEIC293, BHI~21
(e.g., ATCC
3 0 CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell
lines, hepG2 cells,
P3X63Ag8.653, SP2/0-AgI4, 293 cells, HeLa cells and the like, which are
readily available from, for
example, American Type Culture Collection, Manassas, Va. Preferred host cells
include cells of
lymphoid origin such as myeloma and lymphoma cells. Particularly preferred
host cells are
P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC
Accession
17
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Number CRL-1851). In a particularly preferred embodiment, the recombinant cell
is a P3X63Ab8.653
or a SP2/0-Agl4 cell.
Expression vectors for these cells can include one or more of the following
expression control
sequences, such as, but not limited to an origin of replication; a promoter
(e.g., late or early SV40
promoters, the CMV promoter (US Pat.Nos. S,I68,062; 5,385,839), an HSV tk
promoter, a pgk
(phosphoglycerate kinase) promoter, an EF-1 alpha promoter (CJS Pat.No.
5,266,491), at least one human
iinmunoglobulin promoter; an enhancer, and/or processing information sites,
such as ribosome binding
sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly
A addition site), and
transcriptional terminator sequences. See, e.g., Ausubel et al., supra;
Sambrook, et al., supra. Other cells
useful for production of nucleic acids or proteins of the present invention
are known and/or available, for
instance, from the American Type Culture Collection Catalogue of Cell Lines
and Hybridomas
(www.atcc.org) or other known or commercial sources.
When eukaryotic host cells are employed, polyadenlyation or transcription
terminator sequences
are typically incorporated into the vector. An example of a terminator
sequence is the polyadenlyation
sequence from the bovine growth hormone gene. Sequences for accurate splicing
of the transcript can
also be included. An example of a splicing sequence is the VP1 intron from
SV40 (Sprague, et al., J.
Virol. 45:773-781 (I983)). Additionally, gene sequences to control replication
in the host cell can be
incorporated into the vector, as known in the art.
Purification of an CHl-deleted mimetibody or specified portion or variant
Thereof
2 0 A CHl-deleted mimetibody or specified portion or variant can be recovered
and purified from
recombinant cell cultures by well-known methods including, but not limited to,
protein A purification,
ammonium sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. High performance
liquid chromatography
2 5 ("HPLC") can also be employed for purification. See, e.g., Colligan,
Current Protocols in
Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY,
NY, (1997-2002), e.g.,
Chapters l, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.
Mimetibodies or specified portions or variants of the present invention
include naturally
purified products, products of chemical synthetic procedures, and products
produced by recombinant
3 0 techniques from a eukaryotic host, including, for example, yeast, higher
plant, insect and mammalian
cells. Depending upon the host employed in a recombinant production procedure,
the CHl-deleted
mimetibody or specified portion or variant of the present invention can be
glycosylated or can be non-
glycosylated, with glycosylated preferred. Such methods are described in many
standard laboratory
manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra,
Chapters 10, 12, 13, 16, 18
3 5 and 20, Colligan, Protein Science, supra, Chapters 12-14, all entirely
incorporated herein by reference.
is
CA 02490409 2004-12-24
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MIMETIBODIES, SPECIFIED FRAGMENTS AND/OR VARIANTS
The isolated mimetibodies ofthe present invention comprise a CH1-deleted
mimetibody or
specified portion or variant encoded by any one of the polynucleotides of the
present invention as
discussed more fully herein, or any isolated or prepared CHl-deleted
mimetibody or specified portion or
variant thereof.
Preferably, the CHl-deleted mimetibody or ligand-binding portion or variant
binds at least one
protein ligand or receptor, and, thereby provides at least one biological
activity of the corresponding
protein or a fragment thereof. Different therapeutically or diagnostically
significant proteins are well
known in the art and suitable assays or biological activities of such proteins
are also well known in the art.
The following is a general discussion of the variety of proteins, peptides and
biological molecules that
may be used in the in accordance with the teachings herein. These descriptions
do not serve to limit the
scope of the invention, but rather illustrate the breadth of the invention.
Thus, an embodiment of the present invention may target one or more growth
factors, or,
conversely, derive the target-binding moiety from one or more growth factors.
Briefly, growth factors
axe hormones or cytokine proteins that bind to receptors on the cell surface,
with the primary result of
activating cellular proliferation and/or differentiation. Many growth factors
are quite versatile,
stimulating cellular division in numerous different cell types; while others
are specific to a particular
cell-type. The following Table 1 presents several factors, but is not intended
to be comprehensive or
complete, yet introduces some of the more commonly known factors and their
principal activities.
Table 1: Growth Factors
Factor Princi al Source Primal Activi Comments
Platelet DerivedPlatelets, endothelialPromotes proliferationDimer required
of for
Growth Factorcells, placenta. connective tissue, receptor binding.
glial and
(PDGF) smooth muscle cells. Two different
PDGF protein
receptor has intrinsicchains, A and
tyrosine B,
kinase activity. form 3 distinct
dimer
forms.
Epidermal Submaxillary gland,promotes proliferationEGF receptor
of has
Growth FactorBrunners gland. mesenchymal, glial tyrosine kinase
and
(EGF) epithelial cells. activity, activated
in
response to
EGF
bindin .
Fibroblast Wide range of cells;Promotes proliferationFour distinct
Growth of
Factor (FGF) protein is associatedmany cells including receptors, all
with skeletal with
the ECM; nineteen and nervous system; tyrosine kinase
family inhibits
members. Receptorssome stem cells; inducesactivity. FGF
widely distributedmesodermal differentiation.implicated in
in mouse
bone, implicated Non-proliferative mammary tumors
in effects and
several bone-relatedinclude regulation Kaposi's sarcoma.
of pituitary
diseases. and ovarian cell function.
Z9
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Factor Princi al Source Primal Activi Comments
NGF Promotes neurite outgrowthSeveral related
and neural cell survival.proteins first
identified as
proto-
oncogenes; trkA
(trackA), trkB,
trkC.
ErythropoietinKidney. Promotes proliferationAlso considered
and a
(Epo) differentiation of 'blood protein,'
erythrocytes. and a
colony stimulating
factor.
Transforming Common in transformedPotent keratinocyte Related to EGF.
growth
Growth Factorcells, found in factor.
a
(TGF-a) macrophages and
keratinocytes.
Transforming Tumor cells, activatedAnti-inflammatory Large family
(suppresses of
Growth FactorTHl cells (T-helper)cytokine production proteins including
v and and class
(TGF-b) natural killer II MHC expression), activin, inhibin
(NK) cells. and
proliferative effectsbone morpho-genetic
on many
mesenchymal and epithelialprotein. Several
,
cell types, may inhibitclasses arid
macrophage and lymphocytesubclasses of
cell-
roliferation. surface rece
tors.
Insulin-Like Primarily liver, Promotes proliferationRelated to IGF-II
produced of and
Growth Factor-Iin response to many cell types, autocrineproinsulin,
GH and and also
(IGF-I) then induces subsequentparacrine activities called Somatomedin
in addition
cellular activities,to the initially observedC. IGF-I receptor,
particularly on endocrine activities like the insulin
bone on bone.
growth. receptor, has
intrinsic
tyrosine kinase
activity. IGF-I
can
bind to the
insulin
rece tor.
Insulin-Like Expressed almost Promotes proliferationIGF-II receptor
of is
Growth Factor-IIexclusively in many cell types primarilyidentical to
embryonic of the
(IGF-II) and neonatal tissues.fetal origin. Relatedmannose-6-phosphate
to IGF-I
and proinsulin. receptor that
is
responsible
for the
integration
of
I sosomal en
es.
Additional growth factors that may be produced in accordance with the present
invention
include Activin (Vale et al., 321 Nature 776 (1986); Ling et aL, 321 Nature
779 (1986)), Inhibin (U.S.
Patent Nos. 4,737,578; 4,740,587), and Bone Morphongenic Proteins (BMPs) (U.S.
Patent No.
5,846,931; Wozney, Cellular & Molecular Biology of Bone 131-167 (1993)).
In addition to the growth factors discussed above, the present invention may
target or use other
cytokines. Secreted primarily from leukocytes, cytokines stimulate both the
humoral and cellular
immune responses, as well as the activation of phagocytic cells. Cytokines
that are secreted from
ao
CA 02490409 2004-12-24
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lymphocytes are termed lymphokines, whereas those secreted by monocytes or
macrophages are
termed monokines. A large family of cytokines are produced by various cells of
the body. Many of
the lymphokines are also known as interleukins ()Z,s), because they axe not
only secreted by leukocytes,
but are also able to affect the cellular responses of leukocytes. More
specifically, interleukins are
S growth factors targeted to cells of hematopoietic origin. The list of
identified interleukins grows
continuously. See, e.g., U.S. Patent No. 6,174,995; U.S. Patent No. 6,143,289;
Sallusto et al., 18 Annu.
Rev. Immunol. 593 (2000) Kunkel et al., 59 J. Leukocyto Biol. 81 (1996).
Additional growth factor/cytokines encompassed in the present invention
include pituitary
hormones such as human growth hormone (HGH), follicle stimulating hormones
(FSH, FSHa,, and
FSH(3), Human Chorionic Gonadotrophins (HCG, HCGa, HCG[3), uFSH
(urofollitropin), Gonatropin
releasing hormone (GRH), Growth Hormone (GH), leuteinizing hormones (LH, LHa,
LH[3),
somatostatin, prolactin, thyrotropin (TSH, TSHa,, TSH(3), thyrotropin
releasing hormone (TRH),
parathyroid hormones, estrogens, progesterones, testosterones, or structural
or functional analog
thereof. All of these proteins and peptides are known in the art.
The cytokine family also includes tumor necrosis factors, colony stimulating
factors, and
interferons. See, e.g., Cosman, 7 Blood Cell (1996); Gruss et al., 85 Blood
3378 (1995); Beutler et al.,
7 Annu. Rev. Immunol. 625 (1989); Aggarwal et al., 260 J. Biol. Chem. 2345
(I985); Pennica et al.,
312 Nature 724 (1984); R & D Systems, Cytokine Mini-Reviews, at
http://www.rndsystems.com.
Several cytokines are introduced, briefly, in Table 2 below.
Table 2: Cytokines
C okine Princi al Source_ Primal Activi
Interleukins Primarily macrophages but Costimulation of APCs and
also T cells;
IL1-a and neutrophils, endothelial stimulates IL-2 receptor
-b cells, smooth production and
muscle cells, filial cells,expression of interferon-y;
astrocytes, B- may induce
and T-cells, fibroblasts, proliferation in non-lymphoid
and cells.
keratinoc es
IL-2 CD4+ T-helper cells, activatedMajor interleukin responsible
THI for clonal
cells, NK cells T-cell proliferation. IL-2
also exerts
effects on B-cells, macrophages,
and
natural killer (NK) cells.
IL,-2 receptor is
not expressed on the surface
of resting T-
cells, but expressed constitutively
on NK
cells, that will secrete
TNF-a, IFN-y and
GM-CSF in response to IL-2,
which in
turn activate macro ha es.
IL-3 Primarily T-cells Also known as multi-CSF,
as it stimulates
stem cells to produce all
forms of
hemato oietic cells.
21
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C okine Princi al Source Primat Activi
IL-4 THz and mast cells B cell proliferation, eosinophil
and mast
cell growth and function,
IgE and class II
MHC expression on B cells,
inhibition of
monokine roduction
II,-5 THZ and mast cells eosino hil owth and function
IL-6 Macrophages, fibroblasts, IL-6 acts in synergy with
endothelial IL-1 and TNF-a
cells and activated T-helperin many immune responses,
cells. including T-
Does not induce cytokine cell activation; primary
expression. inducer of the
acute-phase response in
liver; enhances
the differentiation of B-cells
and their
consequent production of
immunoglobulin; enhances
Glucocorticoid s nthesis.
IL-7 th mic and marrow stromal T and B 1 ho oiesis
cells
IL-8 Monocytes, neutrophils, Chemoattractant (chemokine)
macrophages, for
and NK cells neutrophils, basophils and
T-cells;
activates neutro hits to
de ranulate.
IL-9 T cells hemato oietic and th mo
oietic effects
IL-10 activated THz cells, CD8+ inhibits cytokine production,
T and B promotes B
cells, macrophages cell proliferation and antibody
production, suppresses cellular
immunity,
mast cell owth
IL-11 stromal cells synergisitc hematopoietic
and
thrombo oietic effects
IL-12 B cells, macrophages proliferation of NIA cells,
INF-g
production, promotes cell-mediated
immune functions
IL-13 THZ cells IL-4-like activities
IL-18 macrophages/ICupffer cells,Interferon-gamma-inducing
factor with
keratinocytes, glucocorticoid-secretingpotent pro-inflammatory
activity
adrenal cortex cells, and
osteoblasts
IL-21 Activated T cells IL21 has a role in proliferation
and
maturation of natural killer
(NIA) cell
populations from bane marrow,
in the
proliferation of mature
B-cell populations
co-stimulated with anti-CD40,
and in the
proliferation of T cells
co-stimulated with
anti-CD3.
IL-23 Activated dendritic cells A complex of p19 and the
p40 subunit of
IL-12. IL-23 binds to IL-12R
beta 1 but
not IL-12R beta 2; activates
Stat4 in PHA
blast T cells; induces strong
proliferation
of mouse memory T cells;
stimulates IFN-
gamma production and proliferation
in
PHA blast T cells, as well
as in CD45R0
(memo ) T cells.
22
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C okine Principal Source Primal Activi
TumorNecrosisPrimarily activated macrophages.Once called cachectin; induces
the
Factor expression of other autocrine
growth
TNF-a factors, increases cellular
responsiveness
to growth factors; induces
signaling
pathways that lead to proliferation;
induces expression of a
number of
nuclear proto-oncogenes
as well as of
several interleukins.
(TNF-(3) T-lymphocytes, particularlyAlso called lymphotoxin;
cytotoxic kills a number
T-lymphocytes (CTL cells);of different cell types,
induced induces terminal
by IL-2 and antigen-T-Celldifferentiation in others;
receptor inhibits
interactions. lipoprotein lipase present
on the surface
of vascular endothelial
cells.
Interferons macrophages, neutrophils Known as type I interferons;
and some antiviral
INF-a and somatic cells effect; induction of class
-b I MHC on all
somatic cells; activation
of NK cells and
macro ha es.
Interferon Primarily CD8+ T-cells, Type II interferon; induces
activated THl of class I
INF-y and NK cells MHC on all somatic cells,
induces class II
MHC on APCs and somatic
cells,
activates macrophages, neutrophils,
NK
cells, promotes cell-mediated
immunity,
enhances ability of cells
to present
anti ens to T-cells; antiviral
effects.
Monocyte Peripheral blood Attracts monocytes to sites
of vascular
Chemoattractantmonocytes/macrophages endothelial cell injury,
implicated in
Protein-1 atherosclerosis.
(MCP 1 )
Colony Stimulate the proliferation
of specific
Stimulating pluripotent stem cells of
the bone marrow
Factors (CSFs) in adults.
Granulocyte-CSF Specific for proliferative
effects on cells
(G-CSF) of the granulocyte lineage;
proliferative
effects on both classes
of 1 hold cells.
Macrophage-CSF Specific for cells of the
macrophage
(M-CSF) linea e.
Granulocyte- Proliferative effects on
cells of both the
MacrophageCSF macrophage and granulocyte
lineages.
(GM-CSF
Other cytokines of interest that may be produced by the invention described
herein include
adhesion molecules (R & D Systems, Adhesion Molecule I (1996), at
http://www.rndsystems.com);
angiogenin (IJ.S. Patent No. 4,721,672; Moener et al., 226 Eur. J. Biochem.
483 (1994)); annexin V
(Cookson et al., 20 Genomics 463 (I994); Grundmann et al., 85 Proc. Natl.
Acad. Sci. USA 3708
(1988); U.S. Patent No. 5,767,247); caspases (U.S. Patent No. 6,214,858;
Thornberry et al., 281
Science 1312 (I998)); chemokines (U.S. PatentNos. 6,174,995; 6,143,289;
Sallusto et al., 18 Annu.
Rev. Immunol. 593 (2000) Kunkel et al., 59 J. Leukocyte Biol. 81 (1996));
endothelin (U.S. Patent
23
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Nos. 6,242,485; 5,294,569; 5,231,166); eotaxin (U.S. Patent No. 6,271,347;
Ponath et aL, 97(3) J. Clin.
Invest. 604-612 (1996)); Flt-3 (U.S. Patent No. 6,190,655); heregulins (U.S.
PatentNos. 6,284,535;
6,143,740; 6,136,558; 5,859,206; 5,840,525); Leptin (Leroy et al., 271(5) J.
Biol. Chem. 2365 (1996);
Maffei et al., 92 Proc. Natl. Acad. Sci. USA 6957 (1995); Zhang Y. et al.
(1994) Nature 372: 425-432);
S Macrophage Stimulating Protein (MSP) (U.S. Patent Nos. 6,248,560; 6,030,949;
5,315,000);
Neurotrophic Factors (U.S. Patent Nos. 6,005,081; 5,288,622);
Pleiotrophin/Midkine (PTN/MK)
(Pedraza et al., 117 J. Biochem. 845 (1995); Tamura et al., 3 Endocrine 21
(1995); U.S. Patent No.
5,210,026; I~adomatsu et al., 151 Biochem. Biophys. Res. Commun. 1312 (1988));
STAT proteins
(U.S. Patent Nos. 6,030808; 6,030,780; Darnell et al., 277 Science 1630-1635
(1997)); Tumor Necrosis
Factor Family (Cosman, 7 Blood Cell (1996); Gruss et al., 85 Blood 3378
(1995); Beutler et al., 7
Annu. Rev. Immunol. 625 (1989); Aggarwal et al., 260 J. Biol. Chem. 2345
(1985); Pennica et al., 312
Nature 724 (1984)).
Also of interest regarding cytokines are proteins or chemical moieties that
interact with
cytokines, such as Matrix Metalloproteinases (MMPs) (U.S. Patent No.
6,307,089; Nagase, Matrix
Metalloproteinases in Zinc Metalloproteinases in Health and Disease (1996)),
and Nitric Oxide
Synthases (NOS) (Fukuto, 34 Adv. Pharm 1 (1995); U.S. Patent No. 5,268,465).
The present invention may also be used to affect blood proteins, a generic
name for a vast
group of proteins generally circulating in blood plasma, and important for
regulating coagulation and
clot dissolution. See, e.g., Haematologic Technologies, Inc., HTI CATALOG, at
www.haemtech.com.
2 0 Table 3 introduces, in a non-limiting fashion, some of the blood proteins
contemplated by the
present invention.
Table 3: Blood Proteins
Protein Principle Activity Reference
Factor V In coagulation, this glycoproteinMann et al., 57 ANN. REV.
pro- BIOCHEM.
cofactor, is converted 915 (1988); see also Nesheim
to active cofactor, et al., 254
factor Va, via the serine J. BIOL. CHEM. 508 (1979);
protease a- Tracy et al.,
thrombin, and less efficiently60 BLOOD 59 (1982); Nesheim
by its et al., 80
serine protease cofactor METHODS ENZYMOL. 249 (1981);
Xa. The Jenny
prothrombinase complex et al., 84 PROC. NATL. ACRD.
rapidly SCI. USA
converts zymogen prothrombin4846 (1987).
to the
active serine protease,
a-thrombin.
Down regulation of prothrombinase
complex occurs via inactivation
of Va
b activated rotein C.
24
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Protein Princi le Activi Reference
_
Factor VII Single chain glycoprotein See generally, Broze et
zymogen in aL, 80
its native form. ProteolyticMETHODS ENZYMOL. 228 (1981);
activation Bajaj
yields enzyme factor VIIa,et aL, 256 J. BIOL. CHEM.
which binds 253 (1981);
to integral membrane proteinWilliams et al., 264 J.
tissue BIOL. CHEM.
factor, forming an enzyme 7536 (1989); Kisiel et al.,
complex that 22
proteolytically converts THROMBOSIS RES. 375 (1981);
factor X to Xa.
Also known as extrinsic Seligsohn et al., 64 J.
factor Xase CLIN. INVEST.
complex. Conversion of 1056 (1979); Lawson et al.,
VII to VIIa 268 J. BIOL.
catalyzed by a number of CHEM. 767 (1993).
proteases
including thrombin, factors
IXa, Xa,
XIa, and XIIa. Rapid activation
also
occurs when VII combines
with tissue
factor in the presence
of Ca, likely
initiated by a small amount
of pre-
existing VIIa. Not readily
inhibited by
antithrombin III/heparin
alone, but is
inhibited when tissue factor
added.
Factor IX Zymogen factor IX , a singleThompson, 67 BLOOD, 565
chain (1986);
vitamin K-dependent glycoprotein,Hedner et al., HEMOSTASIS
AND
made in liver. Binds to THROMBOSIS 39-47 (R.W. Colman,
negatively J.
charged phospholipid surfaces.Hirsh, V.J. Marder, E.W.
Salzman ed.,
Activated by factor XIa 2"d ed. J.P. Lippincott
or the factor Co., Philadelphia)
VIIa/tissue factor/phospholipid1987; Fujikawa et al., 45
METHODS IN
complex. Cleavage at one ENZYMOLOGY 74 (1974).
site yields the
intermediate IXa, subsequently
converted to fully active
form IXa(3 by
cleavage at another site.
Factor IXa(3 is
the catalytic component
of the "intrinsic
factor Xase complex" (factor
VIIIaIIXa/Ca2+/phospholipid)
that
proteolytically activates
factor X to
factor Xa.
Factor X Vitamin K-dependent proteinSee Davie et al., 48 ADV.
zymogen, ENZYMOL 277
made in liver, circulates (1979); Jackson, 49 ANN.
in plasma as a REV.
two chain molecule linked BIOCHEM. 765 (1980); see
by a disulfide also
bond. Factor Xa (activatedFujikawa et al., 11 BIOCHEM.
X) serves as 4882
the enzyme component of (1972); Discipio et aL,
16 BIOCHEM.
prothrombinase complex, 698 (1977); Discipio et
responsible al., 18
for rapid conversion of BIOCHEM. 899 (1979); Jackson
prothrombin to et al., 7
thrombin. BIOCHEM. 4506 (1968); McMullen
et
al., 22 BIOCHEM. 2875 (1983).
CA 02490409 2004-12-24
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Protein Princi le Activi Reference
Factor Liver-made glycoprotein Thompson et al., 60 J. CLIN.
XI homodimer INVEST.
circulates, in a non-covalent1376 (1977); I~urachi et
complex al., 16
with high molecular weightBIOCHEM. 5831 (1977); Bouma
kininogen, et al.,
as a zymogen, requiring 252 J. BIOL. CHEM. 6432
proteolytic (1977);
activation to acquire serineWuepper, 31 FED, PROC. 624
protease (1972);
activity. Conversion of Saito et al., 50 BLOOD 377
factor XI to (1977);
factor XIa is catalyzed Fujikawa et al., 25 BIOCHEM.
by factor XIIa. 2417
XIa unique among the serine(1986); Kurachi et al.,
proteases, 19 BIOCHEM.
since it contains two active1330 (1980); Scott et al.,
sites per 69 J. CLIN.
molecule. Works in the INVEST. 844 (1982).
intrinsic
coagulation pathway by
catalyzing
conversion of factor IX
to factor IXa.
Complex form, factor XIa/I~vIWI~,
activates factor XII to
factor XIIa and
prekallikrein to kallikrein.
Major
inhibitor of XIa is al-antitrypsin
and to
lesser extent, antithrombin-III.
Lack of
factor XT procoagulant
activity causes
bleeding disorder; plasma
thrombo lastin antecedent
deficienc .
Factor Glycoprotein zymogen. ReciprocalSchmaier et al., 18-38,
XII and Davie, 242-
(Hageman activation of XII to active267 HEMOSTASIS & THROMBOSIS
serine
Factor) protease factor XIIa by (Colman et al., eds., J.B.
kallikrein is Lippincott Co.,
central to start of intrinsicPhiladelphia, 1987).
coagulation
pathway. Surface bound
a-XITa activates
factor XI to XIa. Secondary
cleavage of
a-XlIa by kallikrein yields
~3-XIIa, and
catalyzes solution phase
activation of
kallikrein, factor VII
and the classical
com lement cascade.
26
CA 02490409 2004-12-24
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Protein Princi le Activi Reference
_
Factor XITI Zymogenic form of glutaminyl-peptideSee McDonaugh, 340-3S7
fIEMOSTASIS
y-glutamyl transferase factor& THROMBOSIS (Colman et
XIIIa al., eds.,
(fibrinoligase, plasma transglutaminase,J.B. Lippincott Co., Philadelphia,
1987);
fibrin stabilizing factor).Folk et al., 113 METHODS
Made in the ENZYMOL.
liver, found extracellularly364 (1985); Greenberg et
in plasma al., 69 BLOOD
and intracellularly in platelets,867 (1987). Other proteins
known to be
megakaryocytes, monocytes, substrates for Factor XIIIa,
placenta, that may be
uterus, liver and prostratehemostatically important,
tissues. include
Circulates as a tetx-amer fibronectin (Iwanaga et
of 2 pairs of al., 312 ANN.
nonidentical subunits (AZBZ).NY AcAD. ScI. 56 (1978)),
Full a2-
expression of activity is antiplasmin (Sakata et al.,
achieved only 65 J. CLIN.
after the Caz+- and fibrin(ogen)-INVEST. 290 (1980)), collagen
(Mosher
dependent dissociation of et al., 64 J. CLIN. INVEST.
B subunit 781 (1979)),
dimer from AZ' dimer. Last factor V (Francis et al.,
of the 261 J. BIOL.
zymogens to become activatedCHEM. 9787 (1986)), von
in the Willebrand
coagulation cascade, the Factor (Mosher et al., 64
only enzyme in J. CLIN.
this system that is not INVEST. 781 (1979)) and
a serine protease.
XIfIa stabilizes the fibrinthrombospondin (Bale et
clot by al., 260 J.
crosslinking the a and y-chainsBIOL. CHEM. 7502 (I98S);
of fibrin. Bohn, 20
Serves in cell proliferationMOL. CELL BIOCHEM. 67 (1978)).
in wound
healing, tissue remodeling,
atherosclerosis, and tumor
owth.
27
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Protein Princi le Activi Reference
Fibrinogen Plasma fibrinogen, a large FURLAN, Fibrinogen, IN HUMAN
glycoprotein,
disulfide linked dimer madePROTEIN DATA, (Haeberli,
of 3 pairs ed., VCH
of non-identical chains Publishers, N.Y.,1995);
(Aa, Bb and g), Doolittle, in
made in liver. Aa has N-terminalHAEMOSTASIS & THROMBOSIS,
peptide 491-513
(fibrinopeptide A (FPA), (3rd ed., Bloom et al.,
factor X>TIa eds., Churchill
crosslinking sites, and Livingstone, 1994); HANTGAN,
2 et al., in
phosphorylation sites. Bb HAEMOSTASIS & THROMBOSIS
has 269-89
fibrinopeptide B (FPB), (2d ed., Forties et al.,
1 of 3 N-linked eds., Churchill
carbohydrate moieties, and Livingstone, 1991).
an N-
terminal pyroglutamic acid.
The g chain
contains the other N-linked
glycos. site,
and factor X)TIa cross-linking
sites. Two
elongated subunits ((AaBbg)z)
align in
an antiparallel way forming
a trinodular
arrangement of the 6 chains.
Nodes
formed by disulfide rings
between the 3
parallel chains. Central
node (n-
disulfide knot, E domain)
formed by N-
termini of all 6 chains
held together by
11 disulfide bonds, contains
the 2 IIa-
sensitive sites. Release
of FPA by
cleavage generates Fbn 1,
exposing a
polymerization site on Aa
chain. These
sites bind to regions on
the D domain of
Fbn to form proto-fibrils.
Subsequent IIa
cleavage of FPB from the
Bb chain
exposes additional polymerization
sites,
promoting lateral growth
of Fbn
network. Each of the 2 domains
between
the central node and the
C-terminal
nodes (domains D and E)
has parallel a-
helical regions of the Aa,
Bb and g
chains having protease-
(plasmin-)
sensitive sites. Another
major plasmin
sensitive site is in hydrophilic
preturbance of a-chain from
C-terminal
node. Controlled plasmin
degradation
converts Fb into fra ents
D and E.
as
CA 02490409 2004-12-24
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Protein Princi le Activi Reference
Fibronectin High molecular weight, Skorstengaard et aL, I61
adhesive, Eur. J.
glycoprotein found in plasmaBIOCHEM. 44I (1986); Kornblihtt
and et al.,
extracellular matrix in 4 EMBO J. 17SS (1985); Odermatt
slightly different et
forms. Two peptide chains al., 82 PNAS 6571 (1985);
Hynes, R.O.,
interconnected by 2 disulfideANN. REV. CELL BIOL., 1,
bonds, has 67 (1985);
3 different types of repeatingMosher 3S ANN. REV. MED.
561
homologous sequence units.(1984); Rouslahti et al.,
Mediates 44 Cell 517
cell attachment by interacting(1986); Hynes 48 CELL 549
with cell (1987);
surface receptors and extracellularMosher 250 BIOL. CHEM. 6614
(1975).
matrix components. Contains
an Arg-
Gly-Asp-Ser (RGDS) cell
attachment-
promoting sequence, recognized
by
specific cell receptors,
such as those on
platelets. Fibrin-fibronectin
complexes
stabilized by factor XIIIa-catalyzed
covalent cross-linking
of fibronectin to
the fibrin a chain.
b2-GlycoproteinAlso called bzI and Apolipoproteinsee, e.g., Lozier et al.,
I H. 81 PNAS 2640-
Highly glycosylated single44 (1984); Kato & Enjyoi
chain protein 30 BIOCHEM.
made in liver. Five repeating11687-94 (1997); Wurm, 16
mutually INT'L J.
homologous domains consistingBIOCHEM. S11-15 (1984);
of Bendixen et
approximately 60 amino al., 31 BIOCHEM. 3611-17
acids disulfide (1992);
bonded to form Short ConsensusSteinkasserer et al., 277
BIOCHEM. J.
Repeats (SCR) or Sushi 387-91 (1991); Nimpf et
domains. al., 884
Associated with lipoproteins,BIOCHEM. BIOPHYS. ACTH 142-49
binds
anionic surfaces like anionic(1986); Kroll et.al. 434
vesicles, BIOCHEM.
platelets, DNA, mitochondria,BIOPHYS. Acta 490-501 (1986);
and Polz et
heparin. Binding can inhibital., 11 INT'L J. BIOCHEM.
contact 265-73
activation pathway in blood(1976); McNeil et al., 87
coagulation. PNAS 4120-24
Binding to activated platelets(1990); Galli et al;. I
inhibits LANCET 1544-47
platelet associated prothrombinase(1990); Matsuuna et al.,
and II LANCET 177-
adenylate cyclase activities.78 (1990); Pengo et al.,
Complexes 73 THROMBOSIS
between b2I and cardiolipin& HAEMOSTASIS 29-34 (1995).
have been
implicated in the anti-phospholipid
related immune disorders
LAC and
SLE.
Osteonectin Acidic, noncollagenous Villarreal et al., 28 BIOCHEM.
glycoprotein 6483
(Mr=29,000) originally (1989); Tracy et al., 29
isolated from INT'L J.
fetal and adult bovine BIOCHEM. 653 (1988); Romberg
bone matrix . May et al.,
regulate bone metabolism 2S BIOCHEM. 1176 (1986);
by binding Sage &
hydroxyapatite to collagen.Bornstein 266 J. BIOL. CHEM.
Identical to 14831
human placental SPARC. (1991); Kelm & Mann 4 J.
An alpha BONE MIN.
granule component of humanRES. 5245 (I989); Kelm et
platelets al., 80
secreted during activation.BLOOD 3112 ( I 992).
A small
portion of secreted osteonectin
expressed on the platelet
cell surface in
an activation-de endent
manner
29
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Protein Princi le Activity Reference
Plasminogen Single chain glycoprotein See Robbins, 45 METHODS
zymogen with IN
24 disulfide bridges, no ENZYMOLOGY 257 (I976); COLLEN,
free sulfhydryls,
and 5 regions of internal 243-258 BLOOD COAL. (Zwaal
sequence et al.,
homology, "kringles", eacheds., New York, Elsevier,
five triple- 1986); see
looped, three disulfide also Castellino et al.,
bridged, and 80 METHODS 1N
homologous to kringle domainsENZYMOLOGY 365 (1981); Wohl
in t-PA, et al.,
u-PA and prothrombin. Interaction27 THROMB. IZES. 523 (1982);
of Barlow
plasminogen with fibrin et al., 23 BIOCHEM. 2384
and a2- (1984);
antiplasmin is mediated SOTTRUP-JENSEN ET AL., 3
by lysine PROGRESS IN
binding sites. Conversion CHEM. FIBRINOLYSIS & THROMBOLYSIS
of
plasminogen to plasmin 197-228 (Davidson et al.,
occurs by eds., Raven
variety of mechanisms, Press, New York 1975).
including
urinary type and tissue
type
plasminogen activators,
streptokinase,
staphylokinase, kallikrein,
factors IXa
and XIIa, but all result
in hydrolysis at
Arg560-Va1561, yielding
two chains
that remain covalently
associated by a
disulfide bond.
tissue t-PA, a serine endopeptidaseSee Plasminogen.
Plasminogen synthesized by endothelial
cells, is the
Activator major physiologic activator
of
plasminogen in clots, catalyzing
conversion of plasminogen
to plasmin
by hydrolising a specific
arginine-
alanine bond. Requires
fibrin for this
activity, unlike the kidney-produced
version, urokinase-PA.
Plasmin See Plasminogen. Plasmin, See Plasminogen.
a serine
protease, cleaves fibrin,
and activates
and/or degrades compounds
of
coagulation, kinin generation,
and
complement systems. Inhibited
by a
number of plasma protease
inhibitors in
vitro. Regulation of plasmin
in vivo
occurs mainly through interaction
with
az-antiplasmin, and to
a lesser extent, a2-
macro lobulin.
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Protein Principle Active Reference
Platelet Low molecular weight, heparin-bindingRucinski et al., 53 BLOOD
Factor-4 47 (1979);
protein secreted from agonist-activatedKaplan et al., 53 BLOOD
604 (1979);
platelets as a homotetramerGeorge 76 BLOOD 859 (1990);
in complex Busch et
with a high molecular weight,al., 19 THROMB. RES. 129
(1980); Rao
proteoglycan, carrier protein.et al., 6I BLOOD 1208 (1983);
Lysine- Brindley,
rich, COOH-terminal region et al., 72 J. CLIN. INVEST.
interacts 1218 (1983);
with cell surface expressedDeuel et al., 74 PNAS 2256
heparin-like (1981);
glycosaminoglycans on endothelialOsterman et al., 107 BIOCHEM.
cells, PF-4 neutralizes BIOPHYS. RES. CoMMLIN. 130
anticoagulant (1982);
activity of heparin exerts Capitanio et al., 839 BIOCHEM.
procoagulant
effect, and stimulates releaseBIOPHYS. ACTH 161 (1985).
of
histamine from basophils.
Chemotactic
activity toward neutrophils
and
monocytes. Binding sites
on the platelet
surface have been identified
and may be
im ortant for latelet a
a ation.
Protein C Vitamin K-dependent zymogen,See Esmon, 10 PROGRESS IN
protein THROMB.
C, made in liver as a single& HEMOSTS. 25 (1984); Stenflo,
chain 10
polypeptide then converted SEM1N. IN THROMB. & HEMOSTAS.
to a 109
disulfide linked heterodimer.(1984); Griffen et aL, 60
Cleaving BLOOD 261
the heavy chain of human (1982); Kisiel et al., 80
protein C METHODS
converts the zymogen into ENZYMOL. 320 (1981); Discipio
the serine et al.,
protease, activated protein18 BIOCHEM. 899 (1979).
C. Cleavage
catalyzed by a complex of
a-thrombin
and thrombomodulin. Unlike
other
vitamin K dependent coagulation
factors, activated protein
C is an
anticoagulant that catalyzes
the
proteolytic inactivation
of factors Va
and VIIIa, and contributes
to the
fibrinolytic response by
complex
formation with plasminogen
activator
inhibitors.
Protein S Single chain vitamin K-dependentWalker 10 SEMIN. THROMB.
protein functions in coagulationHEMOSTAS. 131 (1984); Dahlback
and et al.,
complement cascades. Does 10 SEMIN. THROMB. HEMOSTAS.,
not possess 139
the catalytic triad. Complexes(1984); Walker 261 J. BIOL.
to C4b CHEM.
binding protein (C4BP) and 10941 (1986).
to
negatively charged phospholipids,
concentrating C4BP at cell
surfaces
following injury. Unbound
S serves as
anticoagulant cofactor protein
with
activated Protein C. A single
cleavage
by thrombin abolishes protein
S cofactor
activi b removin la domain.
31
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Protein Princi le Activi Reference
Protein Z Vitamin K-dependent, single-chainSejima et al., 171 BIOCHEM.
protein made in the liver. BIOPHYSICS RES. COMM. 661
Direct (I990);
requirement for the bindingHogg et al., 266 J. BIOL.
of thrombin CHEM. 10953
to endothelial phospholipids.( 1991 ); Hogg et al., 17
Domain BIOCHEM.
structure similar to that BIOPHYSICS RES. COMM. 801
of other vitamin (1991);
K-dependant zymogens like Han et al., 38 BIOCHEM.
factors VII, 11073 (1999);
IX, X, and protein C. N-terminalKemkes-Matthes et al., 79
region THROMB.
contains carboxyglutamic RES. 49 (1995).
acid domain
enabling phospholipid membrane
binding. C-terminal region
lacks
"typical" serine protease
activation site.
Cofactor for inhibition
of coagulation
factor Xa by serpin called
protein Z-
dependant protease inhibitor.
Patients
diagnosed with protein Z
deficiency
have abnormal bleeding diathesis
during
and after sur ical events.
Prothrombin Vitamin K-dependent, single-chainMann et al., 45 METHODS
IN
protein made in the liver. ENZYMOLOGY 156 (1976); Magnusson
Binds to
negatively charged phospholipidet al., PROTEASES IN BIOLOGICAL
membranes. Contains two CONTROL 123-I49 (Reich et
"kringle" al., eds.
structures. Mature protein Cold Spring Harbor Labs.,
circulates in New York
plasma as a zymogen and, 1975); Discipio et al.,
during 18 BIOCHEM. 899
coagulation, is proteolytically( 1979).
activated
to the potent serine protease
a-thrombin.
a-Thrombin See Prothrombin. During 45 METHODS ENZYMOL. 156
coagulation, (1976).
thrombin cleaves fibrinogen
to form
fibrin, the terminal proteolytic
step in
coagulation, forming the
fibrin clot.
Thrombin also responsible
for feedback
activation of procofactors
V and VIII.
Activates factor XIII and
platelets,
functions as vasoconstrictor
protein.
Procoagulant activity arrested
by
heparin cofactor II or the
antithrombin
III/heparin complex, or
complex
formation with thrombomodulin.
Formation of thrombin/thrombomodulin
complex results in inability
of thrombin
to cleave fibrinogen and
activate factors
V and VIII, but increases
the efficiency
of thrombin for activation
of the
anticoa ulant, rotein C.
b-Thrombo- Low molecular weight, heparin-binding,See, e.g., George 76 BLOOD
859 (1990);
globulin platelet-derived tetramer Holt & Niewiarowski 632
protein, BIOCHIM.
consisting of four identicalBIOPHYS. ACTH 284 (1980);
peptide
chains. Lower affinity for Niewiarowski et al., 55
heparin than BLOOD 453
PF-4. Chemotactic activity (1980); Varma et al., 701
for human BIOCHIM.
fibroblasts, other functionsBIOPHYS. ACTH 7 (1982);
unknown. Senior et al.,
96 J. CELL. BIOL. 382 (1983).
32
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Protein Principle Activity Reference
ThrombopoietinHuman TPO (Thrombopoietin, Horikawa et al., 90(10)
Mpl- BLOOD 4031-38
ligand, MGDF) stimulates (1997); de Sauvage et al.,
the 369 NATURE
proliferation and maturation533-58 (1995).
of
megakaryocytes and promotes
increased
circulating levels of platelets
in vivo.
Binds to c-M 1 rece tor.
Thrombo-spondinHigh-molecular weight, heparin-bindingDawes et al., 29 THROMB.
RES. 569
glycoprotein constituent (1983); Switalska et al.,
of platelets, 106 J. LAB.
consisting of three, identical,CLIN, MED. 690 (1985);
disulfide- Lawler et al.
linked polypeptide chains. 260 J. BIOL. CHEM. 3762
Binds to (1985); Wolff
surface of resting and activatedet al., 261 J. BIOL. CHEM.
6840 (1986);
platelets, may effect plateletAsch et al., 79 J. CLIN.
adherence CHEM. 1054
and aggregation. An integral(1987); Jaffe et al., 295
component NATURE 246
of basement membrane in ( 1982); Wright et al.,
different 33 J. HISTOCHEM.
tissues. Interacts with CYTOCHEM. 295 (1985); Dixit
a variety of et al.,
extracellular macromolecules259 J. BIOL. CHEM. 10100
including (1984);
heparin, collagen, fibrinogenMumby et al., 98 J. CELL.
and BIOL. 646
fibronectin, plasminogen, (1984); Lahav et al, 145
plasminogen EUR. J.
activator, and osteonectin.BIOCHEM. 151 (1984); Silverstein
May et a1,
modulate cell-matrix interactions.260 J. BIOL. CHEM. 10346
(1985);
Clezardin et al. 175 EUR.
J. BIOCHEM.
275 (1988); Sa a & Bornstein
(1991).
Von WillebrandMultimeric plasma glycoproteinHoyer 58 BLOOD 1 (1981);
made Ruggeri ~c
Factor of identical subunits held Zimmerman 65 J. CLIN. INVEST.
together by 1318
disulfide bonds. During (1980); Hoyer & Shainoff
normal SS BLOOD
hemostasis, larger multimers1056 (1980); Meyer et al.,
of vWF 95 J. LAB.
cause platelet plug formationCLIN. INVEST. 590 (1980);
by Santoro 21
forming a bridge between THROMB. RES. 689 (1981);
platelet Santoro, &
glycoprotein IB and exposedCowan 2 COLLAGEN RELAT.
collagen in RES. 31
the subendothelium. Also (1982); Morton et al.,
binds and 32 THROMB. RES.
transports factor VIII (antihemophilic545 (1983); Tuddenham et
al., 52 BRIT.
factor) in lasma. J. HAEMATOL. 259 (1982).
Additional blood proteins contemplated herein include the following human
serum proteins,
which may also be placed in another category of protein (such as hormone or
antigen): Actin, Actinin,
Amyloid Serum P, Apolipoprotein E, B2-Microglobulin, C-Reactive Protein (CRP),
Cholesterylester
transfer protein (CETP), Complement C3B, Ceruplasmin, Creative I~inase,
Cystatin, Cytokeratin 8,
Cytokeratin 14, Cytokeratin 18, Cytokeratin 19, Cytokeratin 20, Desmin,
Desmocollin 3, FAS (CD95),
Fatty Acid Binding Protein, Ferritin, Filamin, Glial Filament Acidic Protein,
Glycogen Phosphorylase
Isoenzyme BB (GPBB), Haptoglobulin, Human Myoglobin, Myelin Basic Protein,
Neurofilament,
Placental Lactogen, Human SHBG, Human Thyroid Peroxidase, Receptor Associated
Protein, Human
Cardiac Troponin C, Human Cardiac Troponin I, Human Cardiac Troponin T, Human
Skeletal
Troponin I, Human Skeletal Troponin T, Vimentin, Vinculin, Transferrin
Receptor, Prealbumin,
Albumin, Alpha-1-Acid Glycoprotein, Alpha-I-Antichymotrypsin, Alpha-1-
Antitrypsin, Alpha-
33
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Fetoprotein, Alpha-1-Microglobulin, Beta-2-microglobulin, C-Reactive Protein,
Haptoglobulin,
Myoglobulin, Prealbumin, PSA, Prostatic Acid Phosphatase, Retinol Binding
Protein, Thyroglobulin,
Thyroid Microsomal Antigen, Thyroxine Binding Globulin, Transferrin , Troponin
I, Troponin T,
Prostatic Acid Phosphatase, Retinol Binding Globulin (RBP). All of these
proteins, and sources
thereof, are known in the art. Many of these proteins are available
commercially from, for example,
Research Diagnostics, Inc. (Flanders, N.J.).
The target in the present invention may also incorporate ox target
neurotransmitters, or
functional portions thereof. Neurotransmitters are chemicals made by neurons
and used by them to
transmit signals to the other neurons or non-neuronal cells (e.g., skeletal
muscle; myocardium, pineal
glandular cells) that they innervate. Neurotransmitters produce their effects
by being released into
synapses when their neuron of origin fires (i,e., becomes depolarized) and
then attaching to receptors in
the membrane of the post-synaptic cells. This causes changes in the fluxes of
particular ions across
that membrane, making cells more likely to become depolarized, if the
neurotransmitter happens to be
excitatory, or less likely if it is inhibitory. Neurotransmitters can also
produce their effects by
modulating the production of other signal-transducing molecules ("second
messengers") in the post-
synaptic cells. See generally COOPER, BLOOM & ROTH, THE BIOCHEMICAL BASIS OF
NEUROPHARMACOLOGY (7th Ed. Oxford Univ. Press, NYC, 1996);
http://web.indstate.edu/thcme/mwking/nerves. Neurotransmitters contemplated in
the present
invention include, but are not limited to, Acetylcholine, Serotonin, y-
aminobutyrate (GABA),
2 0 Glutamate, Aspartate, Glycine, Histamine, Epinephrine, Norepinephrine,
Dopamine, Adenosine, ATP,
Nitric oxide, and any of the peptide neurotransmitters such as those derived
from pre-opiomelanocortin
(POMC), as well as antagonists and agonists of any of the foregoing.
Numerous other proteins or peptides may serve as either targets, or as a
source of target-
binding moieties as described herein. Table 4 presents a non-limiting list and
description of some
2 5 pharmacologically active peptides that may serve as, or serve as a source
of a functional derivative of,
the target of the present invention.
Table 4: Pharmacologically active peptides
Binding partner/ Pharmacological Reference
activity
Protein of interest
(form of a tide)
c-Mpl TPO-mimetic Cwirla et al., 276 SCIENCE
1696-9 (1997);
(linear) U.S. Pat. No. 5,869,451,
issued Feb.
9,1999; U.S. Pat. No. 5,932,946,
issued
Au . 3,1999.
c-Mpl TPO-mimetic Cwirla et al., 276 SCIENCE
1696-9 (1997).
(C-terminally
cross-
linked dimer)
34
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Binding partner/ Pharmacological Reference
activity
Protein of interest
form of a tide)
(disulfide-linked stimulation of Paukovits et al., 364 HOPPE-SEYLERS
Z.
dimer) hematopoesis PHYSIOL. CHEM. 30311 (1984);
("G-CSF-mimetic") Laerurngal., 16 EXP. HEMAT.
274-80
(1988).
(alkylene-linked G-CSF-mimetic Batnagar et al., 39 J. MEI7.
dimer) CHEM. 38149
(1996); Cuthbertson et al.,
40 J. MED.
CHEM. 2876-82 (1997); King
et al., 19
ExP. HEMATOL. 481 ( 1991
); King et al.,
86 Su 1. 1) BLOOD 309 (1995).
IL-1 receptor inflammatory and U.S. Pat. No. 5,608,035;
U.S. Pat. No.
(linear) autoimmune diseases5,786,331; U.S Pat. No.
("IL-1 5,880,096;
antagonist" or "IL-1Yanofsky et al., 93 PNAS
ra- 7381-6 (1996);
mimetic") Akeson et al., 271 J. BIOL.
CHEM. 30517-
23 (1996); Wiekzorek et
al., 49 PoL. J.
PHARMACOL. 107-17 (1997);
Yanofsky,
93 PNAS 7381-7386 (1996).
Facteur thyrnique stimulation of lymphocytesTnagaki-Ohara et al., 171
CELLULAR
(linear) (FTS-mimetic) IMMUNOL. 30-40 (1996); Yoshida,
6 J.
IMMUNOPHARMACOL 141-6 (1984).
CTLA4 MAb CTLA4-mimetic Fukumoto et al., 16 NATURE
BIOTECH.
(intrapeptide di-sulfide 267-70 (1998).
bonded
TNF-a receptor TNF-a antagonist Takasaki et al., 15 NATURE
BIOTECH.
(exo-cyclic) 1266-70 (1997); WO 98/53842,
published
December 3, 1998.
TNF-a receptor TNF-a antagonist Chirinos-Rojas, J. IMM.,
5621-26.
(linear)
C3b inhibition of complementSahu et al., 157 IMMUNOL.
884-91 (1996);
(intrapeptide di-sulfideactivation; autoimmuneMorikis et al., 7 PROTEIN
SCI. 619-27
bonded) diseases (C3b ante (1998).
onist)
vinculin cell adhesion processes,Adey et al., 324 BIOCHEM.
cell J. 523-8
(linear) growth, differentiation( 1997).
wound healing, tumor
metastasis ("vinculin
bindin ")
C4 binding protein anti-thrombotic Linse et al. 272 BIOL. CHEM.
(C413P) 14658-65
(linear) ( 1997).
urokinase receptor processes associatedGoodson et al., 91 PNAS
with 7129-33 (1994);
(linear) urokinase interactionInternational patent application
with its WO
receptor (e.g. angiogenesis,97/35969, published October
2, 1997.
tumor cell invasion
and
metastasis; (URK
ante onist)
Mdm2, Hdm2 Inhibition of inactivationPicksley et al., 9 ONCOGENE
of 2523-9
(linear) p53 mediated by (1994); Bottger et al. 269
Mdm2 or J. MoL. BIOL.
hdm2; anti-tumor 744-56 (1997); Bottger et
al., 13
("Mdm/hdm ante onist")ONCOGENE 13: 2141-7 (1996).
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Binding partner/ Pharmacological Reference
activity
Protein of interest
form of a tide
p21 W~1 anti-tumor by mimickingBall et aL, 7 CURB. BIOL.
the 7I-80 (1997).
(linear) activi of 21~''~1
farnesyl transferaseanti-cancer by preventingGibbs et al., 77 CELL 175-I78
(1994).
(linear) activation of ras
onco ene
Ras effector domain anti-cancer by inhibitingMoodie et at., 10 TRENDS
GEL1EL 44-48
(linear) biological function(1994); Rodriguez et al.,
of the ras 370 NATURE
onco ene 527-532 (1994).
SH2/SH3 domains anti-cancer by inhibitingPawson et al, 3 CURB. BIOL.
434-432
(linear) tumor growth with (1993); Yu et al., 76 CELL
activated 933-945
osine kinases (1994).
p 16'4 anti-cancer by mimickingFahraeus et al., 6 CURB.
BIOL. 84-91
(linear) activity of p16; (1996).
e.g.,
inhibiting cyclin
D-Cdk
corn lex (" ,16-mimetic")
Src, Lyn inhibition of Mast Stauffer et al., 36 BIOCHEM.
cell 9388-94
(linear) activation, IgE-related( 1997).
conditions, type
I
hypersensitivity
("Mast cell
anta onist").
Mast cell protease treatment of inflammatoryInternational patent application
WO
(linear) disorders mediated 98/33812, published August
by 6, 1998.
release of tryptase-6
("Mast
cell rotease inhibitors")
SH3 domains treatment of SH3-mediatedRickles et al., 13 EMBO
J.
(linear) disease states ("SH35598-5604 (1994); Sparks
et al.,
antagonist") 269 J. BIOL. CHEM. 238536
(1994); Sparks et al., 93
PNAS
1540-44 ( 1996).
HBV core antigen treatment of HBV Dyson & Muray, PNAS 2194-98
(HBcAg) viral
(linear) antigen (HBcAg) (1995).
infections
("anti-HBV")
selectins neutrophil adhesionMartens et al., 270 J. BIOL.
(linear) inflammatory diseasesCHEM. 21129-36 (1995);
("selectin antagonist")European Pat. App. EP 0
714
912, ublished June 5, 1996.
calmodulin calmodulin Pierce et al., 1 MOLEC.
(linear, cyclized) antagonist DIVEMILY 25965 (1995);
Dedman et al., 267 J. BIOL.
CHEM. 23025-30 (1993); Adey
& I~ay, 169 GENE 133-34
( 1996).
36
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Binding partner/ Pharmacological Reference
activity
Protein of interest
form of a tide
integrins tumor-homing; treatmentInternational patent applications
fo WO
(linear, cyclized) conditions related 95/14714, published June
to 1, I99S; WO
integrin-mediated 97/08203, published March
cellular 6,1997; WO
events, including 98/20795, published March
platelet 19,1998; WO
aggregation, thrombosis,99/24462, published May
20, 1999; Kraft
wound healing, osteoporosis,et al., 274 J. BIOL. CHEM.
1979-8S
tissue repair, angiogenesis(1999).
(e.g., for treatment
of
cancer) and tumor
invasion
("rote in-bindin
")
fibronectin and extracellulartreatment of inflammatoryInternational patent
application
WO
matrix components and autoimmune conditions98/09985, published March
of T-cells 12, 1998.
and macrophages
(c clic, linear)
somatostatin and treatment or preventionEuropean patent application
cortistatin of EP 0 912
(linear) hormone-producing 393, published Apr. 28,
tumors, 2999.
acromegaly, giantism,
dementia, gastric
ulcer,
tumor growth, inhibition
of
hormone secretion,
modulation of sleep
or
neural activi
bacterial lipopoly-saccharideantibiotic; septic U.S. Pat. No. 5,877,151,
shock; issued March 2,
(linear) disorders modulatable1999.
by
CAP37
parclaxin, mellitin antipathogenic International patent application
WO
(linear or c clic) 97/31019, ublished 28 Au
st 1997.
VIP impotence, neuro- International patent application
WO
(linear, c clic) de enerative disorders97/40070, ublished October
30, 1997.
CTLs cancer European patent application
EP 0 770
(linear) 624, ublished Ma 2,1997.
THF-gamrna2 Burnstein, 27 BIOCHEM. 4066-71
(1988).
(linear)
Amylin Cooper, 84 PNAS 8628-32
(1987).
(linear)
Adreno-medullin Kitamura, 192 BBRC SS3-60
(1993).
(linear
VEGF anti-angiogenic; Fairbrother, 37 BIOCHEM.
cancer, 17754-64
(cyclic, linear) rheumatoid arthritis,(1998).
diabetic
retinopathy, psoriasis
("VEGF anta onist"'
MMP inflammation and Koivunen, 27 NATURE BIOTECH.
768-74
(cyclic) autoimmune disorders;(1999).
tumor growth ("MMP
inhibitor")
HGH fragment U.S. Pat. No. 5,869,452,
issued
(linear) Feb. 9, 1999.
37
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Binding partner/ Pharmacological Reference
activity
Protein of interest
form of a tide
Echistatin inhibition of plateletGan, 263 J. BIOL. 19827-32
(1988).
a a ation
SLE autoantibody SLE International patent application
WO
(linear 96/30057, ublished Oct.
3, 1996.
GD1 alpha suppression of tumorIshikawa et al., 1 FEBS
LETT. 20-4
metastasis 1998).
anti-phospholipid endothelial cell Blank Mal., 96 PNAS 5164-8
(3-2 activation, (1999).
glycoprotein-1 ((32GPI)anti-phospholipid
syndrome
antibodies (APS), thromboembolic
phenomena,
thrombocytopenia,
and
recurrent fetal
loss
T-Cell Receptor (3 diabetes International patent application
chain WO
(linear) 96/101214, published Apr.
18, 1996.
Binding partner/ Pharmacological Reference
activity
Protein of interest
(form of a tide
EPO receptor EPO mimetic Wrighton et al. (1996),
Science 273:
(intrapeptide 4S8-63; U.S. Pat. No. 5,773,569,
issued
disulfide-bonded) June 30, 1998 to Wri hton
et al.
EPO receptor EPO mimetic Livnah et al. (1996), Science
273: 464-
(C-terminally cross- 71; Wrighton et al. (1997),
Nature
linked dimer) Biotechnology 15:1261-5;
int'1 patent
application WO 96/40772,
published
Dec. 19,1996
EPO receptor EPO mimetic Naranda et al., 96 PNAS
7569-74 (1999)
(linear)
c-Mpl . TPO-mimetic Cwirla et al.(1997) Science
276:1696-9;
(linear) U.S. Pat. No. 5,869,451,
issued Feb.
9,1999; U.S. Pat. No. 5,932,946,
issued
Au . 3,1999
c-Mpl TPO-mimetic Cwirla et al. (1997) Science
276:1696-9
(C-terminally cross-
linked dimer
(disulfide-linked stimulation of Paukovits et al. (1984),
Hoppe-Seylers Z.
dimer) hematopoesis Physiol. Chem. 365: 30311;
Laerurn gal.
("G-CSF-mimetic") (1988), Ex . Hemat. 16:274-80
(alkylene-linked G-CSF-mimetic Batnagar 91-al. (1996),
dimer) linked dimer J.
Med. Chem. 39:38149; Cuthbertson
et al.
(1997), J. Med. Chem. 40:
2876-82; King
et al. (1991), Exp. Hematol.
19:481; King
et al. (1995), Blood 86
(Su 1. 1): 309
38
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Binding partner/ Pharmacological Reference
activity
Protein of interest
form of a tide
IL-1 receptor inflammatory and U.S. Pat. No. 5,608,035;
U.S. Pat. No.
(linear) autoimmune diseases5,786,331; U.S-Pat. No.
("IL-1 5,880,096;
antagonist" or "IL-IYanofsky 91-al. (1996) PNAS
ra- 93:7381-6;
mimetic") Akeson et al. (1996), J.
Biol. Chem. 271:
30S 17-23; Wiekzorek et
al. (1997), Pol. J.
Pharmacol. 49:107-17; Yanofsky
(1996),
PNAs, 93:7381-7386.
Facteur thyrnique stimulation of lymphocytesInagaki-Ohara et al. (1996),
Cellular
(linear) (FTS-mimetic) Immunol. 171: 30-40; Yoshida
(1984), J.
Immuno harmacol, 6:141-6.
CTLA4 MAb CTLA4-mimetic Fukumoto et al. (1998),
Nature Biotech.
(intrapeptide di-sulfide 16:267-70
bonded)
TNF-a receptor TNF-a antagonist Takasaki et al. (1997),
Nature Biotech.
(exo-cyclic) 15:1266-70; WO 98/53842,
published
December 3, 1998,
TNF-a receptor TNF-a antagonist Chirinos-Rojas J. Imm.,
5621-26.
(linear)
C3b inhibition of complementSahu et al. (1996), Immunol.
157:884-91;
(intrapeptide di-sulfideactivation; autoimmuneMorikis et al. (1998), Protein
Sci. 7:619-
bonded) diseases (C3b anta 27.
onist)
vinculin cell adhesion processes,Adey et al. (1997), Biochem.
cell J. 324:523-8
(linear) growth, differentiation
wound healing, tumor
metastasis ("vinculin
bindin ")
C4 binding protein anti-thrombotic Linse et al. 272 Biol. Chem.
(C413P) 14658-65
(linear) 1997)
urokinase receptor processes associatedGoodson et al. (1994), 91
with PNAS 7129-33;
(linear) urokinase interactionInternational application
with its WO 97/35969,
receptor (e.g. angiogenesis,published October 2, 1997
tumor cell invasion
and
metastasis; (URK
anta onist)
Mdm2, Hdm2 Inhibition of inactivationPicksley et al. (1994),
of Oncogene 9: 2523-
(linear) p53 mediated by 9; Bottger et al. (1997)
Mdm2 or J. Mol. Biol. 269:
hdm2; anti-tumor 744-56; Bottger et al. (1996),
Oncogene
("Mdm/hdm anta onist"13: 2141-7
p21 w'~' anti-tumor by mimickingBall et al.(1997), Curr.
the Biol. 7: 71-80.
(linear) activi of 21W'~1
farnesyl transferaseanti-cancer by preventingGibbs et aI. (1994), Cell
77:175-178
(linear) activation of ras
onco ene
Ras effector domain anti-cancer by inhibitingMoodie et at. (1994), Trends
Genel 10:44
(linear) biological function48 Rodriguez et al. (1994),
of the ras Nature
onto ene 370:527-532.
SH2/SH3 domains anti-cancer by inhibitingPawson et al (1993), Curr.
Biol. 3:434-
(linear) tumor growth with 432, Yu et al. (1994), Cell
activated 76:933-945.
osine kinases
39
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Binding partner/ Pharmacological Reference
activity
Protein of interest
form of a tide)
p16~4 anti-cancer by mimickingFahraeus et al. (1996),
Curr. Biol. 6:84-91
(linear) activity of p16;
e.g.,
inhibiting cyclin
D-Cdk
com lex (" , I 6-mimetic")
Src, Lyn inhibition of Mast Stauffer et al. (1997),
cell Biochem. 36: 9388-
(linear) activation, IgE-related94.
conditions, type
I
hypersensitivity
("Mast cell
ants onist").
Mast cell protease treatment of inflammatoryInternational application
WO 98/33812,
(linear) disorders mediated published August 6, 1998
by
release of tryptase-6
("Mast
cell rotease inhibitors")
SH3 domains treatment of SH3-mediatedRickles et al. (1994), EMBO
J.
(linear) disease states ("SH313:5598-5604;Sparks aLal.
antagonist") (1994), J. Biol. Chem. 269:
238536; Sparks et al. (1996),
PNAS 93:1540-44.
HBV core antigen treatment of HBV Dyson & Muray (1995), Proc.
(HBcAg) viral
(linear) antigen (HBcAg) NatI. Acad. Sci. 92:2194-98.
infections
("anti-HBV")
selectins neutrophil adhesionMartens et aI. (1995), J.
Biol.
(linear) inflammatory diseasesChem. 270: 21129-36; European
("selectin antagonist")pat. app.EP 0 714 912, published
June 5, 1996
calmodulin calmodulin Pierce et al. (1995), Molec.
(linear, cyclized) antagonist Divemily l: 25965; Dedman
et
al. (1993), J. Biol. Chem.
268:
23025-30; Adey & Kay (1996),
Gene 169:133-34.
integrins tumor-homing; treatmentInternational applications
fo WO 95/14714,
(linear, cyclized) conditions related published June 1, 1995;
to WO 97/08203,
integrin-mediated published March 6,1997;
cellular WO 98/10795,
events, including published March 19,1998;
platelet WO 99/24462,
aggregation, thrombosis,published May 20, 1999;
Kraft et al.
wound healing, osteoporosis,(1999), J. Biol. Chem. 274:1979-85.
tissue repair, angiogenesis
(e.g., for treatment
of
cancer) and tumor
invasion
("rote in-bindin
")
fibronectin and extracellulartreatment of inflammatoryWO 98/09985, published
March
12, 1998.
matrix components and autoimmune conditions
of T-cells
and macrophages
(c clic, linear)
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Binding partner/ Pharmacological Reference
activity
Protein ofinterest
form of a tide)
somatostatin and cortistatintreatment or preventionEuropean patent application
of 0 911 393,
(linear) hormone-producing published Apr. 28, 1999.
tumors,
acromegaly, giantism,
dementia, gastric
ulcer,
tumor growth, inhibition
of
hormone secretion,
modulation of sleep
or
neural activi
bacterial lipopoly-saccharideantibiotic; septicU.S. Pat. No. 5,877,151,
shock; issued March
(linear) disorders modulatable2,1999.
by
CAP37
parclaxin, mellitin antipathogenic WO 97/31019, published 28
August
(linear or c clic) 1997.
VIP impotence, neuro- WO 97/40070, published October
30,
(linear, cyclic) de enerative disorders1997.
CTLs cancer EP 0 770 624, published May
2,1997.
(linear)
THF-gamma2 Burnstein (1988), Biochem.,
27:4066-71
(linear)
Amylin Cooper (1987), PNAS 84:8628-32.
(linear
Adreno-medullin Kitamura (1993), BBRC, 192:553-60
(linear
VEGF anti-angiogenic; Fairbrother (1998), Biochem.,
cancer, 37:17754-
(cyclic, linear) rheumatoid arthritis,64.
diabetic
retinopathy, psoriasis
("VEGF anta onist"')
MMP inflammation and Koivunen 17 Nature Biotech.,
768-74
(cyclic) autoimmune disorders;(1999).
tumor growth ("MMP
inhibitor")
HGH fragment U.S. Pat. No. 5,869,452.
(linear)
Echistatin inhibition of plateletGan (1988), J. Biol. 263:19827-32.
a a ation
SLE autoantibody SLE WO 96/30057, published Oct.
3, 1996.
(linear)
GD1 alpha suppression of Ishikawa et al., 1 FEBS Lett.
tumor 20-4 (1998).
metastasis
anti-phospholipid endothelial cell Blank Mal. (1999), PNAS 96:
[3-2 activation, 5164-8.
glycoprotein-1 ([32GPI)anti-phospholipid
syndrome
antibodies (APS), thromboembolic
phenomena,
thrombocytopenia,
and
recurrent fetal
loss
T-Cell Receptor (3 diabetes WO 96/101214, published Apr.
chain 18, 1996.
(linear)
41
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Peptides
Any number of peptides may be used in conjunction with the present invention.
Of particular interest are
peptides that mimic the activity of EPO, TPO, growth hormone, G-CSF, GM-CSF,
IL-lra, leptin, CTLA4,
TRAIL, TGF-a, and TGF-Vii. Peptide antagonists are also of interest,
particularly those antagonistic to the
activity of TNF, leptin, any of the interleukins (Ih-1- IL-23, etc.), and
proteins involved in complement
activation (e.g., C3b). Targeting peptides are also of interest, including
tumor-homing peptides,
membrane-transporting peptides, and the like. All of these classes of peptides
may be discovered by
methods described in the references cited in this specification and other
references.
A particularly preferred group of peptides are those that bind to cytokine
receptors.
Cytokines have recently been classified according to their receptor code. See
Inglot (1997),
Archivum Immunologiae a Therapiae Experimentalis 45; 353-7, which is hereby
incorporated
entirely by reference.
Non-limiting examples of suitable peptides for this invention appear in Tables
5
through 21 below. These peptides may be prepared by methods disclosed and/or
known in the
art. Single letter amino acid abbreviations are used in most cases. The X in
these sequences
(and throughout this specification, unless specified otherwise in a particular
instance) means
that any of the 20 naturally occurring amino acid residues or know derivatives
thereof may be
present, or any know modified amino acid thereof. Any of these peptides may be
linked in
tandem (i.e., sequentially), with or without linkers, and a few tandemlinked
examples are
2 0 provided in the table. Linkers are listed as "4" and may be any of the
linkers described herein.
Tandem repeats and linkers are shown separated by dashes for clarity. Any
peptide containing
a cysteinyl residue may optionally be cross-linked with another Cys-containing
peptide, either
or both of which may be linked to a vehicle. A few crosslinked examples are
provided in the
table. Any peptide having more than one Cys residue may form an intrapeptide
disulfide bond,
2 5 as well; see, for example, EPO-mimetic peptides in Table 5. A few examples
of intrapeptide
disulfide-bonded peptides are specified in the table. Any of these peptides
may be derivatized
as described herein, and a few derivatized examples are provided in the table.
For derivatives
in which the carboxyl terminus may be capped with an amino group, the capping
amino group
is shown as NH2. For derivatives in which amino acid residues are substituted
by moieties
3 0 other than amino acid residues, the substitutions are denoted by a S,
which signifies any of the
moieties known in the art, e.g., as described in Bhatnagar et al. (1996), J.
Med. Chem. 39:
3814-9 and Cuthbertson et al. (1997), J. Med. Chem. 40:2876-82, which are
entirely
incorporated by reference. The J substituent and the Z substituents (Z5, Z6,
... Z4o) are as
defined in U.S. Pat. Nos. 5,608,035, 5,786,331, and 5,880,096, which are
entirely incorporated
42
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
herein by reference. For the EPO-mimetic sequences (Table 5), the substituents
Xz through
X11 and the integer "n" are as defined in WO 96/40772, which is entirely
incorporated by
reference. The substituents "lI!" "O," and "+" are as defined in Sparks et al.
(1996), Proc. Natl.
Acad. Sci. 93: 1540-4, which is entirely incorporated by reference. X4, X5,
X6, and X7 are as
defined in U.S. Pat. No. 5,773,569, which is hereby entirely incorporated by
reference, except
that: for integrin-binding peptides, X1, X2, X3, X4, X5, X6, X7, and X8 (Table
10), are as
defined in PCT applications WO 95/14714, published June 1, 1995 and WO
97/08203,
published March 6,1997, which are also entirely incorporated by reference; and
for VIP-
mimetic peptides (Table 13), Xl, Xl', Xl", Xz, X3, X4, Xs, X6, and Z; and the
integers m and n
are as defined in WO 97/40070, published October 30,1997, which is also
entirely
incorporated herein by reference. Xaa and Yaa below are as defined in WO
98/09985,
published March 12,1998, which is entirely incorporated herein by reference.
AAI, AAz, ABI,
ABz, and AC are as defined in International application WO 98/53842, published
December 3,
1998, which is entirely incorporated by reference. Xl, Xz, X3, and X4 in Table
18 only are as,
defined in European application EP 0 911 393, published April 28,1999,
entirely incorporated
herein by reference. Residues appearing in boldface are D-amino acids, but can
be optionally
L-amino acids. All peptides are linked through peptide bonds unless otherwise
noted.
Abbreviations are listed at the end of this specification. In the "SEQ ID NO."
column, "NR"
means that no sequence listing is required for the given sequence.
2 0 Table 5-EPO-mimetic peptide sequences
Sequence/structure: SEQ ID NO:
YXCXXGPXTWXCXP 1
YXCXXGPXTWXCXP-YXCXXGPXTWXCXP 2
YXCXXGPXTWXCXP-A-YXCXXGPXTWXCXP 3
2 5 YXCXXGPXTWXCXP-~-E-amine) 4
K
YXCXXGPXTWXCXP-0- (a-amine) 4
3 0 GGTYSCHFGPLTWVCKPQGG 5
GGDYHCRMGPLTWVCKPLGG 6
GGVYACRMGPITWVCSPLGG 7
VGNYMCHFGPITWVCRPGGG 8
GGLYLCRFGPVTWDCGYKGG 9
43
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GGTYSCHFGPLTWVCKPQGG- I O
GGTYSCHFGPLTWVCKPQGG -D-GGTYSCHFGPLTWVCKPQGG 11
GGTYSCHFGPLTWVCKPQGGSSK 12
GGTYSCHFGPLTWVCKPQGGSSK 13
GGTYSCHFGPLTWVCKPQGGSSK 14
GGTYSCHFGPLTWVCKPQGGSSK-0-GGTYSCHFGPLTWVCKPQGGSSK
GGTYSCHFGPLTWVCKPQGGSS -D-s-amine)
K
/
GGTYSCHFGPLTWVCKPQGGSS-0- (a-amine) 1 S
GGTYSCHFGPLTWVCKPQGGSSK(-~-biotin) 16
CX4XSGPX6TWX~C 17
GGTYSCHGPLTWVCKPQGG 18
VGNYMAHMGPITWVCRPGG 19
GGPHHVYACRMGPLTWIC 20
GGTYSCHFGPLTWVCKPQ 21
GGLYACHMGPMTWVCQPLRG 22
TIAQYICYMGPETWECRPSPKA 23
2 0 YSCIIFGPLTWVCK 24
YCHFGPLTWVC 25
X3XXSGPXgTWX~Xg 26
YXZX3X4XSGPX6TWX~X$
z~
X1YXZX3XdX5GPX6X~XgX9X1pX11 28
X1YXZCX4XSGPX6TWX~CX9XIOXn 29
GGLYLCRFGPVTWDCGYKGG 30
GGTYSCHFGPLTWVCKPQGG 31
GGDYHCRMGPLTWVCKPLGG 32
VGNYMCHFGPITWVCRPGGG 33
3 0 GGVYACRMGPITWVCSPLGG 34
VGNYMAHMGPITWVCRPGG 35
GGTYSCHFGPLTWVCKPQ 36
GGLYACHMGPMTWVCQPLRG 37
TIAQYICYMGPETWECRPSPKA 3 g
44
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WO 2004/002417 PCT/US2003/020347
YSCHFGPLTWVCK 39
YCHFGPLTWVC 40
SCHFGPLTWVCK 41
(~z)nXsx4xsGPX6TWX7X8 42
Table 6-IL-1 antagonist peptide sequences
SEQUENCE/STRUCTURE SEQ m NO:
Z11Z7Z$ZQZSYZ6Z9Zlo 43
XXQZSYZ6XX 44
Z7XQZSYZ6XX 45
Z7Z$QZSYZ6Z9Zlo 46
Zl l Z7ZSQZSYZ6Z9Z10 47
Z12z13Z14Z15Z16z17~18z19z20Z21z22Z11z7Z8QZ5YZ6Z9Z10L4g
Z23NZ24z39Z25Z26z27Z28Z29z30Z40 49
TANVSSFEWTPYYWQPYALPL 50
SWTDYGYWQPYALPISGL 51
ETPFTWEESNAYYWQPYALPL 52
ENTYSPNWADSMYWQPYALPL 53
SVGEDHNFWTSEYWQPYALPL 54
2 0 DGYDRWRQSGERYWQPYALPL 55
FEWTPGYWQPY 56
FEWTPGYWQHY
FEWTPGWYQJY Sg
AcFEWTPGWYQJY 59
2 5 FEVffPGWpYQJY 60
FAWTPGYWQJY 61
FEWAPGYWQJY 62
FEWVPGYWQJY 63
FEWTPGYWQJY 64
3 0 AcFEWTPGYWQJY 65
FEWTPaWYQTY 66
FEWTPSarWYQJY
FEWTPGYYQPY 6g
FEWTPGWWQPY 69
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FEWTPNYWQPY 70
FEVff PvYWQJY 71
FEWTPecGYWQJY 72
FEWTPAibYWQJY 73
FEVfFSarGYWQJY 74
FEWTPGYWQPY 75
FEWTPGYWQHY 76
FEWTPGWYQJY 77
AcFEWTPGWYQJY 78
FEWTPGW-pY-QJY 79
FAWTPGYWQJY 80
FEWAPGYWQJY 81
FEWVPGYWQJY 82
FEWTPGYWQJY 83
AcFEWTPGYWQJY 84
FEWTPAWYQJY 85
FEWTPSarWYQJY 86
FEWTPGYYQPY 87
FEWTPGWWQPY 88
2 FEWTPNYWQPY 89
0
FEWTPVYWQJY 90
FEWTPecGYWQJY 91
FEWTPAibYWQJY 92
FEWTSarGYWQJY 93
2 FEWTPGYWQPYALPL 94
5
NapEWTPGYYQJY 95
YEWTPGYYQJY 96
FEWVPGYYQJY 97
FEWTPSYYQJY 99
3 FEWTPNYYQJY 99
0
TI~I'R 100
RKSSI~ 101
~QDR 102
~QDR 103
3 RI~QDKR 104
5
46
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
._ .~4>~Ll~, .. . .nu mnin s7n,11 nrn)f ,ar
((r,.rr~ E'r.r'i rvnll Ifnl~~.IIItL
VTKFYF I OS
VTKFY
106
VTDFY
107
S~-yWQPYSVQ
108
TLVYWQpySLQT
109
RGDYWQpySVQS
110
VHVYWQpYSVQT
111
RLVYWQPYSVQT
112
2 0 SRVWFQPYSLQS
113
NM~WQPYSIQT
114
SVVFWQpYSVQT
115
TFVYWQpyALPL
lI6
TLVYWQpySIQR
117
RLVYWQpYSVQR
118
SPVFWQPYSIQI
119
W~WWQPYSVQS
120
SLIYWQpYSLQM
121
T~YWQPYSVQR
122
2 0 RCDYWQpySVQT
123
MRVFWQPYSVQN
124
~YWQPYSVQT
125
~-1'WQPYS VQR
126
ALVWWQPYSEQI
127
2 5 SRV WFQpySLQS
128
~QpYA.I,PLE 129
QLVWWQpySVQR 130
DLRYWQpyS VQV
131
ELVWWQpySLQL
I32
3 0 DLVWWQPYSVQW
133
NGNYWQpYSFQV
134
ELVYWQPYSIQR
135
ELMY)ATQpySVQE
I36
137
r1I-LYWQPYSMQD
_
'~ 5 GYEWyQPYSVQR
138
CEN0302-pCT
139
47
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WO 2004/002417 PCT/US2003/020347
..._ ,. .~ 1 'rr x a V t~H ... .. . ..,.. .~~Er ,,"" ",.,r~
.~ ~~";: rt"d~ .~;,IE u.,~~..,~d''
LSEQYQpySVQR
140
GGGWWQpySVQR
141
VGRWYQPYSVQR
142
V~WQPYSVQR
143
QARWYQPYS VQR 144
V~WQPYS VQT
14S
RSVYWQPYSVQR
146
TRV WFQPYS VQR
147
GRIWFQpySVQR
148
GRVWFQpYSVQR
149
~T~'QPYS VQR
1S0
ARVWWQpYSVQM
1S1
RLMFYQPYSVQR
1S2
ESMWYQpySVQR
1S3
HFGWWQPYSVHM
1S4
ARFWWQPYSVQR
1SS
RLVYWQPYAP1Y
156
RLVYWQPYSYQT
1S7
2 0 RLVYWQpYSLPI
1S8
RLVYWQPYSVQA
1S9
SRV WYQpyAgGL
160
SRV WYQPyAQGL
161
SRVWyQpYAMPL
I62
SRVWYQPYSVQA
163
SRV WYQpySLGL
164
SRV WyQPYAREL
16S
SRVWyQpySRQP
166
SRV WYQPYFVQP
167
3 0 EYEWYQPYALPL
168
iPEYWQPYALPL
169
SAW WQPYALPL
170
DPLFWQPY~,pL
171
SRQWVQPYALPL
172
3
5 IRS W WQPyALPL
173
174
CEN0302-PCT
48
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WO 2004/002417 PCT/US2003/020347
RGYWQPYALPL 175
RLLWVQPYALPL 176
EYRWFQPYALPL 177
DAYWVQPYALPL 178
WSGYFQPYALPL 179
NIEFWQPYALPL 180
TRDWVQPYALPL 181
DSSWYQPYALPL 182
IGNWYQPYALPL 183
NLRWDQPYALPL 184
LPEFWQPYALPL 185
DSYWWQPYALPL 186
RSQYYQPYALPL 187
ARFWLQPYALPL 188
NSYFWQPYALPL 189
RFMYWQPYSVQR 190
AHLFWQPYSVQR 191
WWQPYALPL 192
YYQPYALPL 193
2 YFQPYALGL 194
0
YWYQPYALPL 195
RW WQPYATPL 196
GWYQPYALGF 197
YWYQPYALGL 198
2 IWYQPYAMPL 199
5
SNMQPYQRLS 200
TFVYWQPYAVGLPAAETACN 201
TFVYWQPYSVQMTITGKVTM 202
TFVYWQPYSSHXXVPXGFPL 203
3 TFVYWQPYYGNPQWAIHVRH 204
0
TFVYWQPYVLLELPEGAVRA 205
TFVYWQPYVDYVWPIPIAQV 206
GWYQPYVDGWR 207
RWEQPYVKDGWS 208
3 EWYQPYALGWAR 209
5
c
49
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
GW WQPYARGL 210
LFEQPYAKALGL 211
GWEQPYARGLAG 212
AWVQPYATPLDE 213
MWYQPYSSQPAE 214
GWTQPYSQQGEV 21 S
DWFQPYSIQSDE 216
PWIQPYARGFG 217
RPLYWQPYSVQV 218
TLIYWQPYSVQI 219
RFDYWQPYSDQT 220
WHQFVQPYALPL 221
EWDSVYWQPYSVQTLLR 223
WEQNVYWQPYSVQSFAD 224
SDVVYWQPYSVQSLEM 225
YYDGVYWQPYSVQVMPA 226
SDIWYQPYALPL 227
QRIW WQPYALPL 228
SRIW WQPYALPL 229
2 RSLYWQPYALPL 230
0
TIIWEQPYALPL 231
WETWYQPYALPL 232
SYDWEQPYALPL 233
SRIWCQPYALPL 234
E1MFWQPYALPL 235
DYVWQQPYALPL 236
MDLLVQWYQPYALPL
237
GSI~VILWYQPYALPL 23 8
RQGANIWYQPYALPL 239
3 GGGDEPWYQPYALPL 240
0
SQLERTWYQPYALPL 241
ETWVREWYQPYALPL 242
KKGSTQWYQPYALPL 243
LQARMNWYQPYALPL 244
3 EPRSQI~WYQPYALPL 245
5
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VI~QI~WRWYQPYALPL 246
LRRHDV WYQPYALPL 247
RSTASIWYQPYALPL 248
ESI~EDQWYQPYALPL 249
EGLTMI~WYQPYALPL 250
EGSREGWYQPYALPL 251
VIEWWQPYALPL 252
V WYWEQPYALPL 253
ASEWWQPYALPL 254
FYEWWQPYALPL 255
EGWWVQPYALPL 256
WGEWLQPYALPL 257
DYVWEQPYALPL 258
AHTWWQPYALPL 259
FIEWFQPYALPL 260
WLAWEQPYALPL 261
VMEWWQPYALPL 262
ERMWQPYALPL 263
NXXWXXPYALPL 264
2 WGNWYQPYALPL 265
0
TLYWEQPYALPL 266
V WRWEQPYALPL 267
LLWTQPYALPL 268
SRIWXX PYALPL 269
2 SDIWYQPYALPL 270
5
WGYYXX PYALPL 271
TSGWYQPYALPL 272
VHPYXXPYALPL 273
EHSYFQPYALPL 274
3 XXIWYQPYALPL 275
0
AQLHSQPYALPL 276
WANWFQPYALPL 277
SRLYSQPYALPL 278
GVTFSQPYALPL 279
3 SIVWSQPYALPL 280
5
51
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
SRDLVQPYALPL 281
HWGHVYWQPYSVQDDLG 282
SWHSVYWQPYSVQSVPE 283
WRDSVYWQPYSVQPESA 284
TWDAVYWQPYSVQKWLD 285
TPPWVYWQPYSVQSLDP 286
YWSSVYWQPYSVQSVHS 287
YWYQPYALGL 288
YWYQPYALPL 289
EWIQPYATGL 290
NWEQPYAKPL 291
AFYQPYALPL 292
FLYQPYALPL 293
VCKQPYLEWC 294
ETPFTWEESNAYYWQPYALPL 295
QGWLTWQDSVDMYWQPYALPL 296
FSEAGYTWPENTYWQPYALPL 297
TESPGGLDWAKIYWQPYALPL 298
DGYDRWRQSGERYWQPYALPL 299
2 TANVSSFEWTPGYWQPYALPL 300
0
SVGEDHNFWTSE YWQPYALPL 301
MNDQTSEV STFPYWQPYALPL 3 02
S W SEAFEQPRNLYWQPYALPL 3 03
QYAEPSALNDWGYWQPYALPL 304
NGDWATADWSNYYWQPYALPL 305
THDEHIYWQPYALPL 306
MLEKTYTTWTPG YWQPYALPL 307
WSDPLTRDADLYWQPYALPL 308
SDAFTTQDSQAMYWQPYALPL 309
3 GDDAAWRTDSLTYWQPYALPL 310
0
ABRQLYRW SEMYWQPYALPL 311
ENTYSPNWADSMYWQPYALPL 312
MNDQTSEVSTFPYWQPYALPL 313
SVGEDHNFWTSEYWQPYALPL 314
3 QTPFTWEESNAYYWQPYALPL 315
5
52
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
ENPFTWQESNAYYWQPYALPL 316
VTPFTWEDSNVF YWQPYALPL 317
QIPFTWEQSNAYYWQPYALPL 318
QAPLTWQESAAYYWQPYALPL 319
EPTFTWEESKAT YWQPYALPL 320
TTTLTWEESNAYYWQPYALPL 321
ESPLTWEESSALYWQPYALPL 322
ETPLTWEESNAYYWQPYALPL 323
EATFTWAESNAYYWQPYALPL 324
EALFTWKESTAYYWQPYALPL 325
STP-TWEESNAYYWQPYALPL 326
ETPFTWEESNAYYWQPYALPL 327
KAPFTWEESQAYYWQPYALPL 328
STSFTWEESNAYYWQPYALPL 329
DSTFTWEESNAYYWQPYALPL 330
YIPFTWEESNAYYWQPYALPL 331
QTAFTWEESNAYYWQPYALPL 332
ETLFTWEESNAT YWQPYALPL 333
VSSFTWEESNAYYWQPYALPL 334
2 QPYALPL 335
0
Py-1-NapPYQJYALPL 336
TANVSSFEWTPG YWQPYALPL 337
FEWTPGYWQPYALPL 3 3 8
FEWTPGYWQJYALPL 339
2 FEWTPGYYQJYALPL 340
5
ETPFTWEESNAYYWQPYALPL 341
FTWEESNAYYWQJYALPL 342
ADVLYWQPYAPVTLWV 343
GDVAEYWQPYALPLTSL 344
3 SWTDYGYWQPYALPISGL 345
0
FEWTPGYWQPYALPL 346
FEWTPGYWQJYALPL 347
FEWTPGWYQPYALPL 348
FEWTPGWYQJYALPL 349
3 FEWTPGYYQPYALPL 350
5
53
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
FEWTPGYYQJYALPL 351
TANVSSFEWTPGYWQPYALPL 352
SWTDYGYWQPYALPISGL 353
ETPFTWEESNAWAIQPYALPL 354
ENTYSPNWADSMYWQPYALPL 355
SVGEDHNFWTSEYWQPYALPL 356
DGYDRWRQSGERYWQPYALPL 357
FEWTPGYWQPYALPL 358
FEWTPGYWQPY 359
FEWTPGYWQJY 360
EWTPGYWQPY 361
FEWTPGWYQJY 362
AEWTPGYWQJY 363
FAWTPGYWQJY 364
FEATPGYWQJY 365
FEWAPGYWQJY 366
FEWTAGYWQJY 367
FEWTPAYWQJY 368
FEWTPGAWQJY 369
2 FEWTPGYAQJY 370
0
FEWTPGYWQJA 371
FEWTGGYWQJY 372
FEWTPGYWQJY 373
FEWTJGYWQJY 374
2 FEVff PecGYWQJY 375
5
FEWTPAibYWQJY 376
FEWTPSarWYQJY 377
FEWTSarGYWQJY 378
FEWTPNYWQJY 379
3 FEWTPVYWQJY 380
0
FEWTVPYWQJY 381
AcFEWTPGVVYQJY 382
AcFEVffPGYWQJY 383
INap-EVff PGYYQJY 384
35 YEWTPGYYQJY 385
54
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
FEWVPGYYQJY 386
FEVff PGYYQJY 387
FEVff PsYYQJY 388
FEWTPnYYQJY 3 89
SHLY Nap-QPYSVQM 390
TLVY-Nap-LDPYSLQT 391
RGDY-Nap-QPYSVQS 392
NMVY-Nap-QPYSIQT 393
VYWQPYSVQ 394
VY-Nap-QPYSVQ 395
TFVYWQJYALPL 396
FEWTPGYYQJ-Bpa 397
XaaFEWTPGYYQJ-Bpa 398
FEWTPGY-Bpa-QJY 399
AeFEWTPGY-Bpa-QJY 400
FEWTPG-Bpa-YQJY 401
AcFEWTPG-Bpa-YQJY 402
AcFE-Bpa-TPGYYQJY 403
AcFE-Bpa-TPGYYQJY 404
2 Bpa-EWTPGYYQJY 405
0
AcBpa-EWTPGYYQJY 406
VYWQPYSVQ 407
RLVYWQPYSVQR 408
RLVY-Nap-QPYSVQR 409
2 RLDYWQPYSVQR 410
5
RLVWFQPYSVQR 411
RLVYWQPYSIQR 412
DNSSWYDSFLL 413
DNTAWYESFLA 414
3 DNTAWYENFLL 415
0
PAREDNTAWYDSFLIWC 416
TSEYDNTTWYEKFLASQ 417
SQIPDNTAWYQSFLLHG 418
SPFIDNTAWYENFLLTY 419
3 EQIYDNTAWYDHFLLSY 420
5
CA 02490409 2004-12-24
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TPFmNTAWYENFLLTY 421
TYTYDNTAWYERFLMSY 422
TMTQDNTAWYENFLLSY 423
TmNTAWYANLVQTYPQ 424
TIDNTAWYERFLAQYPD 425
I~NTAWYENFLLTYTP 426
SQDNTAWYENFLLSYKA 427
QmNTAWYERFLLQYNA 428
NQDNTAWYESFLLQYNT 429
TIDNTAWYENFLLNHNL 430
HYDNTAWYERFLQQGWH 431
ETPFTWEESNAYYWQPYALPL 432
YIPFTWEESNAYYWQPYALPL 433
DGYDRWRQSGERYWQPYALPL 434
pY-INap-pY-QJYALPL 435
TANVSSFEWTPGYWQPYALPL 436
FEWTPGYWQJYALPL 437
FEWTPGYWQPYALPLSD 43 8
FEWTPGYYQJYALPL 439
2 FEWTPGYWQJY 440
0
AcFEWTPGYWQJY 441
AcFEWTPGWYQJY 442
AcFEWTPGYYQJY 443
AcFEWTPaYWQJY 444
2 AcFEWTPaWYQJY 445
5
AcFEWTPaYYQJY 446
FEWTPGYYQJYALPL 447
FEWTPGYWQJYALPL 448
FEWTPGWYQJYALPL 449
3 TANVSSFEWTPGYWQPYALPL 450
0
AcFEWTPGYWQJY 451
AcFEWTPGWYQJY 452
AcFEWTPGYYQJY 453
AcFEWTPAYWQJY 454
3 AcFEWTPAWYQJY 455
5
c
56
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
AcFEWTPAYYQJY 456
Table 7-TPO-mimetic peptide sequences
Sequence/structure SEQ-ID-NO:
EGPTLRQWLAARA 457
IEGPTLRQWLAAKA 458
IEGPTLREWLAAR.A 459
IEGPTLRQWLAARA-A- 460
IEGPTLRQWLAARA
IEGPTLRQWLAAKA-A- 461
IEGPTLRQWLAAKA
IEGPTLRQCLAARA-A- 462
IEGPTLRQCLAARA
IEGPTLRQWLAARA-A-K(BrAc)-A- 463
IEGPTLRQWLAARA
IEGPTLRQWLAARA-A-K(PEG)-A- 464
IEGPTLRQWLAAR A
IEGPTLRQCLAARA-A- 465
IEGPTLRQWLAARA
IEGPTLRQCLAARA-4- 466
IEGPTLRQCLAARA
IEGPTLRQWLAARA-A- 467
IEGPTLRQULA/AtIA
VRDQIXXXL 468
TLREWL 469
GRVRI~QVAGW 470
GRVKDQIAQL 471
GVRDQVSWAL 472
ESVREQVMKY 473
SVRSQISASL 474
GVRETVYRHM 475
GVREV1VMHML 476
GRVRDQIWAAL 477
AGVRDQIL,IWL 478
GRVRDQIIVff,SL 479
57
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Sequence/structure SEQ-Ir7 NO:
GRVRDQI(X)3L 480
CTLRQWLQGC 48I
CTLQEFLEGC 482
CTRTEWLHGC 483
CTLREWLHGGFC 484
CTLREWVFAGLC 48S
CTLRQWLILLGMC 486
CTLAEFLASGVEQC 487
CSLQEFLSHGGYVC 488
CTLREFLDPTTAVC 489
CTLKEWLVSHEVWC 490
CTLREWL(X)2_6C 491-495
REGPTLRQWM 496
EGPTLRQWLA 497
ERGPFWAKAC 498
REGPRCVMWM 499
CGTEGPTLSTWLDC 500
CEQDGPTLLEWLKC 501
CELVGPSLMSWLTC S02
CLTGPFVTQWLYEC 503
CRAGPTLLEWLTLC 504
CADGPTLREWISFC 505
C(X)1_ZEGPTLREWL(X)1_ZC 506-510
GGCTLREWLHGGFCGG 511
GGCADGPTLREWISFCGG 512
GNADGPTLRQWLEGRRPKN 513
LAIEGPTLRQWLHGNGRDT 514
HGRVGPTLREWKTQVATKK 515
TIKGPTLRQWLKSREHTS S 16
ISDGPTLKEWLSVTRGAS 517
SIEGPTLREWLTSRTPHS S 18
58
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Table 8-G-CSF-mimetic peptide sequences
Sequence/structure SEQ ID NO:
EEDCK 519
EEDaK 520
pGluEDa,K 521
PicSDaK 522
EEDCK-0-EEDCK 523
EEDXK-0-EEDXK 524
Table 9-TNF-antagonist peptide sequences
Sequence/structure SEQ m NO:
YCFTASENHCY 525
YCFTNSENHCY 526
YCFTRSENHCY 527
FCASENHCY 528
YCASENHCY 529
FCNSENHCY 530
2 0 FCNSENRCY 531
FCNSVENRCY 532
YCSQSVSNDCF 533
FCVSNDRCY 534
YCRKELGQVCY 535
2 5 YCKEPGQCY 536
YCRKEMGCY 537
FCRKEMGCY 538
YCWSQNLCY 539
YCELSQYLCY 540
3 0 YCWSQNYCY 541
YCWSQYLCY 542
DFLPHYKNTSLGHRP 543
Table 10-Integrin-binding peptide sequences
59
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Sequence/structure SEQ ID NO:
RXIETX2WX3 544
RX1ETXZWX3 545
RGDGX 546
CRGDGXC 547
CXIXZRLDX3X4C 548
CARRLDAPC 549
CPSRLDSPC 550
Z X1XZX3RGDX4XSX6 551
O
CXZCRGDCXSC 552
CDCRGDCFC 553
CDCRGDCLC 554
CLCRGDCIC 555
X1XZDDXXSX~X$ 556
XlXzX3DDX4X5X6X~X8 557
CWDDGWL 558
CWDDLWWLC 559
CWDDGLMC 560
2 CWDDGWMC 561
0
CSWDDGWLC 562
CPDDLWWLC 563
NGR
GSL
RGD
CGRECPRLCQSSC 564
CNGRCVSGCAGRC 565
CLSGSLSC 566
RGD
NGR NR
GSL
NGRAHA 567
CNGRC 568
CDCRGDCFC 569
3 CGSLVRC 570
5
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
DLXXL 571
RTDLDSLRTYTL 572
RTDLDSLRTY 573
RTDLDSLRT 574
RTDLDSLR 575
GDLDLLKLRLTL 576
GDLHSLRQLLSR 577
RDDLHMLRLQLW 578
SSDLHALI~RYG 579
RGDLKQLSELTW 580
RGDLAALSAPPV 581
Table 11-Selectin antagonist peptide sequences
Sequence/structure SEQ ID NO:
DITWDQLWDLMK 5 82
DITWDELWKIMN 583
DYTWFELWDMMQ 584
QITWAQLWNMMK 585
DMTWHDLWTLMS 586
DYSWHDLWEMMS 587
EITWDQLWEVMN Sgg
HVSWEQLWDININ 589
HITWDQLWR1MT 590
2 RNMSWLELWEHMI~ 591
5
AEWTWDQLWHVMNPAESQ 592
HRAEWLALWEQMSP 593
T_~KFDWLALWR1MSV 594
ITWDQLWDLMK 595
3 DITWDQLWDLMI~ 596
0
DITWDQLWDLMK 597
DITWDQLWDLMK S9g
CQNRYTDLVAIQNKNE 599
AENWADNEPNNKRNNED 600
3 RKNNI~TWTWVGTKKALTNE 601
5
61
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
KKALTNEAENWAD 602
CQXRYTDLVAIQNKXE 603
RKXNXXWTWVGTXKXLTEE 604
AENWADGEPNNKXNXED 605
CXXXYTXLVAIQNKXE 606
RKXXXXWXWVGTXKXLTXE 607
AXNWXXXEPNNXXXED 608
XKXKTXEAXNWXX 609
Table 12-Antipathogenic peptide sequences
Sequence/structure SEQ ID NO:
GFFALIPKIISSPLFKTLLSAVGSALSSSGGQQ 610
GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE 611
GFFALIPKIISSPLFKTLLSAV 612
GFFALIPKIISSPLFKTLLSAV 613
KGFFALIPKIISSPLFKTLLSAV 614
KKGFFALIPKIISSPLFKTLLSAV 615
KKGFFALIPKIIS SPLFKTLLSAV 616
2 GFFALIPKIIS 617
0
GIGAVLKVLTTGLPALIS WIKRKRQQ 618
GIGAVLKVLTTGLPALIS WIKRKRQQ 619
GIGAVLKVLTTGLPALISWIKRKRQQ 620
GIGAVLKVLTTGLPALIS WIKR 621
AVLKVLTTGLPALISWIKR 622
KLLLLLKLLLLK 623
KLLLKLLLKLLK 624
KLLLKLKLKLLK 625
KKLLKLKLKLKK 626
3 KLLLKLLLKLLK 627
0
KLLLKLKLKLLK 628
KLLLLK 629
KLLLKLLK 63 0
KLLLKLKLKLLK 631
3 KLLLKLKLKLLK 632
5
62
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
KLLLKLKLKLLK 633
634
KVWKVWKVVK 635
KWVKVKVKWK 636
KVVVKVKVKVK 637
KVWKVKVKWK 638
639
KVLHLL 640
LKLRLL 641
KPLHLL 642
KLILKLVR 643
KVFHLLHL 644
~~L~ 645
KPFHILHL 646
KIiIKIKIKIIK 647
~~R~ 648
649
KIPIKIKIKIPK 650
~p~K 651
2 RIIIRIRIRIIR 652
0
~i 653
~~~R 654
RIVIRIRIRLIR 655
656
2 RIGIRLRVRIIR 657
5
KIVT_RTR_TRT,IR 658
RIAVKWRLRFIK 659
KIGWKLRVRIIR 660
KKIGWL1IRVRR 661
3 RIVIRIRIRLIKIR 662
0
RIIVRIRLRIIKVR 663
RIGIRLRVRIIRRV 664
KIVIRIRARLIRIRIR 665
666
3 KIGIKARVRBRVKII 667
5
63
CA 02490409 2004-12-24
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RIIVHIRLRIII~IIRL 668
HIGIKAHVRIIRVHII 669
RIYVKIHLRYIKKIRL 670
HIG~ 671
RIYVKPHPRYII~KIRL 672
KPGHKARPHIIRYKII 673
R~~RIRI,~RIg~ 674
RIIVI~TRT ,RBKKIRI,IKK 675
KIGWKLRVRIIRVKIGRLR 676
KLVIRIRIRLIRIRIRKIVKVKRIR 677
RFAVKIRLRIIKKIRLIKKIRKRVIK 678
KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK 679
RIYVKPHPRYII~KTRT 680
KPGHKARPHIIRYKII 681
KIVIRIRIRLIRIRIRKIV 682
RIIVKIRLRTTKKTRT,IKK 683
RIYV SKISIYIKKIRL, 684
KIVIFTRIRLTSIRIRSIV 685
KPIHKARPTIIRYKMI 686
2 cyclicCKGFFALIPKIISSPLFKTLLSAVC 687
0
CKKGFFALIPKIISSPLFKTLLSAVC 688
CKKKGFFALIPKIISSPLFKTLLSAVC 689
CyclicCRIVIRIRIRLIRIRC 690
CyclicCKPGHKARPHIIRYKIIC 691
2 CyclicCRFAVKIRLRIIK1~IRLIKKIRKRVIKC 692
5
KLLLKLLL KLLKC 693
KLLLKLLLKLLK 694
KLLLKLKLKLLKC 695
KLLLKLLLKLLK 696
Table 13-VIP-mimetic peptide sequences
Sequence/structure SEQ ID NO:
HSDAVFYDNYTR LRKQMAVKKYLN SILN 697
Me HSDAVFYDNYTR LRKQMAVKKYLN SILN 698
3 5 Xl Xl' Xl" XZ 699
64
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
X3SX4LN 700
701
NSILN 702
703
KKYA 704
AVKKYL 705
NSILN 706
707
SILauN 7pg
KKYLNIe 709
NSYLN 710
NSIYN 711
KKYLPPNSILN 712
LauKKYL 713
CapKKYL 714
KYL
KKYNIe 715
V~~ 716
LNSILN 717
2 YLNSILN 718
0
~~-N 719
KKYLNS 720
KKYLNSI 721
KKYLNSIL 722
2 KKYL 723
5
KKYDA 724
A~~ 725
NSILN 726
727
3 SILauN 728
0
NSYLN 729
NSIYN 73 0
KKYLNIe 731
KI~YLPPNSILN 732
3 KKYL 733
5
CA 02490409 2004-12-24
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KKYDA 734
AVKKYL 735
NSILN 736
~yy'T 737
SIL,auN 73 8
LauKKYL 739
CapKKYL 740
KYL
KYL NR
KKYNIe 741
VKKYL 742
LNSILN 743
YLNSILN 744
KKYLNIe 745
KKYLN 746
KKYLNS 747
KKYLNSI 748
KKYLNSIL 749
KKKYLD 750
2 cycIicCKKYLC 751
0
CKKYLK 752
KKYA 753
WWTDTGLW 754
WWTDDGLW 755
WWDTRGLWVWTI 756
FWGNDGIWLESG 757
DWDQFGLWRGAA 758
RWDDNGLWVVVL 759
SGMWSHYGIWMG 760
3 GGRWDQAGLWVA 761
0
KLWSEQGIWMGE 762
CWSMHGLWLC 763
GCWDNTGIWVPC 764
DWDTRGLWVY 765
3 SLWDENGAWI 766
5
c
66
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
I~WDDRGLWMH 767
QAWNERGLWT
768
QWDTRGLWVA 769
WNVHGIWQE 770
SWDTRGLWVE 771
DWDTRGLWVA 772
SWGRDGLWIE 773
EWTDNGLWAL 774
SWDEKGLWSA 775
1 SWDSSGLWMD 776
o
Table 14-Mdm/hdm antagonist peptide sequences
Sequence/structure SEQ ID NO:
TFSDLW 777
QETFSDLWKLLP 778
QPTFSDLWKLLP 779
QETFSDYWKLLP
780
QPTFSDYWKLLP
781
MPRFMDYWEGLN 782
2 VQNFIDYWTQQF 783
0
TGPAFTHYWATF 784
IDRAPTFRDHWFALV 785
PRPALVFADYWETLY 786
PAFSRFWSDLSAGAH 787
PAFSRFWSKLSAGAH 788
PXFXDYW~~:L 789
QETFSDLWKLLP
790
QPTFSDLWKLLP
791
QETFSDYWKLLP
792
3 QPTFSDYWKLLP 793
0
Table 15-Calmodulin antagonist peptide sequences
Sequence/structure SEQ ID NO:
3 5 SCVKWGKKEFCGS 794
67
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
SCWKYWGKECGS 795
SCYEWGKLRWCGS 796
SCLRWGKWSNCGS 797
SCWRWGKYQICGS 79g
SCVSWGALKLCGS 799
SCIRWGQNTFCGS 800
SCWQWGNLKICGS 801
SCVRWGQLSICGS 802
LKKFNARRKLKGAIL,TTMLAK 803
RRWKKNFIAVSAANRFKK 804
RKWQKTGHAVRAIGRLSS 805
INLKALAALAKKIL 806
KIWSILAPLGTTLVKLVA g07
LKKLLKLLKKLLKL gOg
LKWKKLLKLLKKLLKKLL 809
AEWPSLTEIKTLSHFSV ~ 810
AEWPSPTRVISTTYFGS 811
AELAHWPPVKTVLRSFT 812
AEGSWLQLLNLMKQMNN 813
2 0 AEWPSLTEIK 814
Table I6-Mast cell antagonists/Mast cell protease inhibitor peptide sequences
Sequence/structure SEQ ID NO:
2 5 SGSGVLKRPLPILPVTR 815
RWLSSRPLPPLPLPPRT 816
GSGSYDTLALPSLPLHPMSS g 17
GSGSYDTRALPSLPLHPMSS g 1 g
GSGSSGVTMYPKLPPHWSMA 819
3 0 GSGSSGVR_MYPKLPPHWSMA 820
GSGSSSMRMVPTIPGSAKHG 821
RNR NR
QT
RQK NR
35 NRQ
68
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
RQK
RNRQKT 822
823
~QK 824
NRQKT 825
RQKT 826
Table 17-SH3 antagonist peptide sequences
Sequencelstructure SEQ II? NO:
RPLPPLP 827
RELPPLP 828
SPLPPLP 829
GPLPPLP 830
RPLPIPP 831
RPLPIPP 832
RRLPPTP 834
RQLPPTP 83 S
RPLPSRP 836
RPLPTRP g37
SRLPPLP ' 838
RALPSPP 839
RRLPRTP 840
RPVPPIT 841
2 ILAPPVP 842
5
RPLPMLP 843
RPLPILP 844
RPLPSLP 845
RPLPSLP 846
3 RPLPMIP 847
0
RPLPLIP 848
RPLPPTP 849
RSLPPLP 850
RPQPPPP
851
3 RQLPIPP 852
5
69
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
XXXRPLPPLPXP 853
XXXRPLPPIPXX 854
XXXRPLPPLPXX 855
RXXRPLPPLPXP 856
RXXRPLPPLPPP 857
PPPYPPPPIPXX 85 8
PPPYPPPPVPXX 859
LXXRPLPXTP g60
'?lXXRPLPXLP 861
PPXOXPPP~I'P 862
+PP'llPXKPXWL 863
RPX~PLYR+SXP 864
PPVPPRPXXTL 865
~YP~I'LP'PI~ 866
+ODXPLPXLP 867
Table 18-Somatostatin or cortistatin mimetic peptide sequences
Sequence/structure SEQID NO:
2 0 X1X2 -Asn-Phe-Phe-Trp-Lys-Thr-Phe-X3-Ser-X4 868
Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr 869
Phe Ser Ser Cys Lys
Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser 870
Ser Cys Lys
Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 871
Lys
Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr 872
Phe Ser Ser Cys
2 5 Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe 873
Ser Ser Cys
Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 874
Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr 875
Phe Ser Ser Cys
Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser 876
Ser Cys Lys
Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 877
Lys
3 0 Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys 878
Thr Phe Ser Ser Cys
Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser 879
Ser Cys
Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 880
Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr 881
Phe Thr Ser Cys Lys
Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr 882
Ser Cys Lys
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 883
Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe 884
Thr Ser Cys
Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser 885
Cys
Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys gg6
Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe 887
Thr Ser Cys Lys
Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser 889
Cys Lys
Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 890
Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe 891
Thr Ser Cys
Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser 892
Cys
Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 893
Table 19-UKR antagonist peptide sequences
Sequence/structure SEQID NO:
AEPMPHSLNFSQYLWYT 894
AEHTYSSLWDTYSPLAF ggs
AELDLWMRHYPLSFSNR 896
AESSLWTRYAWPSMPSY gg7
AEWHPGLSFGSYLWSKT ggg
2 AEPALLNWSFFFNPGLH 899
0
AEWSFYNLHLPEPQT1F 900
AEPLDLWSLYSLPPLAM 901
AEPTLWQLYQFPLRLSG 902
AEISFSELMWLRSTPAF 903
2 AELSEADLWTTWFGMGS 904
5
AESSLWRIFSPSALMMS 905
AESLPTLTSILWGKESV 906
AETLFMDLWHDKHILLT 907
AEILNFPLWHEPLWSTE 908
3 AESQTGTLNTLFWNTLR 909
0
AEPVYQYELDSYLRSYY 910
AELDLSTFYDIQYLLRT 911
AEFFKLGPNGYVYLHSA 912
FKLXXXGYVYL 913
3 AESTYHHLSLGYMYTLN 914
5
~1
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YHXLXXGYMYT 915
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Table 20-Macrophage and/or T-cell inhibiting peptide sequences
Sequence/structure SEQ ID NO:
Xaa-Yaa-Arg
Arg-Yaa-Xaa
Xaa-Arg-Yaa
Yaa-Arg-Xaa
Ala-Arg
Arg-Arg
Asn-Arg
Asp-Arg NR
Cys-Arg NR
GIn-Arg
Glu-Arg
Gly-Arg NR
His-Arg NR
Ile-Arg NR
Leu-Arg NR
Lys-Arg NR
2 Met-Arg
0
Phe-Arg
Ser-Arg NR
Thr-Arg NR
Trp-Arg
2 Tyr-Arg
5
V al-Arg NR
Ala-Glu-Arg NR
Arg-Glu-Arg
Asn-Glu-Arg
3 Asp-Glu-Arg
0
Cys-Glu-Arg
Gln-Glu-Arg
Glu-Glu-Arg NR
Gly-Glu-Arg
3 His-Glu-Arg
5
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Ile-Glu-Arg NR
Leu-Glu-Arg
Lys-Glu-Arg
NR
Met-Glu-Arg
Phe-Glu-Arg
Pro-Glu-Arg
Ser-Glu-Arg
Thr-GIu-Arg
Trp-Glu-Arg
Tyr-Glu-Arg
Val-Glu-Arg
Arg-Ala NR
Arg-Asp
Arg-Cys
Arg-Gln
Arg-Glu
Arg-Gly
Arg-His NR
Arg-Ile
2 Arg-Leu
0
Arg-Lys NR
Arg-Met
Arg-Phe NR
Arg-Pro
2 Arg-Ser
S
Arg-Thr NR
Arg-Trp
Arg-Tyr
Arg-Val
3 Arg-Glu-Ala
0
Arg-Glu-Asn
Arg-Glu-Asp NR
Arg-Glu-Cys
Arg-Glu-Gln
3 Arg-Glu-Glu
5
!'~TTTn~ nn rvrvrt,
74
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Arg-Glu-Gly
Arg-Glu-His
Arg-Glu-Ile
Arg-Glu-Leu
Arg-Glu-Lys
Arg-Glu-Met
Arg-Glu-Phe
Arg-Glu-Pro
Arg-Glu-Ser
Arg-Glu-Thr
Arg-Glu-Trp NR
Arg-Glu-Tyr
Arg-Glu-Val
Ala-Arg-Glu
Arg-Arg-Glu
Asn-Arg-Glu NR
Asp-Arg-Glu Ng
Cys-Arg-Glu
Gln-Arg-Glu
2 Glu-Arg-Glu Ng
0
Gly-Arg-Glu
His-Arg-Glu
IIe-Arg-Glu
Leu-Arg-Glu Ng
2 Lys-Arg-Glu Ng
5
Met-Arg-Glu Ng
Phe-Arg-GIu Ng
Pro-Arg-Glu Ng
Ser-Arg-Glu NR
3 Thr-Arg-Glu
0
Trp-Arg-Glu
Tyr-Arg-GIu
Val-Arg-Glu NR
Glu-Arg-Ala
3 Glu-Arg-Arg
5
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Glu-Arg-Asn
Glu-Arg-Asp
Glu-Arg-Cys
Glu-Arg-Gln
Glu-Arg-Gly
Glu-Arg-His
Glu-Arg-IIe
GIu-Arg-Leu
Glu-Arg-Lys
Glu-Arg-Met
Glu-Arg-Phe
Glu-Arg-Pro NR
Glu-Arg-Ser NR
Glu-Arg-Thr
Glu-Arg-Trp
Glu-Arg-Tyr NR
Glu-Arg-Val
Table 21-Additional Exemplary Pharmacologically Active Peptides
Sequence/Structure SEQ ID NO: Activity
VEPNCDIHVMWEWECFERL 916 VEGF-antagonist
GERWCFDGPLTWVCGEES 917 VEGF-antagonist
RGWVEICVADDNGMCVTEAQ 918 VEGF-antagonist
GWDECDVARMWEWECFAGV 919 VEGF- antagonist
GERWCFDGPRAWVCGWEI 920 VEGF- antagonist
EELWCFDGPRAWVCGYVK 921 VEGF- antagonist
RGWVEICAADDYGRCLTEAQ 922 VEGF- antagonist
RGWVEICESDVWGRCL 923 VEGF-antagonist
RGWVEICESDVWGRCL 924 VEGF- antagonist
GGNECDIARMWEWECFERL 925 VEGF- antagonist
RGW VEICAADDYGRCL 926 VEGF-antagonist
CTTHWGFTLC 927 MMP inhibitor
3 5 CLRSGXGC 928 MMP inhibitor
nr~wrn-, n., r.r,e~
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CXXHWGFXXC 929 MMP inhibitor
CXPXC 930 MMP inhibitor
CRRHWGFEFC 931 MMP inhibitor
STTHWGFTLS 932 MMP inhibitor
CSLHWGFWWC 933 CTLA4-mimetic
GFVCSGIFAVGVGRC 934 CTLA4-mimetic
APGVRLGCAVLGRYC 935 CTLA4-mimetic
LLGRMK 936 Antiviral (HBV)
ICVVQDWGHHIZCTAGHMANLTSHASAI 937 C3b antagonist
ICWQDWGHHRCT 938 C3b antagonist
CVVQDWGHHAC 939 C3b antagonist
STGGFDDVYDWARGVSSALTTTLVATR 940 Vinculin-binding
STGGFDDVYDWARRVSSALTTTLVATR 941 Vinculin-binding
SRGVNFSEWLYDMSAAMKEASNVFPSRRSR 942 Vinculin-binding
SSQNWDMEAGVEDLTAANB~GLLSTIHSSSR 943 Vinculin-binding
SSPSLYTQFLVNYESAATRIQDLLIASRPSR 944 Vinculin-binding
SUGMIDILLGAILQRAADATRTSIPIPSLQNSIR
945 Vinculin-binding
DVYTKKELIECARRVSEK 946 Vinculin-binding
EKGSYYPGSGIAQFHIDYNNVS 947 C4BP-binding
2 SGIAQFHIDYNNVSSAEGWfIVN 948 C41BP-binding
0
LVTVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVN
949 4BP-binding
SGIAQFHIDYNNVS 950 C4BP-binding
LLGRMK 951 anti-HBV
ALLGRMKG 952 anti-HBV
2 LDPAFIR 953 anti-HBV
5
CXXRGDC 954 Inhibition of platelet
aggrepation
RPLPPLP 955 Src antagonist
PPVPPR 956 Src antagonist
~~X~~,XX 957 Anti-cancer
3 KACRRLFGPVDSEQLSRDCD 958 pl 6-mimetic
0
RERWNFDFVTETPLEGDFAW 959 p16-mimetic
KRRQTSMTDFYHSKRRLIFS 960 p16-mimetic
TSMTDFYHSKRRLIFSKRKP 961 p16-mimetic
RRLIF 962 p16-mimetic
3 KRRQTSATDFYHSKRRLIFSRQIKIWFQNRRMKWKK
5 963 p16-mimetic
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KRRLIFSKRQIKIWFQNRRMKWKK 964 pl 6-mimetic
Asn Gin Gly Arg His Phe Cys Gly Gly Ala Leu Ile His Ala Arq Phe Val Met Thr
Ala Ala Ser
Cys Phe Gln 965 CAP37 mimetic/LPs bindin
Arg His Phe Cys Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr Ala Ala Ser
Cys 499
CAP37 mimetic/LPS binding
Gly Thr Arg Cys Gin Val Ala Gly Trp Gly Ser Gln Arg Sex Gly Gly Arg Leu Ser
Arg Phe Pro
Arg Phe Val Asn Val 966 CAP37 mimetic/LPS binding
VVHWRIIRIPLQLAAGR 967 carbohydrate (GID 1 alpha) mimetic
LKTPRV 968 I32GPI Ab binding
NTLKTPRV 969 I32GPI Ab binding
NTLKTPRVGGC 970 02GPI Ab binding
KDKATF 971 02GPI Ab binding
KDKATFGCHD 972 P2GPI Ab binding
KDKATFGCHDGC 973 02GPI Ab binding
TLRVYK 974 02GPI Ab binding
ATLRVYKG 975 02GPI Ab binding
CATLRVYKGG 976 132GPI Ab binding
INLKALAALAKI~IL 977 Membrane transporting
GWT NR Membrane transporting
2 0 GWTLNSAGYLLG 978 Membrane transporting
GWTLNSAGYLLGKINLKALAALAKKIL 979 Membrane transporting
The present invention is also particularly useful with peptides having
activity in
treatment of: a VEGF related condition, e.g., but not limited to, cancer,
wherein the peptide is a
2 S VEGF-mimetic or a VEGF receptor antagonist, a HER2 agonist or antagonist,
a CD20
antagonist and the like; asthma, wherein the protein of interest is a CKR3
antagonist, an IL-5
receptor antagonist, and the like; thrombosis, wherein the protein of interest
is a GPIIb
antagonist, a GPTIla antagonist, and the like; autoimmune diseases and other
conditions
involving immune modulation, wherein the protein of interest is an IL-2
receptor antagonist, a
3 0 CD40 agonist or antagonist, a CD40L agonist or antagonist, a thymopoietin
mimetic and the
like.
For example, EPO biological activities are well known in the art. See, e.g.,
Anagnostou A et al
Erythropoietin has a mitogenic and positive chemotactic effect on endothelial
cells. Proceedings of the
National Academy of Science (LTSA) 87: 5978-82 (1990); Fandrey J and Jelkman
WE Tnterleukin 1 and
3 5 tumor necrosis factor-alpha inhibit erythropoietin production in vitro.
Annals of the New York Academy
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of Science 628: 250-5 (1991); Geissler K et al Recombinant human
erythropoietin: A multipotential
hemopoietic growth factor in vivo and in vitro. Contrib. Nephrol. 87: 1-10
(1990); Gregory CJ
Erythropoietin sensitivity as a differentiation marker in the hemopoietic
system. Studies of three
erythropoietic colony responses in culture. Journal of Cellular Physiology 89:
289-301 (1976); Jelkman W
et al Monokines inhibiting erythropoietin production in human hepatoma
cultures and in isolated perfused
rat kidneys. Life Sci. 50: 301-8 (1992); Kirnata H et al Human recombinant
erythropoietin directly
stimulates B cell immunoglobulin production and proliferation in serum-free
medium. Clinical and
Experimental linmunology 85: 151-6 (1991); Kimata H et a1 Erythropoietin
enhances immunoglobulin
production and proliferation by human plasma cells in a serum-free medium.
Clin. Immunology
Immunopathol. 59: 495-SOI (1991); Kimata H et al Effect of recombinant human
erythropoietin on
human IgE production in vitro Clinical and Experimental Immunology 83: 483-7
(1991); Koury MJ and
Bondurant MC Erythropoietin retards DNA breakdown and prevents programmed cell
death in erythroid
progenitor cells. Science 248: 378-81 (1990); Lim VS et al Effect of
recombinant human erythropoietin
on renal function in humans. Kidney International 37: 131-6 (1990); Mitjavila
MT et al Autocrine
stimulation by erythropoietin and autonomous growth of human erythroid
leukemic cells in vitro. Journal
of Clinical Investigation 88: 789-97 (1991); Andre M et al Performance of an
imrnunoradiometric assay
of erythropoietin and results for specimens from anemic and polycythemic
patients. Clinical Chemistry
38: 758-63 (1992); Hankins WD et al Erythropoietin-dependent and
erythropoietin-producing cell lines.
Implications for research and for leukemia therapy. Annals of the New York
Academy of Science 554:
2 0 21-8 (1989); Kendall RGT et al Storage and preparation of samples for
erythropoietin radioimmunoassay.
Clin. Lab. Haematology 13: 189-96 (1991); Krumvieh D et al Comparison of
relevant biological assays
for the determination of biological active erythropoietin. Dev. Biol. Stand.
69: 15-22 (1988); Ma DD et a1
Assessment of an EIA for measuring human serum erythropoietin as compared with
RIA and an in-vitro
bioassay. British Journal of Haematology 80: 431-6 (1992); Noe G et al A
sensitive sandwich ELISA for
2 5 measuring erythropoietin in human serum British Journal of Haematology 80:
285-92 (1992); Pauly JU et
al Highly specific and highly sensitive enzyme immunoassays for antibodies to
human interleukin 3 (IL3)
and human erythropoietin (EPO) in serum. Behring Institut Mitteilungen 90: 112-
25 (1991); Sakata S and
Enoki Y Improved microbioassay for plasma erythropoietin based on CFU-E colony
formation. Ann.
Hematology 64: 224-30 (1992); Sanengen T et al hnmunoreactive erythropoietin
and erythropoiesis
3 0 stimulating factors) in plasma from hypertransfused neonatal and adult
mice. Studies with a
radioimmunoassay and a cell culture assay for erythropoietin. Acta Physiol.
Scand. 135: 11-6 (1989);
Widness JA et al A sensitive and specific erythropoietin immunoprecipitation
assay: application to
pharmacokinetic studies. Journal of Lab. Clin. Med. 119: 285-94 (1992); for
further information see also
individual cell lines used in individual bioassays. Each of the above
references are entirely incorporated
3 5 herein by reference. EPO can be assayed by employing cell lines such as
HCD57 , NFS-60 , TF-1 and
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UT-7 , which respond to the factor . EPO activity can be assessed also in a
Colony formation assay by
determining the number of CFU-E from bone marrow cells. An alternative and
entirely different detection
method is RT-PCR quantitation of cytokines.
A CHl-deleted mimetibody, or specified portion or variant thereof, that
partially or preferably
substantially provides at least one biological activity of at least one
protein or fragment, can bind the
protein or fragment ligand and thereby provide at least one activity that is
otherwise mediated through
the binding of protein to at least one protein ligand or receptor or through
other protein-dependent or
mediated mechanisms. As used herein, the term "CH1-deleted mimetibody
activity" refers to a CHl-
deleted mimetibody that can modulate or cause at least one protein-dependent
activity by about 20-
10,000%, preferably by at least about 60, 70, 80, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550,
600, 700, 800, 900, 1000,
2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 % or more depending on the
assay.
The capacity of a CHl-deleted mimetibody or specified portion or variant to
provide at least
one protein-dependent activity is preferably assessed by at least one suitable
protein biological assay,
as described herein and/or as known in the art. A human CHl-deleted mimetibody
or specified portion
or variant of the invention can be similar to any class (IgG, IgA, IgM, etc.)
or isotype and can comprise
at least a portion of a kappa or lambda light chain, wherein at least one of
the LBRs is replaced by at
least one LBR as described herein. In one embodiment, the human CHl-deleted
mimetibody or
specified portion or variant comprises an IgG heavy chain or defined fragment,
for example, at least
2 0 one of isotypes, IgGl, IgG2, IgG3 or IgG4. In another embodiment, the
human protein human CHl-
deleted mimetibody or specified portion or variant thereof comprises an IgGl
heavy chain and an IgGl
Light chain.
At least one CHl-deleted mimetibody or specified portion or variant of the
invention binds at
least one specified ligand specific to at least one protein, subunit,
fragment, portion or any
2 5 combination thereof. The at least one LBR of at least one CHl-deleted
mimetibody, specified portion
or variant of the present invention can optionally bind at least one specified
ligand epitope of the
ligand. The binding epitope can comprise any combination of at least one amino
acid sequence of at
least 1-3 amino acids to the entire specified portion of contiguous amino
acids of the sequences
selected from the group consisting of a protein ligand, such as a receptor or
portion thereof.
3 0 Generally, the CHl-deleted mimetibody or ligand-binding fragment of the
present invention
can comprise a ligand binding region (LBR) (e.g., LBRl, LBR2 and LBR3) or
variant provided in at
least one heavy chain variable region and at least one ligand binding region
(LBRI, LBR2 and LBR3)
or variant provided in at least one light chain variable region. As a non-
limiting example, the CHl-
deleted mimetibody or ligand-binding portion or variant can comprise at least
one of the heavy chain
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LBR3, and/or a light chain LBR3. In a particular embodiment, the CHl-deleted
mimetibody or ligand-
binding fragment can have an ligand-binding region that comprises at least a
portion of at least one
heavy chain LBR (i.e., LBRl, LBRZ and/or LBR3) having the amino acid sequence
of the
corresponding LBRs 1, 2 and/or 3. In another particular embodiment, the CHl-
deleted mimetibody or
ligand-binding portion or variant can have an ligand-binding region that
comprises at least a portion of
at least one light chain LBR (i.e., LBRl, LBR2 and/or LBR3) having the amino
acid sequence of the
corresponding LBRs 1, 2 and/or 3. Such mimetibodies can be prepared by joining
together the various
portions (e.g., LBRs, framework) of the CHl-deleted mimetibody using known
techniques, by
preparing and expressing at least one (i.e., one or more) nucleic acid
molecules that encode the CHl-
deleted mimetibody, using known techniques of recombinant DNA technology or by
using any other
suitable method, such as chemical synthesis.
The CHl-deleted mimetibody can comprise at least one of a heavy or light chain
variable
region having a defined amino acid sequence. Mimetibodies that bind to human
protein ligands or
receptors and that comprise a defined heavy or light chain variable region can
be prepared using
1S suitable methods, such as phage display (Katsube, Y., et al., Iht JMoI.
Med, 1(5):863-868 (1998)) or
methods that employ transgenic animals, as known in the art and/or as
described herein. The CH1-
deleted mimetibody, specified portion or variant can be expressed using the
encoding nucleic acid or
portion thereof in a suitable host cell.
The invention also relates to mimetibodies, ligand-binding fragments,
immunoglobulin chains
2 0 and LBRs comprising amino acids in a sequence that is substantially the
same as an amino acid
sequence described herein. Preferably, such mimetibodies or ligand-binding
fragments and
mimetibodies comprising such chains or LBRs can bind human protein ligands
with high affinity (e.g.,
KD less than or equal to about 10-9 M). Amino acid sequences that are
substantially the same as the
sequences described herein include sequences comprising conservative amino
acid substitutions, as
2 5 well as amino acid deletions and/or insertions. A conservative amino acid
substitution refers to the
replacement of a first amino acid by a second amino acid that has chemical
and/or physical properties
(e.g., charge, structure, polarity, hydrophobicity/ hydrophilicity) that are
similar to those of the first
amino acid. Conservative substitutions include replacement of one amino acid
by another within the
following groups: lysine (K), arginine (R) and histidine (H); aspartate (D)
and glutamate (E);
3 0 asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K,
R, H, D and E; alanine (A),
valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F),
tryptophan (W), methionine (M),
cysteine (C) and glycine (G); F, W and Y; C, S and T.
Amino Acid Codes
The amino acids that make up mimetibodies or specified portions or variants of
the present
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invention are often abbreviated. The amino acid designations can be indicated
by designating the
amino acid by its single letter code, its three letter code, name, or three
nucleotide codon(s) as is well
understood in the art (see Alberts, B., et al., Molecular Biology of The Cell,
Third Ed., Garland
Publishing, Inc., New York, 1994):
~ING ~E,~ETTER THREEI~FTTER ~ NAME ~ THREC ON S~OTIDE
A Ala Alanine GCA, GCC, GCG, GCU
C C s C steine UGC, UGU
D As As artic acid GAC, GAU
E Glu Glutamic acid GAA, GAG
F Phe Phen lanine UUC, UUU
G Gl Gl cine GGA, GGC, GGG, GGU
H His Histidine CAC, CAU
I Ile Isoleucine AUA, AUC, AUU
K L s L sine AAA, AAG
L Leu Leucine UUA, UIJG, CUA, CUC,
CUG, CUU
M Met Methionine AUG
N Asn As ara ine AAC, AAU
p Pro Proline CCA, CCC, CCG, CCU
Q Gln Glutamine CAA, CAG
R Arg Arginine AGA, AGG, CGA, CGC,
CGG, CGU
S Ser Serine AGC, AGU, UCA, UCC,
UCG, UCU
T Thr Threonine ACA, ACC, ACG, ACU
V Val Valine GUA, GUC, GUG, GUU
W T T to han UGG
Y Tyr Tyrosine ~ UAC, UAU
A CH1-deleted mimetibody or specified portion or variant of the present
invention can include
one or more amino acid substitutions, deletions or additions, either from
natural mutations or human
manipulation, as specified herein.
Of course, the number of amino acid substitutions a skilled artisan would make
depends on
many factors, including those described above. Generally speaking, the number
of amino acid
substitutions, insertions or deletions for at least one of a CH1-deleted
mimetibody LBR, variable,
constant, light or heavy chain, or Ig will not be more than 40, 30, 20,19, 18,
17, 16, 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids, such as 1-30 or any range or value
therein, as specified herein.
Amino acids in a CH1-deleted mimetibody or specified portion or variant of the
present
invention that are essential for function can be identified by methods known
in the art, such as site-
directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra,
Chapters 8, 15;
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Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure
introduces single
alanine mutations at every residue in the molecule. The resulting mutant
molecules are then tested for
biological activity, such as, but not limited to at least one protein related
activity, as specified herein or
as known in the art. Sites that are critical for CHl-deleted mimetibody or
specified portion or variant
binding can also be identified by structural analysis such as crystallization,
nuclear magnetic resonance
or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and
de Vos, et al., Science
255:306-312 (1992)).
Mimetibodies or specified portions or variants of the present invention can
comprise as the Pep
portion of Formula (I), but are not limited to, at least one portion, sequence
or combination selected
from 3 to all the of at least one of SEQ ID NOS:1-979. Non-limiting variants
that can enhance or
maintain at least one of the listed activities include, but are not limited
to, any of the above
polypeptides, further comprising at least one mutation corresponding to at
least one substitution,
insertion or deletion that does not significantly affect the suitable
biological activtities or functions of
said CHl-deleted mimetibody.
A CHl-deleted mimetibody or specified portion or variant can further
optionally comprise at
least one functional portion of at least one polypeptide as Pep portion of
Formula (I), at least one of 90-
100% of SEQID NOS:1-1109. A CH1-deleted rnimetibody can further optionally
comprise an amino
acid sequence for the Pep portion of Formula (I), selected from one or more of
SEQID NOS:1-1109.
In one embodiment, the Pep amino acid sequence of an immunoglobulin chain, or
portion
2 0 thereof (e.g., comprising at least one specified variable region, LBR) has
about 90-100% identity (i.e.,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein) to
the corresponding amino
acid sequence of the corresponding portion of at least one of SEQ ID NOS: 1-
979. Preferably, 90-
100% amino acid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or
any range or value therein)
is determined using a suitable computer algorithm, as known in the art.
2 5 Mimetibodies or specified portions or variants of the present invention
can comprise any number
of contiguous amino acid residues from a CHl-deleted mimetibody or specified
portion or variant of the
present invention, wherein that number is selected from the group of integers
consisting of from 10-100%
of the number of contiguous residues in a CHl-deleted mimetibody. Optionally,
this subsequence of
contiguous amino acids is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21,
3 0 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190,
200, 210, 220, 230, 240, 250 or more amino acids in length, or any range or
value therein. Further, the
number of such subsequences can be any integer selected from the group
consisting of from 1 to 20, such
as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20,
or more.
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As those of skill will appreciate, the present invention includes at least one
biologically active
CHl-deleted mimetibody or specified portion or variant of the present
invention. Biologically active
mimetibodies or specified portions or variants have a specific activity at
least 20%, 30%, or 40%, and
preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%,
or 95%-1000% of that of
the native (non-synthetic), endogenous or related and known inserted or fused
protein or specified portion
or variant. Methods of assaying and quantifying measures of enzymatic activity
and substrate specificity
are well known to those of skill in the art.
In another aspect, the invention relates to human mimetibodies and ligand-
binding fragments,
as described herein, which are modified by the covalent attachment of an
organic moiety. Such
modification can produce a CHl-deleted mimetibody or ligand-binding fragment
with improved
pharmacokinetic properties (e.g., increased in vivo serum half life). The
organic moiety can be a linear
or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester
group. In particular
embodiments, the hydrophilic polymeric group can have a molecular weight of
about 800 to about
120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol
(PEG), polypropylene glycol
(PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and
the fatty acid or fatty
acid ester group can comprise from about eight to about forty carbon atoms.
The modified mimetibodies and ligand-binding fragments of the invention can
comprise one or
more organic moieties that are covalently bonded, directly or indirectly, to
the CHl-deleted
mimetibody or specified portion or variant. Each organic moiety that is bonded
to a CHl-deleted
2 0 mimetibody or ligand-binding fragment of the invention can independently
be a hydrophilic polymeric
group, a fatty acid group or a fatty acid ester group. As used herein, the
term "fatty acid" encompasses
mono-carboxylic acids and di-carboxylic acids. A "hydrophilic polymeric
group," as the term is used
herein, refers to an organic polymer that is more soluble in water than in
octane. For example,
polylysine is more soluble in water than in octane. Thus, a CHl-deleted
mimetibody modified by the
2 5 covalent attachment of polylysine is encompassed by the invention.
Hydrophilic polymers suitable for
modifying mirnetibodies of the invention can be linear or branched and
include, for example,
polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and
the like),
carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and
the like), polymers of
hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the
like), polyalkane oxides
3 0 (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl
pyrolidone. Preferably, the
hydrophilic polymer that modifies the GH1-deleted mimetibody of the invention
has a molecular
weight of about 800 to about 150,000 Daltons as a separate molecular entity.
For example, PEGZSOO~
PEGsooo, PEG~soo~ I'EG9ooo~ PEGioooo, PEGiasoo~ PEGlsooo, and PEGZO,ooo,
wherein the subscript is the
average molecular weight of the polymer in Daltons, can be used.
3 5 The hydrophilic polymeric group can be substituted with one to about six
alkyl, fatty acid or
84
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
fatty acid ester groups. Hydrophilic polymers that are substituted with a
fatty acid or fatty acid ester
group can be prepared by employing suitable methods. For example, a polymer
comprising an amine
group can be coupled to a carboxylate of the fatty acid or fatty acid ester,
and an activated carboxylate
(e.g., activated with N,N-carbonyl diimidazole) on a fatty acid or fatty acid
ester can be coupled to a
hydroxyl group on a polymer.
Fatty acids and fatty acid esters suitable for modifying mirnetibodies of the
invention can be
saturated or can contain one or more units of unsaturation. Fatty acids that
are suitable for modifying
mimetibodies of the invention include, for example, n-dodecanoate (C12,
laurate), n-tetradecanoate
(C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (Czo,
arachidate), n-docosanoate (Cz2,
behenate), n-triacontanoate (C3o), n-tetracontanoate (C4o), cis-D9-
octadecanoate (C18, oleate), all cis-~
5, 8,11,14-eicosatetraenoate (CZO, arachidonate), octanedioic acid,
tetradecanedioic acid,
octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid
esters include mono-esters
of dicarboxylic acids that comprise a linear or branched lower alkyl group.
The lower alkyl group can
comprise from one to about twelve, preferably one to about six, carbon atoms.
The modified human mimetibodies and ligand-binding fragments can be prepared
using
suitable methods, such as by reaction with one or more modifying agents. A
"modifying agent" as the
term is used herein, refers to a suitable organic group (e.g., hydrophilic
polymer, a fatty acid, a fatty
acid ester) that comprises an activating group. An "activating group" is a
chemical moiety or
functional group that can, under appropriate conditions, react with a second
chemical group thereby
2 0 forming a covalent bond between the modifying agent and the second
chemical group. For example,
amine-reactive activating groups include electrophilic groups such as
tosylate, mesylate, halo (chloro,
bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.
Activating groups that can
react with thiols include, for example, maleimide, iodoacetyl, acrylolyl,
pyridyl disulfides, 5-thiol-2-
nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde functional
group can be coupled to
2 5 amine- or hydrazide-containing molecules, and an azide group can react
with a trivalent phosphorous
group to form phosphoramidate or phosphorimide linkages. Suitable methods to
introduce activating
groups into molecules are known in the art (see for example, Hermanson, G. T.,
Bioco~jugate
Techniques, Academic Press: San Diego, CA (1996)). An activating group can be
bonded directly to
the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester),
or through a linker moiety, for
3 0 example a divalent C1-Clz group wherein one or more carbon atoms can be
replaced by a heteroatom
such as oxygen, nitrogen or sulfur. Suitable linker moieties include, for
example, tetraethylene glycol,
-(CHz)3-, NH-(CHZ)6 NH-, -(CHZ)Z NH- and -CHZ-O-CHZ-CHz-O-CHZ-CHZ-Q-CH-NH-.
Modifying
agents that comprise a linker moiety can be produced, for example, by reacting
a mono-Boc-
alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a
fatty acid in the
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an
amide bond between
the free amine and the fatty acid carboxylate. The Boc protecting group can be
removed from the
product by treatment with trifluoroacetic acid (TFA) to expose a primary amine
that can be coupled to
another carboxylate as described, or can be reacted with malefic anhydride and
the resulting product
cyclized to produce an activated maleimido derivative of the fatty acid. (See,
for example, Thompson,
et al., WO 92/16221 the entire teachings of which are incorporated herein by
reference.)
The modif ed mimetibodies of the invention can be produced by reacting an
human CHl-
deleted mimetibody or ligand-binding fragment with a modifying agent. For
example, the organic
moieties can be bonded to the CHl-deleted mimetibody in a non-site specific
manner by employing an
amine-reactive modifying agent, for example, an NHS ester of PEG. Modified
human mimetibodies or
ligand-binding fragments can also be prepared by reducing disulfide bonds
(e.g., infra-chain disulfide
bonds) of a CHl-deleted mimetibody or ligand-binding fragment. The reduced CHl-
deleted
mimetibody or ligand-binding fragment can then be reacted with a thiol-
reactive modifying agent to
produce the modified CHl-deleted mimetibody of the invention. Modified human
mimetibodies and
ligand-binding fragments comprising an organic moiety that is bonded to
specific sites of a CH1-
deleted mimetibody or specified portion or variant of the present invention
can be prepared using
suitable methods, such as reverse proteolysis (Fisch et al., Biocor~jugate
Chenz., 3:147-153 (1992);
Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein
Sci. 6(10):2233-2241
(1997); Itoh et al., Bioorg. Claern., 24(1): 59-68 (1996); Capellas et al.,
Biotechnol. Bioeng., 56(4):456-
2 0 463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate
Techniques, Academic
Press: San Diego, CA (1996).
CH1-DELETED MIMETIEODY COMPOSITIONS
The present invention also provides at least one CH1-deleted mimetibody or
specified portion
or variant composition comprising at least one, at least two, at least three,
at least four, at least five, at
2 5 least six or more mimetibodies or specified portions or variants thereof,
as described herein and/or as
known in the art that are provided in a non-naturally occurring composition,
mixture or form. Such
composition percentages are by weight, volume, concentration, molarity, or
molality as liquid or dry
solutions, mixtures, suspension, emulsions or colloids, as known in the art or
as described herein.
Such compositions can comprise 0.00001-99.9999 percent by weight, volume,
concentration, molarity,
3 0 or molality as liquid, gas, or dry solutions, mixtures, suspension,
emulsions or colloids, as known in the
art or as described herein, on any range or value therein, such as but not
limited to 0.00001, 0.00003,
0.00005, 0.00009, 0.0001, 0.0003, 0.0005, 0.0009, 0.001, 0.003, 0.005, 0.009,
0.01, 0.02, 0.03, 0.05,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,
2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.3,4.5
,4.6,4.7,4.8,
35 4.9, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 6S, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80,
86
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
99.1, 99.2, 99.3, 99.4, 99.5,
99.6, 99.7, 99.8, 99.9 %. Such compositions of the present invention thus
include but are not limited to
0.00001-100 mg/ml and/or 0.00001-100 mg/g.
The composition can optionally further comprise an effective amount of at
least one compound
or protein selected from at least one of an anti-infective drug, a
cardiovascular (CV) system drug, a
central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a
respiratory tract drug,
a gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or
electrolyte balance, a
hematologic drug, an antineoplactic, an immunomodulation drug, an ophthalmic,
otic or nasal drug, a
topical drug, a nutritional drug or the like. Such drugs are well known in the
art, including
formulations, indications, dosing and administration for each presented herein
(see, e.g., Nursing 2001
Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, PA, 2001;
Health Professional's
Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle
River, NJ;
Pharmcotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, CT,
each entirely
incorporated herein by reference).
The anti-infective drug can be at least one selected from amebicides or at
least one
antiprotozoals, anthelmintics, antifungals, antimalarials, antituberculotics
or at least one antileprotics,
aminoglycosides, penicillins, cephalosporins, tetracyclines, sulfonamides,
fluoroquinolones, antivirals,
macrolide anti-infectives, miscellaneous anti-infectives. The CV drug can be
at least one selected from
inotropics, antiarrhythmics, antianginals, antihypertensives, antilipemics,
and miscellaneous ,
2 0 cardiovascular drugs. The CNS drug can be at least one selected from
nonnarcotic analgesics or at
least one selected from antipyretics, nonsteroidal anti-inflammatory drugs,
narcotic or at least one
opiod analgesics, sedative-hypnotics, anticonvulsants, antidepressants,
antianxiety drugs,
antipsychotics, central nervous system stimulants, antiparkinsonians,
miscellaneous central nervous
system drugs. The ANS drug can be at least one selected from cholinergics
(parasympathomimetics),
2 5 anticholinergics, adrenergics (sympathomimetics), adrenergic blockers
(sympatholytics), skeletal
muscle relaxants, neuromuscular blockers. The respiratory tract drug can be at
least one selected from
antihistamines, bronchodilators, expectorants or at least one antitussives,
miscellaneous respiratory
drugs. The GI tract drug can be at least one selected from antacids or at
least one adsorbents or at least
one antiflatulents, digestive enzymes or at least one gallstone solubilizers,
antidiarrheals, laxatives,
3 0 antiemetics, antiulcer drugs. The hormonal drug can be at least one
selected from corticosteroids,
androgens or at least one anabolic steroids, estrogens or at least one
progestins, gonadotropins,
antidiabetic drugs or at least one glucagon, thyroid hormones, thyroid hormone
antagonists, pituitary
hormones, parathyroid-like drugs. The drug for fluid and electrolyte balance
can be at least one
selected from diuretics, electrolytes or at least one replacement solutions,
acidifiers or at least one
3 5 alkalinizers. The hematologic drug can be at least one selected from
hematinics, anticoagulants, blood
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
derivatives, thrombolytic enzymes. The antineoplastics can be at least one
selected from alkylating
drugs, antimetabolites, antibiotic antineoplastics, antineoplastics that alter
hormone balance,
miscellaneous antineoplastics. The immunomodulation drug can be at least one
selected from
immunosuppressants, vaccines or at least one toxoids, antitoxins or at least
one antivenins, immune
serums, biological response modifiers. The ophthalmic, otic, and nasal drugs
can be at least one
selected from ophthalmic anti-infectives, ophthalmic anti-inflammatories,
miotics, mydriatics,
ophthalmic vasoconstrictors, miscellaneous ophthalmics, otics, nasal drugs.
The topical drug can be at
least one selected from local anti-infectives, scabicides or at least one
pediculicides, topical
corticosteroids. The nutritional drug can be at least one selected from
vitamins, minerals, or calorics.
See, e.g., contents ofNursing 2001 Drug Handbook, supra.
The at least one amebicide or antiprotozoal can be at least one selected from
atovaquone,
chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole
hydrochloride,
pentamidine isethionate. The at least one anthelmintic can be at least one
selected from mebendazoIe,
pyrantel pamoate, thiabendazole. The at least one antifungal can be at least
one selected from
amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B
lipid complex,
amphotericin B liposomal, fluconazole, flucytosine, griseofulvin microsize,
griseofulvin
ultramicrosize, itraconazole, ketoconazole, nystatin, terbinafine
hydrochloride. The at least one
antimalarial can be at least one selected from chloroquine hydrochloride,
chloroquine phosphate,
doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine
phosphate,
2 0 pyrimethamine, pyrimethamine with sulfadoxine. The at least one
antituberculotic or antileprotic can
be at least one selected from clofazimine, cycloserine, dapsone, ethambutol
hydrochloride, isoniazid,
pyrazinamide, rifabutin, rifampin, rifapentine, streptomycin sulfate. The at
least one aminoglycoside
can be at least one selected from amikacin sulfate, gentamicin sulfate,
neomycin sulfate, streptomycin
sulfate, tobramycin sulfate. The at least one penicillin can be at least one
selected from
2 5 amoxcillin/clavulanate potassium, amoxicillin trihydrate, ampicillin,
ampicillin sodium, ampicillin
trihydrate, ampicillin sodium/sulbactam sodium, cloxacillin sodium,
dicloxacillin sodium, mezlocillin
sodium, nafcillin sodium, oxacillin sodium, penicillin G benzathine,
penicillin G potassium, penicillin
G procaine, penicillin G sodium, penicillin V potassium, piperacillin sodium,
piperacillin
sodium/tazobactam sodium, ticarcillin disodium, ticarcillin
disodium/clavulanate potassium. The at
3 0 least one cephalosporin can be at least one selected from at least one of
cefaclor, cefadroxil, cefazolin
sodium, cefdinir, cefepime hydrochloride, cefixime, cefmetazole sodium,
cefonicid sodium,
cefoperazone sodium, cefotaxime sodium, cefotetan disodium, cefoxitin sodium,
cefpodoxime proxetil,
cefprozil, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium,
cefuroxime axetil,
cefuroxime sodium, cephalexin hydrochloride, cephalexin monohydrate,
cephradine, loracarbef. The
3 5 at least one tetracycline can be at least one selected from demeclocycline
hydrochloride, doxycycline
$a
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
calcium, doxycycline hyclate, doxycycline hydrochloride, doxycycline
monohydrate, minocycline
hydrochloride, tetracycline hydrochloride. The at least one sulfonamide can be
at least one selected
from co-trimoxazole, sulfadiazine, sulfamethoxazole, sulfisoxazole,
sulfisoxazole acetyl. The at least
one fluoroquinolone can be at least one selected from alatrofloxacin mesylate,
ciprofloxacin, enoxacin,
S levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin,
ofloxacin, sparfloxacin,
trovafloxacin mesylate. The at least one fluoroquinolone can be at least one
selected from
alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin
hydrochloride, nalidixic
acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin mesylate. The at
least one antiviral can be at
least one selected from abacavir sulfate, acyclovir sodium, amantadine
hydrochloride, amprenavir,
cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir,
fomivirsen sodium, foscarnet
sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine,
nelfmavir mesylate,
nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride,
ritonavir, saquinavir,
saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine,
zanamivir, zidovudine. The at
least one macroline anti-infective can be at least one selected from
azithromycin, clarithromycin,
dirithromycin, erythromycin base, erythromycin estolate, erythromycin
ethylsuccinate, erythromycin
lactobionate, erythromycin stearate. The at least one miscellaneous anti-
infective can be at least one
selected from aztreonam, bacitracin, chloramphenicol sodium sucinate,
clindamycin hydrochloride,
clindamycin palmitate hydrochloride, clindamycin phosphate, imipenem and
cilastatin sodium,
meropenem, nitrofurantoin macrocrystals, nitrofurantoin microcrystals,
quinupristin/dalfopristin,
2 0 spectinomycin hydrochloride, trimethoprim, vancomycin hydrochloride. (See,
e.g., pp. 24-214 of
Nursi~ag 2001 Drug Handbook.)
The at least one inotropic can be at least one selected from amrinone lactate,
digoxin,
milrinone lactate.. The at least one antiarrhythmic can be at least one
selected from adenosine,
arniodarone hydrochloride, atropine sulfate, bretylium tosylate, diltiazem
hydrochloride, disopyramide,
2 5 disopyramide phosphate, esmolol hydrochloride, flecainide acetate,
ibutilide fumarate, lidocaine
hydrochloride, mexiletine hydrochloride, moricizine hydrochloride, phenytoin,
phenytoin sodium,
procainamide hydrochloride, propafenone hydrochloride, propranolol
hydrochloride, quinidine
bisulfate, quinidine gluconate, quinidine polygalacturonate, quinidine
sulfate, sotalol, tocainide
hydrochloride, verapamil hydrochloride. The at least one antianginal can be at
least one selected from
3 0 amlodipidine besylate, amyl nitrite, bepridil hydrochloride, diltiazem
hydrochloride, isosorbide
dinitrate, isosorbide mononitrate, nadolol, nicardipine hydrochloride,
nifedipine, nitroglycerin,
propranolol hydrochloride, verapamil, verapamil hydrochloride. The at least
one antihypertensive can
be at least one selected from acebutolol hydrochloride, amlodipine besylate,
atenolol, benazepril
hydrochloride, betaxolol hydrochloride, bisoprolol fumarate, candesartan
cilexetil, captopril, carteolol
3 5 hydrochloride, carvedilol, clonidine, clonidine hydrochloride, diazoxide,
diltiazem hydrochloride,
89
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WO 2004/002417 PCT/US2003/020347
doxazosin mesylate, enalaprilat, enalapril maleate, eprosartan mesylate,
felodipine, fenoldopam
mesylate, fosinopril sodium, guanabenz acetate, guanadrel sulfate, guanfacine
hydrochloride,
hydralazine hydrochloride, irbesartan, isradipine, labetaloI hydrchloride,
lisinopril, losartan potassium,
methyldopa, methyldopate hydrochloride, metoprolol succinate, metoprolol
tartrate, minoxidil,
moexipril hydrochloride, nadolol, nicardipine hydrochloride, nifedipine,
nisoldipine, nitroprusside
sodium, penbutolol sulfate, perindopril erbumine, phentolamine mesylate,
pindolol, prazosin
hydrochloride, propranolol hydrochloride, quinapril hydrochloride, ramipril,
telmisartan, terazosin
hydrochloride, timolol maleate, trandolapril, valsartan, verapamil
hydrochloride The at least one
antilipemic can be at least one selected from atorvastatin calcium,
cerivastatin sodium, cholestyramine,
colestipol hydrochloride, fenofibrate (micronized), fluvastatin sodium,
gemfibrozil, lovastatin, niacin,
pravastatin sodium, simvastatin. The at least one miscellaneous CV drug can be
at least one selected
from abciximab, alprostadil, arbutamine hydrochloride, cilostazol, clopidogrel
bisulfate, dipyridamole,
eptifibatide, midodrine hydrochloride, pentoxifylline, ticlopidine
hydrochloride, tirofiban
hydrochloride. (See, e.g., pp. 215-336 of Nursirzg 2001 Drug Handbook.)
The at least one nonnarcotic analgesic or antipyretic can be at least one
selected from
acetaminophen, aspirin, choline magnesium trisalicylate, diflunisal, magnesium
salicylate. The at least
one nonsteroidal anti-inflammatory drug can be at least one selected from
celecoxib, diclofenac
potassium, diclofenac sodium, etodolac, fenoprofen calcium, flurbiprofen,
ibuprofen, indomethacin,
indomethacin sodium trihydrate, ketoprofen, ketorolac tromethamine,
nabumetone, naproxen, naproxen
2 0 sodium, oxaprozin, piroxicam, rofecoxib, sulindac, The at least one
narcotic or opiod analgesic can be
at least one selected from alfentanil hydrochloride, buprenorphine
hydrochloride, butorphanol tartrate,
codeine phosphate, codeine sulfate, fentanyl citrate, fentanyl transdermal
system, fentanyl
transmucosal, hydromorphone hydrochloride, meperidine hydrochloride, methadone
hydrochloride,
morphine hydrochloride, morphine sulfate, morphine tartrate, nalbuphine
hydrochloride, oxycodone
2 5 hydrochloride, oxycodone pectinate, oxymorphone hydrochloride, pentazocine
hydrochloride,
pentazocine hydrochloride and naloxone hydrochloride, pentazocine lactate,
propoxyphene
hydrochloride, propoxyphene napsylate, remifentanil hydrochloride, sufentanil
citrate, tramadol
hydrochloride. The at least one sedative-hypnotic can be at least one selected
from chloral hydrate,
estazolam, flurazepam hydrochloride, pentobarbital, pentobarbital sodium,
Phenobarbital sodium,
3 0 secobarbital sodium, temazepam, triazolam, zaleplon, zolpidem tartrate.
The at least one
anticonvulsant can be at least one selected from acetazolamide sodium,
carbamazepine, clonazepam,
clorazepate dipotassium, diazepam, divalproex sodium, ethosuximde,
fosphenytoin sodium,
gabapentin, lamotrigine, magnesium sulfate, Phenobarbital, Phenobarbital
sodium, phenytoin,
phenytoin sodium, phenytoin sodium (extended), primidone, tiagabine
hydrochloride, topiramate,
3 5 valproate sodium, valproic acid. The at least one antidepressant can be at
least one selected from
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
amitriptyline hydrochloride, amitriptyline pamoate, amoxapine, bupropion
hydrochloride, citalopram
hydrobromide, clomipramine hydrochloride, desipramine hydrochloride, doxepin
hydrochloride,
fluoxetine hydrochloride, imipramine hydrochloride, imipramine pamoate,
mirtazapine, nefazodone
hydrochloride, nortriptyline hydrochloride, paroxetine hydrochloride,
phenelzine sulfate, sertraline
hydrochloride, tranylcypromine sulfate, trimipramine maleate, venlafaxine
hydrochloride. The at least
one antianxiety drug can be at least one selected from alprazolam, buspirone
hydrochloride,
chlordiazepoxide, chlordiazepoxide hydrochloride, clorazepate dipotassium,
diazepam, doxepin
hydrochloride, hydroxyzine embonate, hydroxyzine hydrochloride, hydroxyzine
pamoate, lorazepam,
mephrobamate, midazolam hydrochloride, oxazepam. The at least one
antipsychotic drug can be at
least one selected from chlorpromazine hydrochloride, clozapine, fluphenazine
decanoate,
fluephenazine enanthate, fluphenazine hydrochloride, haloperidol, haloperidol
decanoate, haloperidol
lactate, loxapine hydrochloride, loxapine succinate, mesoridazine besylate,
molindone hydrochloride,
olanzapine, perphenazine, pimozide, prochlorperazine, quetiapine fumarate,
risperidone, thioridazine
hydrochloride, thiothixene, thiothixene hydrochloride, trifluoperazine
hydrochloride. The at least one
central nervous system stimulant can be at least one selected from amphetamine
sulfate, caffeine,
dextroamphetamine sulfate, doxapram hydrochloride, methamphetamine
hydrochloride,
methylphenidate hydrochloride, modafinil, pemoline, phentermine hydrochloride.
The at least one
antiparkinsonian can be at least one selected from amantadine hydrochloride,
benztropine mesylate,
biperiden hydrochloride, biperiden lactate, bromocriptine mesylate, carbidopa-
levodopa, entacapone,
2 0 levodopa, pergolide mesylate, pramipexole dihydrochloride, ropinirole
hydrochloride, selegiline
hydrochloride, tolcapone, trihexyphenidyl hydrochloride. The at least one
miscellaneous central
nervous system drug can be at least one selected from bupropion hydrochloride,
donepezil
hydrochloride, droperidol, fluvoxamine maleate, lithium carbonate, lithium
citrate, naratriptan
hydrochloride, nicotine polacrilex, nicotine transdermal system, propofol,
rizatriptan benzoate,
2 5 sibutramine hydrochloride monohydrate, sumatriptan succinate, tacrine
hydrochloride, zolmitriptan.
(See, e.g., pp. 337-530 of Nursing 2001 Drug Handbook.)
The at least one cholinergic (e.g., parasymathomimetic) can be at least one
selected from
bethanechol chloride, edrophonium chloride, neostigmine bromide, neostigmine
methylsulfate,
physostigmine salicylate, pyridostigmine bromide. The at least one
anticholinergics can be at least one
3 0 selected from atropine sulfate, dicyclomine hydrochloride, glycopyrrolate,
hyoscyamine, hyoscyamine
sulfate, propantheline bromide, scopolamine, scopolamine butylbromide,
scopolamine hydrobromide.
The at least one adrenergics (sympathomimetics) can be at least one selected
from dobutamine
hydrochloride, dopamine hydrochloride, metaraminol bitartrate, norepinephrine
bitartrate,
phenylephrine hydrochloride, pseudoephedrine hydrochloride, pseudoephedrine
sulfate. The at least
3 5 one adrenergic blocker (sympatholytic) can be at least one selected from
dihydroergotamine mesylate,
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ergotamine tartrate, methysergide maleate, propranolol hydrochloride. The at
least one skeletal muscle
relaxant can be at least one selected from baclofen, carisoprodol,
chlorzoxazone, cyclobenzaprine
hydrochloride, dantrolene sodium, methocarbamol, tizanidine hydrochloride. The
at least one
neuromuscular blockers can be at least one selected from atracurium besylate,
cisatracurium besylate,
doxacurium chloride, mivacurium chloride, pancuronium bromide, pipecuronium
bromide,
rapacuronium bromide, rocuronium bromide, succinylcholine chloride,
tubocurarine chloride,
vecuronium bromide. (See, e.g., pp. 531-84 of Nursing 2001 Drug Handbook.)
The at least one antihistamine can be at least one selected from
brompheniramine maleate,
cetirizine hydrochloride, chlorpheniramine maleate, clemastine fumarate,
cyproheptadine
hydrochloride, diphenhydramine hydrochloride, fexofenadine hydrochloride,
loratadine, promethazine
hydrochloride, promethazine theoclate, triprolidine hydrochloride. The at
least one bronchodilators
can be at least one selected from albuterol, albuterol sulfate, aminophylline,
atropine sulfate, ephedrine
sulfate, epinephrine, epinephrine bitartrate, epinephrine hydrochloride,
ipratropium bromide,
isoproterenol, isoproterenol hydrochloride, isoproterenol sulfate,
levalbuterol hydrochloride,
metaproterenol sulfate, oxtriphylline, pirbuterol acetate, salmeterol
xinafoate, terbutaline sulfate,
theophylline. The at least one expectorants or antitussives can be at least
one selected from
benzonatate, codeine phosphate, codeine sulfate, dextramethorphan
hydrobromide, diphenhydramine
hydrochloride, guaifenesin, hydromorphone hydrochloride. The at least one
miscellaneous respiratory
drug can be at least one selected from acetylcysteine, beclomethasone
dipropionate, beractant,
2 0 budesonide, calfactant, cromolyn sodium, dornase alfa, epoprostenol
sodium, flunisolide, fluticasone
propionate, montelukast sodium, nedocromil sodium, palivizumab, triamcinolone
acetonide,
zafirlukast, zileuton. (See, e.g., pp. 585-642 of Nursing 2001 Drug Handbook.)
The at least one antacid, adsorbents, or antiflatulents can be at least one
selected from
aluminum carbonate, aluminum hydroxide, calcium carbonate, magaldrate,
magnesium hydroxide,
2 5 magnesium oxide, simethicone, sodium bicarbonate. The at least one
digestive enymes or gallstone
solubilizers can be at least one selected from pancreatin, pancrelipase,
ursodiol. The at least one
antidiarrheal can be at least one selected from attapulgite, bismuth
subsalicylate, calcium
polycarbophil, diphenoxylate hydrochloride or atropine sulfate, loperamide,
octreotide acetate, opium
tincture, opium tincure (camphorated). The at least one laxative can be at
least one selected from
3 0 bisocodyl, calcium polycarbophil, cascara sagrada, cascara sagrada
aromatic fluidextract, cascara
sagrada fluidextract, castor oil, docusate calcium, docusate sodium, glycerin,
lactulose, magnesium
citrate, magnesium hydroxide, magnesium sulfate, methylcellulose, mineral oil,
polyethylene glycol or
electrolyte solution, psyllium, senna, sodium phosphates. The at least one
antiemetic can be at least
one selected from chlorpromazine hydrochloride, dimenhydrinate, dolasetron
mesylate, dronabinol,
3 5 granisetron hydrochloride, meclizine hydrochloride, metocloproamide
hydrochloride, ondansetron
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hydrochloride, perphenazine, prochlorperazine, prochlorperazine edisylate,
prochlorperazine maleate,
promethazine hydrochloride, scopolamine, thiethylperazine maleate,
trimethobenzamide hydrochloride.
The at least one antiulcer drug can be at least one selected from cimetidine,
cimetidine hydrochloride,
famotidine, lansoprazole, misoprostol, nizatidine, omeprazole, rabeprozole
sodium, rantidine bismuth
citrate, ranitidine hydrochloride, sucralfate. (See, e.g., pp. 643-95 of
Nuf°sing 2001 Drug Handbook.)
The at least one coricosteroids can be at least one selected from
betamethasone, betamethasone
acetate or betamethasone sodium phosphate, betamethasone sodium phosphate,
cortisone acetate,
dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate,
fludrocortisone acetate,
hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate,
hydrocortisone sodium phosphate,
hydrocortisone sodium succinate, methylprednisolone, methylprednisolone
acetate,
methylprednisolone sodium succinate, prednisolone, prednisolone acetate,
prednisolone sodium
phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone
acetonide, triamcinolone
diacetate. The at least one androgen or anabolic steroids can be at least one
selected from danazol,
fluoxymesterone, methyltestosterone, nandrolone decanoate, nandrolone
phenpropionate, testosterone,
testosterone cypionate, testosterone enanthate, testosterone propionate,
testosterone transdermal
system. The at least one estrogen or progestin can be at least one selected
from esterified estrogens,
estradiol, estradiol cypionate, estradiol/norethindrone acetate transdermal
system, estradiol valerate,
estrogens (conjugated), estropipate, ethinyl estradiol, ethinyl estradiol and
desogestrel, ethinyl estradiol
and ethynodiol diacetate, ethinyl estradiol and desogestrel, ethinyl estradiol
and ethynodiol diacetate,
2 0 ethinyl estradiol and levonorgestrel, ethinyl estradiol and norethindrone,
ethinyl estradiol and
norethindrone acetate, ethinyl estradiol and norgestimate, ethinyl estradiol
and norgestrel, ethinyl
estradiol and norethindrone and acetate and ferrous fumarate, levonorgestrel,
medroxyprogesterone
acetate, mestranol and norethindron, norethindrone, norethindrone acetate,
norgestrel, progesterone.
The at least one gonadroptropin can be at least one selected from ganirelix
acetate, gonadoreline
2 5 acetate, histrelin acetate, menotropins. The at least one antidiabetic or
glucaon can be at least one
selected from acarbose, chlorpropamide, glimepiride, glipizide, glucagon,
glyburide, insulins,
metformin hydrochloride, miglitol, pioglitazone hydrochloride, repaglinide,
rosiglitazone maleate,
troglitazone. The at least one thyroid hormone can be at least one selected
from levothyroxine sodium,
liothyronine sodium, liotrix, thyroid. The at least one thyroid hormone
antagonist can be at least one
3 0 selected from methimazole, potassium iodide, potassium iodide (saturated
solution), propylthiouracil,
radioactive iodine (sodium iodide 13y ), strong iodine solution. The at least
one pituitary hormone can
be at least one selected from corticotropin, cosyntropin, desmophressin
acetate, leuprolide acetate,
repository corticotropin, somatrem, somatropin, vasopressin. The at least one
parathyroid-like drug
can be at least one selected from calcifediol, calcitonin (human), calcitonin
(salmon), calcitriol,
3 5 dihydrotachysterol, etidronate disodium. (See, e.g., pp. 696-796 of
Nursing 2001 Drug Handbook.)
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The at least one diuretic can be at least one selected from acetazolamide,
acetazolamide
sodium, amiloride hydrochloride, bumetanide, chlorthalidone, ethacrynate
sodium, ethacrynic acid,
furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone,
spironolactone, torsemide,
triamterene, urea. The at least one electrolyte or replacement solution can be
at least one selected from
calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium
glubionate, calcium
gluceptate, calcium gluconate, calcium lactate, calcium phosphate (dibasic),
calcium phosphate
(tribasic), dextran (high-molecular-weight), dextran (low-molecular-weight),
hetastarch, magnesium
chloride, magnesium sulfate, potassium acetate, potassium bicarbonate,
potassium chloride, potassium
gluconate, Ringer's injection, Ringer's injection (lactated), sodium chloride.
The at least one acidifier
or alkalinizer can be at least one selected from sodium bicarbonate, sodium
lactate, tromethamine.
(See, e.g., pp. 797-833 of Nursing 2001 Drug Handbook.)
The at least one hematinic can be at least one selected from ferrous fumarate,
ferrous
gluconate, ferrous sulfate, ferrous sulfate (dried), iron dextran, iron
sorbitol, polysaccharide-iron
complex, sodium ferric gluconate complex. The at least one anticoagulant can
be at least one selected
from ardeparin sodium, dalteparin sodium, danaparoid sodium, enoxaparin
sodium, heparin calcium,
heparin sodium, warfarin sodium. The at least one blood derivative can be at
least one selected from
albumin 5%, albumin 25%, antihemophilic factor, anti-inhibitor coagulant
complex, antithrombin III
(human), factor IX (human), factor IX complex, plasma protein fractions. The
at least one
thrombolytic enzyme can be at least one selected from alteplase, anistreplase,
reteplase (recombinant),
2 0 streptokinase, urokinase. (See, e.g., pp. 834-66 of Nursing 2001 Drug
Handbook.)
The at Least one alkylating drug can be at least one selected from busulfan,
carboplatin,
carmustine, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, lomustine,
mechlorethamine
hydrochloride, melphalan, melphalan hydrochloride, streptozocin, temozolomide,
thiotepa. The at least
one antimetabolite can be at least one selected from capecitabine, cladribine,
cytarabine, floxuridine,
2 5 fludarabine phosphate, fluorouracil, hydroxyurea, mercaptopurine,
methotrexate, methotrexate sodium,
thioguanine. The at least one antibiotic antineoplastic can be at least one
selected from bleomycin
sulfate, dactinomycin, daunorubicin citrate Iiposomal, daunorubicin
hydrochloride, doxorubicin
hydrochloride, doxorubicin hydrochloride liposomal, epirubicin hydrochloride,
idarubicin
hydrochloride, mitomycin, pentostatin, plicamycin, valrubicin. The at least
one antineoplastics that
3 0 alter hormone balance can be at least one selected from anastrozole,
bicalutamide, estramustine
phosphate sodium, exemestane, flutamide, goserelin acetate, letrozole,
leuprolide acetate, megestrol
acetate, nilutamide, tamoxifen citrate, testolactone, toremifene citrate. The
at least one miscellaneous
antineoplastic can be at least one selected from asparaginase, bacillus
Calmette-Guerin (BCG) (live
intravesical), dacarbazine, docetaxel, etoposide, etoposide phosphate,
gemcitabine hydrochloride,
3 5 irinotecan hydrochloride, mitotane, mitoxantrone hydrochloride,
paclitaxel, pegaspargase, porfimer
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sodium, procarbazine hydrochloride, rituximab, teniposide, topotecan
hydrochloride, trastuzumab,
tretinoin, vinblastine sulfate, vincristine sulfate, vinorelbine tartrate.
(See, e.g., pp. 867-963 of Nursing
2001 Drug Handbook.)
The at least one immunosuppressant can be at least one selected from
azathioprine,
basiliximab, cyclosporine, daclizumab, lymphocyte immune globulin, muromonab-
CD3,
mycophenolate mofetil, mycophenolate mofetil hydrochloride, sirolimus,
tacrolimus. The at least one
vaccine or toxoid can be at least one selected from BCG vaccine, cholera
vaccine, diphtheria and
tetanus toxoids (adsorbed), diphtheria and tetanus toxoids and acellular
pertussis vaccine adsorbed,
diphtheria and tetanus toxoids and whole-cell pertussis vaccine, Haemophilius
b conjugate vaccines,
hepatitis A vaccine (inactivated), hepatisis B vaccine (recombinant),
influenza virus vaccine 1999-2000
trivalent types A & B (purified surface antigen), influenza virus vaccine 1999-
2000 trivalent types A &
B (subvirion or purified subvirion), influenza virus vaccine 1999-2000
trivalent types A & B (whole
virion), Japanese encephalitis virus vaccine (inactivated), Lyme disease
vaccine (recombinant OspA),
measles and mumps and rubella virus vaccine (live), measles and mumps and
rubella virus vaccine
(live attenuated), measles virus vaccine (live attenuated), meningococcal
polysaccharide vaccine,
mumps virus vaccine (live), plague vaccine, pneumococcal vaccine (polyvalent),
poliovirus vaccine
(inactivated), poliovirus vaccine (live, oral, trivalent), rabies vaccine
(adsorbed), rabies vaccine (human
diploid cell), rubella and mumps virus vaccine (live), rubella virus vaccine
(live, attenuated), tetanus
toxoid (adsorbed), tetanus toxoid (fluid), typhoid vaccine (oral), typhoid
vaccine (parenteral), typhoid
2 0 Vi polysaccharide vaccine, varicella virus vaccine, yellow fever vaccine.
The at least one antitoxin or
antivenin can be at least one selected from black widow spider antivenin,
Crotalidae antivenom
(polyvalent), diphtheria antitoxin (equine), Micrurus fulvius antivenin). The
at least one immune
serum can be at least one selected from cytomegalovirus immune globulin
(intraveneous), hepatitis B
immune globulin (human), immune globulin intramuscular, immune globulin
intravenous, rabies
2 5 immune globulin (human), respiratory syncytial virus immune globulin
intravenous (human), Rh°(D)
immune globulin (human), Rho(D) immune globulin intravenous (human), tetanus
immune globulin
(human), varicella-zoster immune globulin. The at least one biological
response modifiers can be at
least one selected from aldesleukin, epoetin alfa, filgrastim, glatiramer
acetate for injection, interferon
alfacon-1, interferon alfa-2a (recombinant), interferon alfa-2b (recombinant),
interferon beta-la,
3 0 interferon beta-lb (recombinant), interferon gamma-lb, levamisole
hydrochloride, oprelvekin,
sargramostim. (See, e.g., pp. 964-1040 of Nursing 2001 Drug Handbook.)
The at least one ophthalmic anti-infectives can be selected form bacitracin,
chloramphenicol,
ciprofloxacin hydrochloride, erythromycin, gentamicin sulfate, ofloxacin 0.3%,
polymyxin B sulfate,
sulfacetamide sodium 10%, sulfacetamide sodium 15%, sulfacetamide sodium 30%,
tobramycin,
3 5 vidarabine. The at least one ophthalmic anti-inflammatories can be at
least one selected from
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dexamethasone, dexamethasone sodium phosphate, diclofenac sodium 0.1%,
fluorometholone,
flurbiprofen sodium, ketorolac tromethamine, prednisolone acetate (suspension)
prednisolone sodium
phosphate (solution). The at least one miotic can be at least one selected
from acetylocholine chloride,
carbachol (intraocular), carbachol (topical), echothiophate iodide,
pilocarpine, pilocarpine
hydrochloride, pilocarpine nitrate. The at least one mydriatic can be at least
one selected from atropine
sulfate, cyclopentolate hydrochloride, epinephrine hydrochloride, epinephryl
borate, homatropine
hydrobromide, phenylephrine hydrochloride, scopolamine hydrobromide,
tropicamide. The at least
one ophthalmic vasoconstrictors can be at least one selected from naphazoline
hydrochloride,
oxymetazoline hydrochloride, tetrahydrozoline hydrochloride. The at least one
miscellaneous
ophthalmics can be at least one selected from apraclonidine hydrochloride,
betaxolol hydrochloride,
brimonidine tartrate, carteolol hydrochloride, dipivefrin hydrochloride,
dorzolamide hydrochloride,
emedastine difumarate, fluorescein sodium, ketotifen fumarate, latanoprost,
levobunolol hydrochloride,
metipranolol hydrochloride, sodium chloride (hypertonic), timolol maleate. The
at least one otic can
be at least one selected from boric acid, carbamide peroxide, chloramphenicol,
triethanolamine
polypeptide oleate-condensate. The at least one nasal drug can be at least one
selected from
beclomethasone dipropionate, budesonide, ephedrine sulfate, epinephrine
hydrochloride, flunisolide,
fluticasone propionate, naphazoline hydrochloride, oxymetazoline
hydrochloride, phenylephrine
hydrochloride, tetrahydrozoline hydrochloride, triamcinolone acetonide,
xylometazoline hydrochloride.
(See, e.g., pp. 1041-97 of Nursing 2001 Drug Handbook.)
2 0 The at least one local anti-infectives can be at least one selected from
acyclovir, amphotericin
B, azelaic acid cream, bacitracin, butoconazole nitrate, clindamycin
phosphate, clotrimazole, econazole
nitrate, erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate,
metronidazole (topical),
miconazole nitrate, mupirocin, naftifme hydrochloride, neomycin sulfate,
nitrofurazone, nystatin, silver
sulfadiazine, terbinaflne hydrochloride, terconazole, tetracycline
hydrochloride, tioconazole, tolnaftate.
2 5 The at least one scabicide or pediculicide can be at least one selected
from crotamiton, lindane,
permethrin, pyrethrins. The at least one topical corticosteroid can be at
least one selected from
betamethasone dipropionate, betamethasone valerate, clobetasol propionate,
desonide, desoximetasone,
dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate,
fluocinolone acetonide,
fluocinonide, flurandrenolide, fluticasone propionate, halcionide,
hydrocortisone, hydrocortisone
3 0 acetate, hydrocortisone butyrate, hydrocorisone valerate, mometasone
furoate, triamcinolone acetonide.
(See, e.g., pp. 1098-1136 of Nursing 2001 Drug Handbook.)
The at least one vitamin or mineral can be at least one selected from vitamin
A, vitamin B
complex, cyanocobalamin, folic acid, hydroxocobalamin, leucovorin calcium,
niacin, niacinamide,
pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin C,
vitamin D, cholecalciferol,
3 5 ergocalciferol, vitamin D analogue, doxercalciferol, paricalcitol, vitamin
E, vitamin K analogue,
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phytonadione, sodium fluoride, sodium fluoride (topical), trace elements,
chromium, copper, iodine,
manganese, selenium, zinc. The at least one calorics can be at least one
selected from amino acid
infusions (crystalline), amino acid infusions in dextrose, amino acid
infusions with electrolytes, amino
acid infusions with electrolytes in dextrose, amino acid infusions for hepatic
failure, amino acid
infusions for high metabolic stress, amino acid infusions for renal failure,
dextrose, fat emulsions,
medium-chain triglycerides. (See, e.g., pp. 1137-63 of Nursing 2001 D>"ug
Handbook.)
CHl deleted mimetibody antibody or polypeptide compositions of the present
invention can
further comprise at least one of any suitable and/or effective amount of a
composition or
pharmaceutical composition comprising at least one CHl deleted mimetibody
protein or antibody to a
cell, tissue, organ, animal or patient in need of such modulation, treatment
or therapy, optionally
further comprising at least one selected from at least one TNF antagonist
(e.g., but not limited to a TNF
chemical or protein antagonist, TNF monoclonal or polyclonal antibody or
fragment, a soluble TNF
receptor (e.g., p55, p70 or p85) or fragment, fusion polypeptides thereof, or
a small molecule TNF
antagonist, e.g., TNF binding protein I or II (TBP-1 or TBP-II), nerelimonmab,
infliximab, enteracept,
CDP-571, CDP-870, afelimomab, lenercept, and the like), an antirheumatic
(e.g., methotrexate,
auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate,
hydroxychloroquine
sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-
steroid inflammatory drug
(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a
neuromuscular Mocker, an
antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an
antiviral, a carbapenem,
2 0 cephalosporin, a flurorquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another
antimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid, a
diabetes related agent, a
mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone,
an antidiarrheal, an
antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an
erythropieitin (e.g., epoetin
alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF,
Leukine), an inununization, an
2 5 immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine,
daclizumab), a growth
hormone, a hormone replacement drug, an estrogen receptor modulator, a
mydriatic, a cycloplegic, an
alkylating agent, an antimetabolite, a mitotic inhibitor, a
radiopharmaceutical, an antidepressant,
antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant,
donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid,
a leukotriene inhibitor, a
3 0 methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha
(Pulmozyme), a cytokine or a
cytokine antagonist. Non-limiting examples of such cytokines include, but are
not limted to, any of IL-
1 to IL-23. Suitable dosages are well known in the art. See, e.g., Wells et
al., eds., Pharmacotherapy
Handbook, 2"d Edition, Appleton and Lange, Stamford, CT (2000); PDR
Pharmacopoeia, Tarascon
Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA
(2000), each of
3 5 which references are entirely incorporated herein by reference.
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Such compositions can also include toxin molecules that are associated, bound,
co-formulated
or co-administered with at least one antibody or polypeptide of the present
invention. The toxin can
optionally act to selectively kill the pathologic cell or tissue. The
pathologic cell can be a cancer or
other cell. Such toxins can be, but are not limited to, purified or
recombinant toxin or toxin fragment
comprising at least one functional cytotoxic domain of toxin, e.g., selected
from at least one of ricin,
diphtheria toxin, a venom toxin, or a bacterial toxin. The term toxin also
includes both endotoxins and
exotoxins produced by any naturally occurring, mutant or recombinant bacteria
or viruses which may
cause any pathological condition in humans and other mammals, including toxin
shock, which can
result in death. Such toxins may include, but are not limited to,
enterotoxigenic E. coli heat-labile
enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeronzonas
enterotoxins, toxic
shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB),
or C (SEC),
Streptococcal enterotoxins and the like. Such bacteria include, but are not
limited to, strains of a
species of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g.,
strains of serotype
0157:H7), Staphylococcus species (e.g., Staphylococcus aurezrs, Staphylococcus
pyogertes), Shigella
species (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and
Shigella sonrzei), Salmonella
species (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella
enteritidis), Clostridium species
(e.g., Clostridiurrz perfringe>zs, Clostridium dificile, Clostridium
botulirzurn), Canzphlobacter species
(e.g., Carrzphlobacter jejuni, Camphlobacter fetus), Heliobacter species,
(e.g., Heliobacter pylori),
Aeronzorzas species (e.g., Aeromonas sobria, Aeromonas lZydrophila, Aeromonas
caviae), Pleisornorras
2 0 shigelloides, Yersina enterocolitica, lribrios species (e.g., Tlibrios
clzolerae, Tribrios parahernolytieus),
Klebsiella species, Pseudonzonas aeruginosa, and Streptococci. See, e.g.,
Stein, ed., INTERNAL
MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990); Evans et
al., eds., Bacterial
Infections of Humans: Epidemiology and Control, 2d. Ed., pp 239-254, Plenum
Medical Book Co.,
New York (1991); Mandell et al, Principles and Practice of Infectious
Diseases, 3d. Ed., Churchill
2 5 Livingstone, New York (1990); Berkow et al, eds., Tlre Merck Manual, 16th
edition, Merck and Co.,
Rahway, N.J., 1992; Wood et al, FEMS Microbiology Immunology, 76:121-134
(1991); Marrack et al,
Science, 248:705-711 (1990), the contents of which references are incorporated
entirely herein by
reference.
CH1-deleted mimetibody or specified portion or variant compositions of the
present invention
3 0 can further comprise at least one of any suitable auxiliary, such as, but
not limited to, diluent, binder,
stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the
like. Pharmaceutically
acceptable auxiliaries are preferred. Non-limiting examples of, and methods of
preparing such sterile
solutions are well known in the art, such as, but limited to, Gennaro, Ed.,
Renzirzgton's Pharmaceutical
Sciences, 18~' Edition, Mack Publishing Co. (Easton, PA) 1990.
Pharmaceutically acceptable carriers
3 5 can be routinely selected that are suitable for the mode of
administration, solubility and/or stability of
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the CHl-deleted mimetibody composition as well known in the art or as
described herein.
Pharmaceutical excipients and additives useful in the present composition
include but are not
limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g.,
sugars, including
monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars
such as alditols, aldonic
acids, esterified sugars and the like; and polysaccharides or sugar polymers),
which can be present
singly or in combination, comprising alone or in combination 1-99.99% by
weight or volume.
Exemplary protein excipients include serum albumin such as human serum albumin
(HSA),
recombinant human albumin (rHA), gelatin, casein, and the like. Representative
amino acid/CHl-
deleted mimetibody or specified portion or variant components, which can also
function in a buffering
capacity, include alanine, glycine, arginine, betaine, histidine, glutamic
acid, aspartic acid, cysteine,
lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and
the like. One preferred
amino acid is glycine.
Carbohydrate excipients suitable for use in the invention include, for
example,
monosaccharides such as fructose, maltose, galactose, glucose, D-mannose,
sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like;
polysaccharides, such as
raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and
alditols, such as mannitol,
xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the
like. Preferred carbohydrate
excipients for use in the present invention are mannitol, trehalose, and
raffinose.
CHl-deleted mimetibody compositions can also include a buffer or a pH
adjusting agent;
2 0 typically, the buffer is a salt prepared from an organic acid or base.
Representative buffers include
organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid,
carbonic acid, tartaric acid,
succinic acid, acetic acid, or phthalic acid; Tris, tromethamine
hydrochloride, or phosphate buffers.
Preferred buffers for use in the present compositions are organic acid salts
such as citrate.
Additionally, the CH1-deleted mimetibody or specified portion or variant
compositions of the
2 5 invention can include polymeric excipients/additives such as
polyvinylpyrrolidones, ficolls (a
polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-(3-
cyclodextrin), polyethylene
glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants,
antistatic agents, surfactants
(e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g.,
phospholipids, fatty acids),
steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
3 0 These and additional known pharmaceutical excipients and/or additives
suitable for use in the
CH1-deleted mimetibody compositions according to the invention are known in
the art, e.g., as listed in
"Remington: The Science & Practice of Pharmacy", 19th ed., Williams &
Williams, (1995), and in the
"Physician's Desk Reference", S2"d ed., Medical Economics, Montvale, NJ
(1998), the disclosures of
which are entirely incorporated herein by reference. Preferrred carrier or
excipient materials are
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carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or
polymeric agents.
Formulations
As noted above, the invention provides for stable formulations, which can
preferably include a
suitable buffer with saline or a chosen salt, as well as optional preserved
solutions and formulations
containing a preservative as well as multi-use preserved formulations suitable
for pharmaceutical or
veterinary use, comprising at least one CH1-deleted mimetibody or specified
portion or variant in a
pharmaceutically acceptable formulation. Preserved formulations contain at
least one known
preservative or optionally selected from the group consisting of at least one
phenol, m-cresol, p-cresol,
o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite,
phenoxyethanol, formaldehyde,
chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl,
ethyl, propyl, butyl and
the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate
and thimerosal, or
mixtures thereof in an aqueous diluent. Any suitable concentration or mixture
can be used as known in
the art, such as 0.001-S%, or any range or value therein, such as, but not
limited to 0.001, 0.003, O.OOS,
0.009, 0.01, 0.02, 0.03, O.OS, 0.09, 0.1, 0.2, 0.3, 0.4., O.S, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,
3.I, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,
3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-
limiting examples include, no
preservative, O.I-2% m-cresol (e.g., 0.2, 0.3. 0.4, O.S, 0.9, 1.0%), 0.1-3%
benzyl alcohol (e.g., O.S, 0.9,
1.1., l.S, 1.9, 2.0, 2.S%), 0.001-O.S% thimerosal (e.g., 0.005, 0.01), 0.001-
2.0% phenol (e.g., 0.05,
0.25, 0.28, O.S, 0.9, 1.0%), O.OOOS-1.0% alkylparaben(s) (e.g., 0.00075,
0.0009, 0.001, 0.002, O.OOS,
2 0 0.0075, 0.009, 0.01, 0.02, O.OS, 0.075, 0.09, 0.1, 0.2, 0.3, O.S, 0.75,
0.9, I .0%), and the like.
As noted above, the invention provides an article of manufacture, comprising
packaging
material and at least one vial comprising a solution of at least one CHI-
deleted mimetibody or
specified portion or variant with the prescribed buffers and/or preservatives,
optionally in an aqueous
diluent, wherein said packaging material comprises a label that indicates that
such solution can be held
2 5 over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54,
60, 66, 72 hours or greater. The
invention further comprises an article of manufacture, comprising packaging
material, a first vial
comprising lyophilized at least one CHI-deleted mimetibody or specified
portion or variant, and a
second vial comprising an aqueous diluent of prescribed buffer or
preservative, wherein said packaging
material comprises a label that instructs a patient to reconstitute the at
least one CH1-deleted
3 0 mimetibody or specified portion or variant in the aqueous diluent to form
a solution that can be held
over a period of twenty-four hours or greater.
The at least one CHl-deleted mimetibody or specified portion or variant used
in accordance
with the present invention can be produced by recombinant means, including
from mammalian cell or
transgenic preparations, or can be purified from other biological sources, as
described herein or as
3 5 known in the art.
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The range of amounts of at least one CH1-deleted mimetibody or specified
portion or variant in
the product of the present invention includes amounts yielding upon
reconstitution, if in a wet/dry
system, concentrations from about 1.0 p,g/ml to about 1000 mg/ml, although
lower and higher
concentrations are operable and are dependent on the intended delivery
vehicle, e.g., solution
formulations will differ from transdermal patch, pulmonary, transmucosal, or
osmotic or micro pump
methods.
Preferably, the aqueous diluent optionally further comprises a
pharmaceutically acceptable
preservative. Preferred preservatives include those selected from the group
consisting of phenol, m-
cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben
(methyl, ethyl, propyl, butyl and
the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate
and thimerosal, or
mixtures thereof. The concentration of preservative used in the formulation is
a concentration
sufficient to yield an anti-microbial effect. Such concentrations are
dependent on the preservative
selected and are readily determined by the skilled artisan.
Other excipients, e.g. isotonicity agents, buffers, antioxidants, preservative
enhancers, can be
optionally and preferably added to the diluent. An isotonicity agent, such as
glycerin, is commonly
used at known concentrations. A physiologically tolerated buffer is preferably
added to provide
improved pH control. The formulations can cover a wide range of pHs, such as
from about pH 4 to
about pH 10, and preferred ranges from about pH 5 to about pH 9, and a most
preferred range of about
6.0 to about 8Ø Preferably the formulations of the present invention have pH
between about 6.8 and
2 0 about 7.8. Preferred buffers include phosphate buffers, most preferably
sodium phosphate, particularly
phosphate buffered saline (PBS).
Other additives, such as a pharmaceutically acceptable solubilizers like Tween
20
(polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20)
sorbitan
monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic
F68
2 5 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene
glycol) or non-ionic
surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic~
polyls, other block co-
polymers, and chelators such as EDTA and EGTA can optionally be added to the
formulations or
compositions to reduce aggregation. These additives are particularly useful if
a pump or plastic
container is used to administer the formulation. The presence of
pharmaceutically acceptable
3 0 surfactant mitigates the propensity for the protein to aggregate.
The formulations of the present invention can be prepared by a process which
comprises mixing at least one CHl-deleted mimetibody or specified portion or
variant and a
preservative selected from the group consisting of phenol, m-cresol, p-cresol,
o-cresol, chlorocresol,
benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like),
benzalkonium chloride,
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benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures
thereof in an aqueous
diluent. Mixing the at least one CH1-deleted mimetibody or specified portion
or variant and
preservative in an aqueous diluent is carried out using conventional
dissolution and mixing procedures.
To prepare a suitable formulation, for example, a measured amount of at least
one CHl-deleted
mimetibody or specified portion or variant in buffered solution is combined
with the desired
preservative in a buffered solution in quantities sufficient to provide the
protein and preservative at the
desired concentrations. Variations of this pxocess would be recognized by one
of ordinary skill in the
art. For example, the order the components are added, whether additional
additives are used, the
temperature and pH at which the formulation is prepared, are all factors that
may be optimized for the
concentration and means of administration used.
The claimed formulations can be provided to patients as clear solutions or as
dual vials
comprising a vial of lyophilized at least one CH1-deleted mimetibody or
specified portion or variant
that is reconstituted with a second vial containing water, a preservative
and/or excipients, preferably a
phosphate buffer and/or saline and a chosen salt, in an aqueous diluent.
Either a single solution vial or
dual vial requiring reconstitution can be reused multiple times and can
suffice for a single or multiple
cycles of patient treatment and thus can provide a more convenient treatment
regimen than currently
available.
The present claimed articles of manufacture are useful for administration over
a period
of immediately to twenty-four hours or greater. Accordingly, the presently
claimed articles of
2 0 manufacture offer significant advantages to the patient. Formulations of
the invention can optionally be
safely stored at temperatures of from about 2 to about 40°C and retain
the biologically activity of the
protein for extended periods of time, thus, allowing a package label
indicating that the solution can be
held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or
greater. If preserved diluent
is used, such label can include use up to at least one of 1-12 months, one-
half, one and a half, and/or
2 5 two years.
The solutions of at least one CH1-deleted mimetibody or specified portion or
variant in
the invention can be prepared by a process that comprises mixing at least one
CH1-deleted mimetibody
or specified portion or variant in an aqueous diluent. Mixing is carried out
using conventional
dissolution and mixing procedures. To prepare a suitable diluent, for example,
a measured amount of
3 0 at least one CH1-deleted mimetibody or specified portion or variant in
water or buffer is combined in
quantities sufficient to provide the protein and optionally a preservative or
buffer at the desired
concentrations. Variations of this process would be recognized by one of
ordinary skill in the art. For
example, the order the components are added, whether additional additives are
used, the temperature
and pH at which the formulation is prepared, are all factors that may be
optimized for the concentration
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and means of administration used.
The claimed products can be provided to patients as clear solutions or as dual
vials
comprising a vial of lyophilized at least one CH1-deleted mimetibody or
specified portion or variant
that is reconstituted with a second vial containing the aqueous diluent.
Either a single solution vial or
dual vial requiring reconstitution can be reused multiple times and can
suffice for a single or multiple
cycles of patient treatment and thus provides a more convenient treatment
regimen than currently
available.
The claimed products can be provided indirectly to patients by providing to
pharmacies, clinics, or other such institutions and facilities, clear
solutions or dual vials comprising a
vial of lyophilized at least one CHl-deleted mimetibody or specified portion
or variant that is
reconstituted with a second vial containing the aqueous diluent. The clear
solution in this case can be
up to one liter or even larger in size, providing a large reservoir from which
smaller portions of the at
least one CH1-deleted mimetibody or specified portion or variant solution can
be retrieved one or
multiple times for transfer into smaller vials and provided by the pharmacy or
clinic to their customers
and/or patients.
Recognized devices comprising these single vial systems include those pen-
injector
devices for delivery of a solution such as Humaject~'NovoPeri , B-D~Pen,
AutoPen~, and OptiPeri .
Recognized devices comprising a dual vial system include those pen-injector
systems for reconstituting
a lyophilized drug in a cartridge for delivery of the reconstituted solution
such as the HumatroPen~.
2 0 The products presently claimed include packaging material. The packaging
material
provides, in addition to the information required by the regulatory agencies,
the conditions under which
the product can be used. The packaging material of the present invention
provides instructions to the
patient to reconstitute the at least one CH1-deleted mimetibody or specified
portion or variant in the
aqueous diluent to form a solution and to use the solution over a period of 2-
24 hours or greater for the
2 5 two vial, wet/dry, product. For the single vial, solution product, the
label indicates that such solution
can be used over a period of 2-24 hours or greater. The presently claimed
products are useful for
human pharmaceutical product use.
The formulations of the present invention can be prepared by a process that
comprises
mixing at least one CHl-deleted mimetibody or specified portion or variant and
a selected buffer,
3 0 preferably a phosphate buffer containing saline or a chosen salt. Mixing
the at least one CHl-deleted
mimetibody or specified portion or variant and buffer in an aqueous diluent is
carried out using
conventional dissolution and mixing procedures. To prepare a suitable
formulation, for example, a
measured amount of at least one CHl-deleted mimetibody or specified portion or
variant in water or
buffer is combined with the desired buffering agent in water in quantities
sufficient to provide the
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protein and buffer at the desired concentrations. Variations of this process
would be recognized by one
of ordinary skill in the art. For example, the order the components are added,
whether additional
additives are used, the temperature and pH at which the formulation is
prepared, are all factors that can
be optimized for the concentration and means of administration used.
The claimed stable or preserved formulations can be provided to patients as
clear
solutions or as dual vials comprising a vial of lyophilized at least one CHl-
deleted mimetibody or
specified portion or variant that is reconstituted with a second vial
containing a preservative or buffer
and excipients in an aqueous diluent. Either a single solution vial or dual
vial requiring-reconstitution
can be reused multiple times and can suffice for a single or multiple cycles
of patient treatment and
thus provides a more convenient treatment regimen than currently available.
At least one CHl-deleted mimetibody or specified portion or variant in either
the stable or
preserved formulations or solutions described herein, can be administered to a
patient in accordance
with the present invention via a variety of delivery methods including SC or
IM injection; transdermal,
pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or
other means appreciated
by the skilled artisan, as well-known in the art.
Therapeutic Applications
The present invention for mimetibodies also provides a method for modulating
or treating
2 0 anemia, in a cell, tissue, organ, animal, or patient including, but not
limited to, at least one of any
anemia, cancer treatment related anemia, radiotherapy or chemotherapy related
anemia, viral or
bacterial infection treatment related anemia, renal anemia, anemia of
prematurity, pediatric and/or
adult cancer-associated anemia, anemia associated with lymphoma, myeloma,
rnultple myeloma,
AIDS-associated anemia, concomitant treatment for patients with or without
autologous blood donation
2 5 awaiting elective surgery, preoperatve and post operative for surgery,
autologous blood donation or
transfusion, perioperative management, cyclic neutropenia or Kostmann syndrome
(congenital
agranulocytosis), end-stage renal disease, anemia associated with dialysis,
chronic renal insufficiency,
primary hemopoietic diseases, such as congenital hypoplastic anemia,
thalassemia major, or sickle cell
disease, vaso-occlusive complications of sickle cell disease. Furman et al.,
Pediatrics 1992; 90: 716-
3 0 728, Goldberg Science. 1988;242:1412-1415; Paul et al., Exp Hematol.
1984;12:825-830; Erslev et al.,
Arch Intern Med. 1968;122:230-235; Ersley et al., Ann Clin Lab Sci.
1980;10:250-257; Jacobs et al.,
Nature. 1985;313:806-810; Lin et al., Proc Natl Acad Sci USA. 1985;82:7580-
7584; Law et al., Proc
Natl Acad Sci USA. 1986;83:6920-6924; Goldwasser et al., J Biol Chem.
1974;249:4202-4206; Eaves
et a., Blood. 1978;52:1196-1210; Sawyer et al., Blood. 1989;74:103-109;
Winearls et al., Lancet.
3 5 1986;2:1175-1178; Eschbach et aL, N Engl J Med. 1987;316:73-78; Eschbach
et al., Ann Intern Med.
1989;111:992-1000, each reference entirely incoporated herein by reference.
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Mimetibodies of the present invention can also be used for non-renal forms of
anemia induced,
for example, by chronic infections, inflammatory processes, radiation therapy,
and cytostatic drug
treatment, and encouraging results in patients with non-renal anemia have been
reported. See, e.g.,
Abels RI and Rudnick SA Erythropoietin: evolving clinical applications.
Experimental Hematology 19:
842-50 (I991); Graber SE and Krantz SB Erythropoietin: biology and clinical
use. Hematology/Oncol.
Clin. North Amer. 3: 369-400 (1989); Jelkman W and Gross AJ (eds)
Erythropoietin. Springer, Berlin
1989; Koury MJ and Bondurant MC The molecular mechanism of erythropoietin
action. European
Journal of Biochemistry 210: 649-63 (1992); Krantz SB Erythropoietin. Blood
77: 419-34 (1991);
Tabbara IA Erythropoietin. Biology and clinical applications. Archives of
Internal Medicine 153: 298-
304 (1993), each entirely incorporated herein by reference.
The present invention also provides a method for modulating or treating an
anemia or blood
cell related condition, in a cell, tissue, organ, animal, or patient, wherein
said anemia or blood cell
related condition is associated with at least one including, but not limited
to, at least one of immune
related disease, cardiovascular disease, infectious, malignant and/or
neurologic disease. Such a method
can optionally comprise administering an effective amount of at least one
composition or
pharmaceutical composition comprising at least one CHl-deleted mimetibody or
specified portion or
variant to a cell, tissue, organ, animal or patient in need of such
modulation, treatment or therapy.
The present invention also provides a method for modulating or treating
cancerlinfecteous
disease in a cell, tissue, organ, animal or patient, including, but not
limited to, at least one of acute or
2 0 chronic bacterial infection, acute and chronic parasitic or infectious
processes, including bacterial, viral
and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis,
septic arthritis, peritonitis,
pneumonia , epiglottitis, e. coli 0157:h7, hemolytic uremic
syndrome/thrombolytic thrombocytopenic
purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic
shock syndrome,
streptococcal myositis, gas gangrene, mycobacterium tuberculosis,
mycobacterium avium
2 5 intracellulare, pneumocystis earinii pneumonia, pelvic inflammatory
disease, orchitis/epidydimitis,
legionella, lyme disease, influenza a, epstein-barn virus, vital-associated
hemaphagocytic syndrome,
vital encephalitis/aseptic meningitis, and the like; (ii) leukemia, acute
leukemia, acute lymphoblastic
leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML),
chromic myelocytic
leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,
myelodyplastic syndrome
3 0 (MDS), a lymphoma, Hodgkin's disease, a malignamt lymphoma, non-hodgkin's
lymphoma, Burkitt's
lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic
carcinoma,
nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic
syndrome/hypercalcemia of
malignancy, solid tumors, adenocarcinomas, sarcomas, malignant melanoma, and
the like; or (iii)
neurodegenerative diseases, multiple sclerosis, migraine headache, AIDS
dementia complex,
3 5 demyelinating diseases, such as multiple sclerosis and acute transverse
myelitis; extrapyramidal and
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cerebellar disorders' such as lesions of the corticospinal system; disorders
of the basal ganglia or
cerebellar disorders; hyperkinetic movement disorders such as Huntington's
Chorea and senile chorea;
drug-induced movement disorders, such as those induced by drugs which block
CNS dopamine
receptors; hypokinetic movement disorders, such as Parkinson's disease;
Progressive supranucleo
Palsy; structural lesions of the cerebellum; spinocerebellar degenerations,
such as spinal ataxia,
Friedreich's ataxia, cerebellar cortical degenerations, multiple systems
degenerations (Mencel,
Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders (Refsum's
disease,
abetalipoprotemia, ataxia, telangiectasia, and mitochondria) multi.system
disorder); demyelinating core
disorders, such as multiple sclerosis, acute transverse myelitis; and
disorders of the motor unit' such as
neurogenic muscular atrophies (anterior horn cell degeneration, such as
amyotrophic lateral sclerosis,
infantile spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's
Syndrome in middle age; Diffuse Lewy body disease; Senile Dementia of Lewy
body type; Wernicke-
I~orsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute
sclerosing
panencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica, and the
like. Such a method can
optionally comprise administering an effective amount of a composition or
pharmaceutical
composition comprising at least one TNF antibody or specified portion or
variant to a cell, tissue,
organ, animal or patient in need of such modulation, treatment or therapy.
See, e.g., the Merck Manual,
16th Edition, Merck & Company, Rahway, NJ ( 1992)
Such a method can optionally comprise administering an effective amount of at
least one
2 0 composition or pharmaceutical composition comprising at least one CHl-
deleted mimetibody or
specified portion or variant to a cell, tissue, organ, animal or patient in
need of such modulation,
treatment or therapy.
The present invention also provides a method for modulating or treating at
least one
cardiovascular disease in a cell, tissue, organ, animal, or patient,
including, but not limited to, at least
2 5 one of cardiac stun syndrome, myocardial infarction, congestive heart
failure, stroke, ischemic stroke,
hemorrhage, arteriosclerosis, atherosclerosis, diabetic ateriosclerotic
disease, hypertension, arterial
hypertension, renovascular hypertension, syncope, shock, syphilis of the
cardiovascular system, heart
failure, cor pulmonale, primary pulmonary hypertension, cardiac arrhythmias,
atria) ectopic beats,
atria) flutter, atria) fibrillation (sustained or paroxysmal), chaotic or
multifocal atria) tachycardia,
3 0 regular narrow QRS tachycardia, specific arrythmias, ventricular
fibrillation, His bundle anythmias,
atrioventricular block, bundle branch block, myocardial ischemic disorders,
coronary artery disease,
angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive
cardiomyopathy, restrictive
cardiomyopathy, valvular heart diseases, endocarditis, pericardial disease,
cardiac tumors, aordic and
peripheral aneuryisms, aortic dissection, inflammation of the aorta, occulsion
of the abdominal aorta
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and its branches, peripheral vascular disorders, occulsive arterial disorders,
peripheral atherlosclerotic
disease, thromboangitis obliterans, functional peripheral arterial disorders,
Raynaud's phenomenon and
disease, acrocyanosis, erythromelalgia, venous diseases, venous thrombosis,
varicose veins,
arteriovenous fistula, lymphederma, lipedema, unstable angina, reperfusion
injury, post pump
syndrome, ischemia-reperfusion injury, and the like. Such a method can
optionally comprise
administering an effective amount of a composition or pharmaceutical
composition comprising at least
one CHl-deleted mimetibody or specified portion or variant to a cell, tissue,
organ, animal or patient in
need of such modulation, treatment or therapy.
Any method of the present invention can comprise administering an effective
amount of a
composition or pharmaceutical composition comprising at least one CH1-deleted
mimetibody or
specified portion or variant to a cell, tissue, organ, animal or patient in
need of such modulation,
treatment or therapy. Such a method can optionally further comprise co-
administration or combination
therapy for treating such immune diseases, wherein the administering of said
at least one CHl-deleted
mimetibody, specified portion or variant thereof, further comprises
administering, before concurrently,
and/or after, at least one selected from at least one TNF antagonist (e.g.,
but not limited to a TNF
antibody or fragment, a soluble TNF receptor or fragment, fusion proteins
thereof, or a small molecule
TNF antagonist), an antirheumatic, a muscle relaxant, a narcotic, a non-
steroid anti-inflammatory drug
(NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a
neuromuscular blocker, an
antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an
antiviral, a carbapenem,
2 0 cephalosporin, a flurorquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another
antimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid, a
diabetes related agent, a
mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone,
an antidiarrheal, an
antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an
erythropieitin (e.g., epoetin
alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF,
Leukine), an immunization, an
2 5 immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine,
daclizumab), a growth
hormone, a hormone replacement drug, an estrogen receptor modulator, a
mydriatic, a cycloplegic, an
alkylating agent, an antimetabolite, a mitotic inhibitor, a
radiopharmaceutical, an antidepressant,
antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant,
donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid,
a leukotriene inhibitor, a
3 0 methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha
(Pulmozyme), a cytokine or a
cytokine antagonist. Suitable dosages are well known in the art. See, e.g.,
Wells et al., eds.,
Pharmacotherapy Handbook, 2°d Edition, Appleton and Lange, Stamford, CT
(2000); PDR
Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon
Publishing, Loma
Linda, CA (2000), each of which references are entirely incorporated herein by
reference.
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CA 02490409 2004-12-24
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Mimetibodies can also be used ex vivo, such as in autologous marrow culture.
Briefly, bone
marrow is removed from a patient prior to chemotherapy and treated with TPO
and/or EPO, optionally
in combination with mimetibodies, optionally in combination with one or more
additional cytokines.
The treated marrow is then returned to the patient after chemotherapy to speed
the recovery of the
marrow. In addition, TPO, alone and in combination with EPO mimetibodies
and/or EPO, can also be
used for the ex vivo expansion of marrow or peripheral blood progenitor (PBPC)
cells. Prior to
chemotherapy treatment, marrow can be stimulated with stem cell factor (SCF)
or G-CSF to release
early progenitor cells into peripheral circulation. These progenitors are
optionally collected and
concentrated from peripheral blood and then treated in culture with TPO and
mimetibodies, optionally
in combination with one or more other cytokines, including but not limited to
SCF, G-CSF, IL-3, GM-
CSF, IL-6 or IL-11, to differentiate and proliferate into high-density
megakaryocyte cultures, which are
optionally then be returned to the patient following high-dose chemotherapy.
Doses of TPO for ex
vivo treatment of bone marrow will be in the range of 100 pg/ml to 10 ng/ml,
preferably 500 pg/ml to 3
ng/ml. Doses of mimetibodies will be equivalent in activity to EPO which can
be used from 0.1
units/ml to 20 units/ml, preferably from 0.5 units/mI to 2 units/ml, or any
range or value therein.
TNF antagonists suitable for compositions, combination therapy, co-
administration, devices
and/or methods of the present invention (further comprising at least one anti
body, specified portion
and variant thereof, of the present invention), include, but are not limited
to, anti-TNF antibodies,
ligand-binding fragments thereof, and receptor molecules which bind
specifically to TNF; compounds
2 0 which prevent and/or inhibit TNF synthesis, TNF release or its action on
target cells, such as
thalidomide, tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and
rolipram), A2b adenosine
receptor agonists and A2b adenosine receptor enhancers; compounds which
prevent and/or inhibit TNF
receptor signalling, such as mitogen activated protein (MAP) kinase
inhibitors; compounds which
block and/or inhibit membrane TNF cleavage, such as metalloproteinase
inhibitors; compounds which
2 5 block and/or inhibit TNF activity, such as angiotensin converting enzyme
(ACE) inhibitors (e.g.,
captopril); and compounds which block and/or inhibit TNF production and/or
synthesis, such as MAP
kinase inhibitors.
As used herein, a "tumor necrosis factor antibody," "TNF antibody," "TNFa
antibody," or
fragment and the like decreases, blocks, inhibits, abrogates or interferes
with TNFa activity in vitro, in
3 0 situ and/or preferably in vivo. For example, a suitable TNF human antibody
of the present invention
can bind TNFa and includes anti-TNF antibodies, antigen-binding fragments
thereof, and specified
mutants or domains thereof that bind specifically to TNFa. A suitable TNF
antibody or fragment can
also decrease block, abrogate, interfere, prevent and/or inhibit TNF RNA, DNA
or protein synthesis,
TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF
production and/or
~.o$
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
synthesis.
Chimeric antibody cA2 consists of the antigen binding variable region of the
high-affinity
neutralizing mouse anti-human TNFoc IgGl antibody, designated A2, and the
constant regions of a
human IgGl, kappa immunoglobulin. The human IgG1 Fc region improves allogeneic
antibody
effector function, increases the circulating serum half life and decreases the
immunogenicity of the
antibody. The avidity and epitope specificity of the chimeric antibody cA2 is
derived from the variable
region of the murine antibody A2. In a particular embodiment, a preferred
source for nucleic acids
encoding the variable region of the murine antibody A2 is the A2 hybridoma
cell line.
Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural and
recombinant human
TNFa in a dose dependent manner. From binding assays of chimeric antibody cA2
and recombinant
human TNFa, the affinity constant of chimeric antibody cA2 was calculated to
be 1.04x101°M-1.
Preferred methods for determining monoclonal antibody specificity and affinity
by competitive
inhibition can be found in Harlow, et al., Antibodies: A Laboratory Manual,
Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al., eds.,
Current Protocols irz
Imrrzurzology, Greene Publishing Assoc. and Wiley Interscience, New York,
(1992-2002); I~ozbor et
al., Imrnuizol. Today, 4:72-79 (1983); Ausubel et al., eds. Current Protocols
in Molecular' Biology,
Wiley Interscience, New York (1987-2002); and Muller, Meth. Enzymol., 92:589-
601 (1983), which
references are entirely incorporated herein by reference.
In a particular embodiment, murine monoclonal antibody A2 is produced by a
cell line
2 0 designated c134A. Chimeric antibody cA2 is produced by a cell line
designated c168A.
Additional examples of monoclonal anti-TNF antibodies that can be used in the
present
invention are described in the art (see, e.g., U.S. Patent No. 5,231,024;
Moller, A. et al., Cytokirze
2(3):162-169 (1990); U.S. Application No. 07/943,852 (filed September 11,
1992); Rathjen et al.,
International Publication No. WO 91/02078 (published February 21, 1991); Rubin
et al., EPO Patent
2 5 Publication No. 0 218 868 (published April 22, 1987); Yone et al., EPO
Patent Publication No. 0 288
088 (October 26, 1988); Liang, et al., Biochem. Biophys. Res. Cornrrz. 137:847-
854 (1986); Meager, et
al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridonza 6:359-369 (1987);
Bringman, et al.,
Hybridoma 6:489-507 (1987); and Hirai, et al., J. Immunol. Meth. 96:57-62
(1987), which references
are entirely incorporated herein by reference).
3 0 TNF Receptor Molecules
Preferred TNF receptor molecules useful in the present invention are those
that bind TNFa
with high affinity (see, e.g., Feldmann et al., International Publication No.
WO 92/07076 (published
April 30, 1992); Schall et al., Cell 61:361-370 (1990); and Loetscher et al.,
Cell 61:351-359 (1990),
which references are entirely incorporated herein by reference) and optionally
possess low
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CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
immunogenicity. In particular, the 55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-
R) TNF cell surface
receptors are useful in the present invention. Truncated forms of these
receptors, comprising the
extracellular domains (ECD) of the receptors or functional portions thereof
(see, e.g., Corcoran et al.,
Eur. J. Biochem. 223:831-840 (1994)), are also useful in the present
invention. Truncated forms of the
TNF receptors, comprising the ECD, have been detected in urine and serum as 30
kDa and 40 kDa
TNFa inhibitory binding proteins (Engelmann, H. et al., J. Biol. Chem.
265:1531-1536 (1990)). TNF
receptor multimeric molecules and TNF immunoreceptor fusion molecules, and
derivatives and
fragments or portions thereof, are additional examples of TNF receptor
molecules which are useful in
the methods and compositions of the present invention. The TNF receptor
molecules which can be
used in the invention are characterized by their ability to treat patients for
extended periods with good
to excellent alleviation of symptoms and low toxicity. Low immunogenicity
and/or high affinity, as
well as other undefined properties, may contribute to the therapeutic results
achieved.
TNF receptor multimeric molecules useful in the present invention comprise all
or a functional
portion of the ECD of two or more TNF receptors linked via one or more
polypeptide linkers or other
nonpeptide linkers, such as polyethylene glycol (PEG). The multimeric
molecules can further
comprise a signal peptide of a secreted protein to direct expression of the
multimeric molecule. These
multimeric molecules and methods for their production have been described in
U.S. Application No.
08/437,533 (filed May 9, 1995), the content of which is entirely incorporated
herein by reference.
TNF immunoreceptor fusion molecules useful in the methods and compositions of
the present
2 0 invention comprise at least one portion of one or more immunoglobulin
molecules and all or a
functional portion of one or more TNF receptors. These immunoreceptor fusion
molecules can be
assembled as monomers, or hetero- or homo-multimers. The immunoreceptor fusion
molecules can
also be monovalent or multivalent. An example of such a TNF immunoreceptor
fusion molecule is
TNF receptor/IgG fusion protein. TNF immunoreceptor fusion molecules and
methods for their
production have been described in the art (Lesslauer et al., Eur. J. Imrnunol.
21:2883-2886 (1991);
Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Peppel et
al., J. Exp. Med.
174:1483-1489 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219
(1994); Butler et al.,
Cytokine 6(6):616-623 (1994); Baker et al., Eur. J. Irnrnunol. 24:2040-2048
(1994); Beutler et al., U.S.
Patent No. 5,447,851; and U.S. Application No. 08/442,133 (filed May 16,
1995), each of which
3 0 references are entirely incorporated herein by reference). Methods for
producing immunoreceptor
fusion molecules can also be found in Capon et al., U.S. Patent No. 5,116,964;
Capon et al., U.S.
Patent No. 5,225,538; and Capon et al., Nature 337:525-531 (1989), which
references are entirely
incorporated herein by reference.
A functional equivalent, derivative, fragment or region of TNF receptor
molecule refers to the
lio
CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
portion of the TNF receptor molecule, or the portion of the TNF receptor
molecule sequence which
encodes TNF receptor molecule, that is of sufficient size and sequences to
functionally resemble TNF
receptor molecules that can be used in the present invention (e.g., bind TNFoc
with high affinity and
possess low immunogenicity). A functional equivalent of TNF receptor molecule
also includes
modified TNF receptor molecules that functionally resemble TNF receptor
molecules that can be used
in the present invention (e.g., bind TNFa with high affinity and possess low
immunogenicity). For
example, a functional equivalent of TNF receptor molecule can contain a
"SILENT" colon or one or
more amino acid substitutions, deletions or additions (e.g., substitution of
one acidic amino acid for
another acidic amino acid; or substitution of one colon encoding the same or
different hydrophobic
amino acid for another colon encoding a hydrophobic amino acid). See Ausubel
et al., Current
Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-
Interscience, New York ( 1987-
2002).
Cytokines include, but are not limited to all known cytokines. See, e.g.,
CopewithCytokines.com. Cytokine antagonists include, but are not limited to,
any antibody, fragment
or mimetic, any soluble receptor, fragment or mimetic, any small molecule
antagonist, or any
combination thereof.
Any method of the present invention can comprise a method for treating a
protein mediated
disorder, comprising administering an effective amount of a composition or
pharmaceutical
composition comprising at least one CH1-deleted mimetibody or specified
portion or variant to a cell,
2 0 tissue, organ, animal or patient in need of such modulation, treatment or
therapy. Such a method can
optionally further comprise co-administration or combination therapy for
treating such immune
diseases, wherein the administering of said at least one CHI-deleted
mimetibody, specified portion or
variant thereof, further comprises administering, before concurrently, and/or
after, at least one selected
from at least one other cytokines such as IL-3, lL -6 and 1L -1 I; stem cell
factor; G-CSF and GM-CSF.
2 5 Combination therapy with GM-CSF, for example, is indicated in patients
with low neutrophil levels.
Typically, treatment of pathologic conditions is effected by administering an
effective amount or
dosage of at least one CHl-deleted mimetibody composition that total, on
average, a range from at least
about 0.01 to 500 milligrams of at least one CHI-deleted mimetibody or
specified portion or variant
/kilogram of patient per dose, and preferably from at least about 0.1 to 100
milligrams CHl-deleted
3 0 mimetibody or specified portion or variant /kilogram of patient per single
or multiple administration,
depending upon the specific activity of contained in the composition.
Alternatively, the effective serum
concentration can comprise 0,1-5000 p,g/ml serum concentration per single or
multiple adminstration.
Suitable dosages are known to medical practitioners and will, of course,
depend upon the particular
disease state, specific activity of the composition being administered, and
the particular patient
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CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
undergoing treatment. In some instances, to achieve the desired therapeutic
amount, it can be necessary to
provide for repeated administration, i.e., repeated individual administrations
of a particular monitored or
metered dose, where the individual administrations are repeated until the
desired daily dose or effect is
achieved.
Preferred doses can optionally include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,
0.07, 0.08, 009, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, and/or 30 mglkgladministration, or any range, value or
fraction thereof, or to
achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0,
2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5,
4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5,
9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20,
12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5, 6.9,
7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5,
9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0,13.5, 13.9, 14,
14.5, 15, 15.5, 15.9, 16, 16.5, 16.9,
17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 35, 40,
45, 50, S5, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700,
800, 900, 1000, 1500, 2000,
2500, 3000, 3500, 4000, 4500, and/or 5000 ~g/ml serum concentration per single
or multiple
administration, or any range, value or fraction thereof.
Alternatively, the dosage administered can vary depending upon known factors,
such as the
pharmacodynamic characteristics of the particular agent, and its mode and
route of administration; age,
health, and weight of the recipient; nature and extent of symptoms, kind of
concurrent treatment,
frequency of treatment, and the effect desired. Usually a dosage of active
ingredient can be about 0.1
2 0 to 100 milligrams per kilogram of body weight. Ordinarily 0.1 to 50, and
preferably 0.1 to 10
milligrams per kilogram per administration or in sustained release form is
effective to obtain desired
results.
As a non-limiting example, treatment of humans or animals can be provided as a
one-time or
periodic dosage of at least one CH1-deleted mimetibody or specified portion or
variant of the present
2 5 invention 0.01 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5,
6, 7, 8, 9, 10, 1 l, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60,
70, 80, 90 or 100 mg/kg, per
day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or
alternatively, at least one of week
l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or
any combination thereof, using
3 0 single, infusion or repeated doses.
Dosage forms (composition) suitable for internal administration generally
contain from about
0.0001 milligram to about 500 milligrams of active ingredient per unit or
container. In these
pharmaceutical compositions the active ingredient will ordinarily be present
in an amount of about
0.5-95% by weight based on the total weight of the composition.
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WO 2004/002417 PCT/US2003/020347
For parenteral administration, the CHl-deleted mimetibody or specified portion
or variant can
be formulated as a solution, suspension, emulsion or lyophilized powder in
association, or separately
provided, with a pharmaceutically acceptable parenteral vehicle. Examples of
such vehicles are water,
saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
Liposomes and
nonaqueous vehicles such as fixed oils may also be used. The vehicle or
lyophilized powder may
contain additives that maintain isotonicity (e.g., sodium chloride, mannitol)
and chemical stability (e.g.,
buffers and preservatives). The formulation is sterilized by known or suitable
techniques.
Suitable pharmaceutical carriers are described in the most recent edition of
Remington's
Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
Therapeutic Administration
Many known and developed modes of can be used according to the present
invention for ,
administering pharmaceutically effective amounts of at least one CHl-deleted
mimetibody or specified
portion or variant according to the present invention. While pulmonary
administration is used in the
following description, other modes of administration can be used according to
the present invention
with suitable results.
A CH1-deleted mimetibody of the present invention can be delivered in a
carrier, as a solution,
emulsion, colloid, or suspension, or as a powder, using any of a variety of
devices and methods suitable
for administration by inhalation or other modes described here within or known
in the art.
Parenteral Formulations and Administration
Formulations for parenteral administration can contain as common excipients
sterile water or
saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, hydrogenated
naphthalenes and the like. Aqueous or oily suspensions for injection can be
prepared by using an
2 5 appropriate emulsifier or humidifier and a suspending agent, according to
known methods. Agents for
injection can be a non-toxic, non-orally administrable diluting agent such as
aquous solution or a sterile
injectable solution or suspension in a solvent. As the usable vehicle or
solvent, water, Ringer's
solution, isotonic saline, etc. are allowed; as an ordinary solvent, or
suspending solvent, sterile
involatile oil can be used. For these purposes, any kind of involatile oil and
fatty acid can be used,
3 0 including natural or synthetic or semisynthetic fatty oils or fatty acids;
natural or synthetic or
semisynthtetic mono- or di- or tri-glycerides. Parental administration is
known in the art and includes,
but is not limited to, conventional means of injections, a gas pressured
needle-less injection device as
described in U.S. Pat. No. 5,851,198, and a laser perforator device as
described in U.S. Pat. No.
5,839,446 entirely incorporated herein by reference.
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WO 2004/002417 PCT/US2003/020347
Alternative Delivery
The invention further relates to the administration of at least one CHl-
deleted mimetibody or
specified portion or variant by parenteral, subcutaneous, intramuscular,
intravenous, bolus, vaginal,
rectal, buccal, sublingual, intranasal, or transdermal means. Protein, CHl-
deleted mimetibody or
specified portion or variant compositions can be prepared for use for
parenteral (subcutaneous,
intramuscular or intravenous) administration particularly in the form of
liquid solutions or suspensions;
for use in vaginal or rectal administration particularly in semisolid forms
such as creams and
suppositories; for buccal, or sublingual administration particularly in the
form of tablets or capsules; or
intranasally particularly in the form of powders, nasal drops or aerosols or
certain agents; or
transdermally particularly in the form of a gel, ointment, lotion, suspension
or patch delivery system
with chemical enhancers such as dimethyl sulfoxide to either modify the skin
structure or to increase
the drug concentration in the transdermal patch (Junginger, et al. In "Drug
Permeation Enhancement";
Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirely
incorporated herein by
reference), or with oxidizing agents that enable the application of
formulations containing proteins and
peptides onto the skin (WO 98153847), or applications of electric fields to
create transient transport
pathways such as electroporation, or to increase the mobility of charged drugs
through the skin such as
iontophoresis, or application of ultrasound such as sonophoresis (U.S. Pat.
Nos. 4,309,989 and
4,767,402) (the above publications and patents being entirely incorporated
herein by reference).
2 0 Pulmonary/Nasal Administration
For pulmonary administration, preferably at least one CH1-deleted mimetibody
or specified
portion or variant composition is delivered in a particle size effective for
reaching the lower airways of
the lung or sinuses. According to the invention, at least one CHl-deleted
mimetibody or specified
portion or variant can be delivered by any of a variety of inhalation or nasal
devices known in the art
2 5 for administration of a therapeutic agent by inhalation. These devices
capable of depositing
aerosolized formulations in the sinus cavity or alveoli of a patient include
metered dose inhalers,
nebulizers, dry powder generators, sprayers, and the like. Other devices
suitable for directing the
pulmonary or nasal administration of CHl-deleted mimetibody or specified
portion or variants are also
known in the art. All such devices can use of formulations suitable for the
administration for the
3 0 dispensing of CH1-deleted mimetibody or specified portion or variant in an
aerosol. Such aerosols can
be comprised of either solutions (both aqueous and non aqueous) or solid
particles. Metered dose
inhalers like the Ventoliri metered dose inhaler, typically use a propellent
gas and require actuation
during inspiration (See, e.g., WO 94/16970, WO 98/35888). Dry powder inhalers
like Turbuhaler~
(Astray, Rotahaler~ (Glaxo), Diskus° (Glaxo), Spiros~ inhaler (Dura),
devices marketed by Inhale
3 5 Therapeutics, and the Spinhaler° powder inhaler (Fisons), use
breath-actuation of a mixed powder (US
r
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CA 02490409 2004-12-24
WO 2004/002417 PCT/US2003/020347
4668218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, US
5458135 Inhale, WO
94/06498 Fisons, entirely incorporated herein by reference). Nebulizers like
AERx~ Aradigm, the
Ultravent~ nebulizer (Mallinckrodt), and the Acorn II~ nebulizer (Marquest
Medical Products) (US
5404871 Aradigm, WO 97/22376), the above references entirely incorporated
herein by reference,
produce aerosols from solutions, while metered dose inhalers, dry powder
inhalers, etc. generate small
particle aerosols. These specific examples of commercially available
inhalation devices are intended to
be a representative of specific devices suitable for the practice of this
invention, and are not intended as
limiting the scope of the invention. Preferably, a composition comprising at
least one CHl-deleted
mimetibody or specified portion or variant is delivered by a dry powder
inhaler or a sprayer. There are
a several desirable features of an inhalation device for administering at
least one CH1-deleted
mimetibody or specified portion or variant of the present invention. For
example, delivery by the
inhalation device is advantageously reliable, reproducible, and accurate. The
inhalation device can
optionally deliver small dry particles, e.g. less than about 10 pm, preferably
about 1-5 p,m, for good
respirability.
Administration of CH1-deleted mimetibody or specified portion or variant
Compositions as a
Spray
A spray including CH1-deleted mimetibody or specified portion or variant
composition protein
can be produced by forcing a suspension or solution of at least one CH1-
deleted mimetibody or
2 0 specified portion or variant through a nozzle under pressure. The nozzle
size and configuration, the
applied pressure, and the liquid feed rate can be chosen to achieve the
desired output and particle size.
An electrospray can be produced, for example, by an electric field in
connection with a capillary or
nozzle feed. Advantageously, particles of at least one CH1-deleted mimetibody
or specified portion or
variant composition protein delivered by a sprayer have a particle size less
than about 10 pm,
2 5 preferably in the range of about 1 p,m to about 5 pm, and most preferably
about 2 pm to about 3 Vim.
Formulations of at least one CHl-deleted mimetibody or specified portion or
variant
composition protein suitable for use with a sprayer typically include CH1-
deleted mimetibody or
specified portion or variant composition protein in an aqueous solution at a
concentration of about 1
mg to about 20 mg of at least one CHl-deleted mimetibody or specified portion
or variant composition
3 0 protein per ml of solution. The formulation can include agents such as an
excipient, a buffer, an
isotonicity agent, a preservative, a surfactant, and, preferably, zinc. The
formulation can also include
an excipient or agent for stabilization of the CH1-deleted mimetibody or
specified portion or variant
composition protein, such as a buffer, a reducing agent, a bulk protein, or a
carbohydrate. Bulk
proteins useful in formulating CH1-deleted mimetibody or specified portion or
variant composition
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WO 2004/002417 PCT/US2003/020347
proteins include albumin, protamine, or the like. Typical carbohydrates useful
in formulating CH1-
deleted mimetibody or specified portion or variant composition proteins
include sucrose, mannitol,
lactose, trehalose, glucose, or the like. The CHl-deleted mimetibody or
specified portion or variant
composition protein formulation can also include a surfactant, which can
reduce or prevent surface-
s induced aggregation of the CHl-deleted mimetibody or specified portion or
variant composition
protein caused by atomization of the solution in forming an aerosol. Various
conventional surfactants
can be employed, such as polyoxyethylene fatty acid esters and alcohols, and
polyoxyethylene sorbitol
fatty acid esters. Amounts will generally range between 0.001 and 14% by
weight of the formulation.
Especially preferred surfactants for purposes of this invention are
polyoxyethylene sorbitan
monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents
known in the art for
formulation of a protein such as mimetibodies, or specified portions or
variants, can also be included in
the formulation.
Administration of CHl-deleted mimetibody or specified portion or variant
compositions by a
Nebulizer
CHl-deleted mimetibody or specified portion or variant composition protein can
be
administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
Typically, in a jet
nebulizer, a compressed air source is used to create a high-velocity air jet
through an orifice. As the
gas expands beyond the nozzle, a low-pressure region is created, which draws a
solution of CHl-
2 0 deleted mimetibody or specified portion or variant composition protein
through a capillary tube
connected to a liquid reservoir. The liquid stream from the capillary tube is
sheared into unstable
filaments and droplets as it exits the tube, creating the aerosol. A range of
configurations, flow rates,
and baffle types can be employed to achieve the desired performance
characteristics from a given jet
nebulizer. In an ultrasonic nebulizer, high-frequency electrical energy is
used to create vibrational,
2 5 mechanical energy, typically employing a piezoelectric transducer. This
energy is transmitted to the
formulation of CH1-deleted mimetibody or specified portion or variant
composition protein either
directly or through a coupling fluid, creating an aerosol including the CH1-
deleted mimetibody or
specified portion or variant composition protein. Advantageously, particles of
CHl-deleted
mimetibody or specified portion or variant composition protein delivered by a
nebulizer have a particle
3 0 size less than about 10 p,m, preferably in the range of about 1 pm to
about 5 p,m, and most preferably
about 2 ~m to about 3 p,m.
Formulations of at least one CHI-deleted mimetibody or specified portion or
variant suitable
for use with a nebulizer, either jet or ultrasonic, typically include CHl-
deleted mimetibody or specified
portion or variant composition protein in an aqueous solution at a
concentration of about I mg to about
W6
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20 mg of at least one CHl-deleted mimetibody or specified portion or variant
protein per ml of
solution. The formulation can include agents such as an excipient, a buffer,
an isotonicity agent, a
preservative, a surfactant, and, preferably, zinc. The formulation can also
include an excipient or agent
for stabilization of the at least one CH1-deleted mimetibody or specified
portion or variant composition
protein, such as a buffer, a reducing agent, a bulk protein, or a
carbohydrate. Bulk proteins useful in
formulating at least one CHl-deleted mimetibody or specified portion or
variant composition proteins
include albumin, protamine, or the like. Typical carbohydrates useful in
formulating at least one CH1-
deleted mimetibody or specified portion or variant include sucrose, mannitol,
lactose, trehalose,
glucose, or the like. The at least one CH1-deleted mimetibody or specified
portion or variant
formulation can also include a surfactant, which can reduce or prevent surface-
induced aggregation of
the at least one CH1-deleted mimetibody or specified portion or variant caused
by atomization of the
solution in forming an aerosol. Various conventional surfactants can be
employed, such as
polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital
fatty acid esters.
Amounts will generally range between 0.001 and 4% by weight of the
formulation. Especially
preferred surfactants for purposes of this invention axe polyoxyethylene
sorbitan mono-oleate,
polysorbate 80, polysorbate 20, or the like. Additional agents known in the
art for formulation of a
protein such as CHI-deleted mimetibody or specified portion or variant protein
can also be included in
the formulation.
2 0 Administration of CHl-deleted mimetibody or specified portion or variant
compositions By A
Metered Dose Inhaler
In a metered dose inhaler (MDI), a propellant, at least one CH1-deleted
mimetibody or
specified portion or variant, and any excipients or other additives are
contained in a canister as a
2 5 mixture including a liquefied compressed gas. Actuation of the metering
valve releases the mixture as
an aerosol, preferably containing particles in the size range of less than
about 10 ~,m, preferably about
1 p,m to about 5 pm, and most preferably about 2 pm to about 3 pm. The desired
aerosol particle size
can be obtained by employing a formulation of CHl-deleted mimetibody or
specified portion or variant
composition protein produced by various methods known to those of skill in the
art, including jet-
3 0 milling, spray drying, critical point condensation, or the like. Preferred
metered dose inhalers include
those manufactured by 3M or Glaxo and employing a hydrofluorocarbon
propellant.
Formulations of at least one CH1-deleted mimetibody or specified portion or
variant for use
with a metered-dose inhaler device will generally include a finely divided
powder containing at least
one CHl-deleted mimetibody or specified portion or variant as a suspension in
a non-aqueous medium,
3 5 for example, suspended in a propellant with the aid of a surfactant. The
propellant can be any
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conventional material employed for this purpose, such as chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including
trichlorofluoromethane,
dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-
tetrafluoroethane, HFA-134a
(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.
Preferably the propellant is a
hydrofluorocarbon. The surfactant can be chosen to stabilize the at least one
CHl-deleted mimetibody
or specified portion or variant as a suspension in the propellant, to protect
the active agent against
chemical degradation, and the like. Suitable surfactants include sorbitan
trioleate, Soya lecithin, oleic
acid, or the like. In some cases solution aerosols are preferred using
solvents such as ethanol.
Additional agents known in the art for formulation of a protein such as
protein can also be included in
the formulation.
One of ordinary skill in the art will recognize that the methods of the
current invention can be
achieved by pulmonary administration of at least one CH1-deleted mimetibody or
specified portion or
variant compositions via devices not described herein.
Mucosal Formulations and Administration
For absorption through mucosal surfaces, compositions and methods of
administering at least
one CHl-deleted mimetibody or specified portion or variant include an emulsion
comprising a plurality
of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and
an aqueous
continuous phase, which promotes absorption through mucosal surfaces by
achieving mucoadhesion of
the emulsion particles (U.S. Pat. Nos. 5,514,670). Mucous surfaces suitable
for application of the
2 0 emulsions of the present invention can include corneal, conjunctival,
buccal, sublingual, nasal, vaginal,
pulmonary, stomachic, intestinal, and rectal routes of administration.
Formulations for vaginal or
rectal administration, e.g. suppositories, can contain as excipients, for
example, polyalkyleneglycols,
vaseline, cocoa butter, and the like. Formulations for intranasal
administration can be solid and contain
as excipients, for example, lactose or can be aqueous or oily solutions of
nasal drops. For buccal
2 5 administration excipients include sugars, calcium stearate, magnesium
stearate, pregelinatined starch,
and the like (U.S. Pat. No. 5,849,695).
Oral Formulations and Administration
Formulations for oral rely on the co-administration of adjuvants (e.g.,
resorcinols and nonionic
surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene
ether) to increase
3 0 artificially the permeability of the intestinal walls, as well as the co-
administration of enzymatic
inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate
(DFF) and trasylol) to inhibit
enzymatic degradation. The active constituent compound of the solid-type
dosage form for oral
administration can be mixed with at least one additive, including sucrose,
lactose, cellulose, mannitol,
trehalose, raffmose, maltitol, dextran, starches, agar, arginates, chitins,
chitosans, pectins, gum
3 5 tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or
semisynthetic polymer, and
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glyceride. These dosage forms can also contain other types) of additives,
e.g., inactive diluting agent,
lubricant such as magnesium stearate, paraben, preserving agent such as sorbic
acid, ascorbic acid,
alpha-tocopherol, antioxidant such as cysteine, disintegrator, binder,
thickener, buffering agent,
sweetening agent, flavoring agent, perfuming agent, etc.
Tablets and pills can be further processed into enteric-coated preparations.
The liquid
preparations for oral administration include emulsion, syrup, elixir,
suspension and solution
preparations allowable for medical use. These preparations may contain
inactive diluting agents
ordinarily used in said field, e.g., water. Liposomes have also been described
as drug delivery systems
for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres
of artificial polymers
of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals
(U.S. Pat. No.
4,925,673). Furthermore, carrier compounds described in U.S. Pat. No.
5,879,681 and U.S. Pat. No.
5,5,871,753 are used to deliver biologically active agents orally are known in
the art.
Transdermal Formulations and Administration
For transdermal administration, the at least one CHl-deleted mimetibody or
specified portion
or variant is encapsulated in a delivery device such as a liposome or
polymeric nanoparticles,
microparticle, microcapsule, or microspheres (referred to collectively as
microparticles unless
otherwise stated). A number of suitable devices are known, including
microparticles made of synthetic
polymers such as polyhydroxy acids such as polylactic acid, polyglycolic acid
and copolymers thereof,
polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers
such as collagen,
2 0 polyamino acids, albumin and other proteins, alginate and other
polysaccharides, and combinations
thereof (U.S. Pat. No. 5,814,599).
Prolonged Administration and Formulations
It can be sometimes desirable to deliver the compounds of the present
invention to the subject
over prolonged periods of time, for example, for periods of one week to one
year from a single
2 5 administration. Various slow release, depot or implant dosage forms can be
utilized. For example, a
dosage form can contain a pharmaceutically acceptable non-toxic salt of the
compounds that has a low
degree of solubility in body fluids, for example, (a) an acid addition salt
with a polybasic acid such as
phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid,
pamoic acid, alginic acid,
polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic
acid, and the like; (b) a
3 0 salt with a polyvalent metal cation such as zinc, calcium, bismuth,
barium, magnesium, aluminum,
copper, cobalt, nickel, cadmium and the like, or with an organic cation formed
from e.g., N,N'-
dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and
(b) e.g. a zinc tannate
salt. Additionally, the compounds of the present invention or, preferably, a
relatively insoluble salt
such as those just described, can be formulated in a gel, for example, an
aluminum monostearate gel
3 5 with, e.g. sesame oil, suitable for injection. Particularly preferred
salts are zinc salts, zinc tannate salts,
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pamoate salts, and the like. Another type of slow release depot formulation
for injection would contain
the compound or salt dispersed for encapsulated in a slow degrading, non-
toxic, non-antigenic polymer
such as a polylactic acid/polyglycolic acid polymer for example as described
in U.S. Pat. No.
3,773,919. The compounds or, preferably, relatively insoluble salts such as
those described above can
also be formulated in cholesterol matrix silastic pellets, particularly for
use in animals. Additional slow
release, depot or implant formulations, e.g. gas or liquid liposomes are known
in the literature (U.S.
Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery
Systems", J. R. Robinson ed.,
Marcel Dekker, Inc., N.Y., 1978).
Having generally described the invention, the same will be more readily
understood by
reference to the following examples, which are provided by way of illustration
and are not intended as
limiting.
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Example 1: Cloning and Expression of CHl-deleted mimetibody in Mammalian Cells
A typical mammalian expression vector contains at least one promoter element,
which
mediates the initiation of transcription of mRNA, the CH1-deleted mimetibody
or specified portion or
variant coding sequence, and signals required for the termination of
transcription and polyadenylation
of the transcript. Additional elements include enhancers, Kozak sequences and
intervening sequences
flanked by donor and acceptor sites for RNA splicing. Highly efficient
transcription can be achieved
with the early and late promoters from SV40, the long terminal repeats (LTRS)
from Retroviruses, e.g.,
RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However,
cellular elements
can also be used (e.g., the human actin promoter). Suitable expression vectors
for use in practicing the
present invention include, for example, vectors such as pIRESlneo, pRetro-Off,
pRetro-On, PLXSN, or
pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or
pcDNA3.1/Hygro (+/-
(Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC
37152), pSV2dhfr
(ATCC 37146) and pBCI2MI (ATCC 67109). Mammalian host cells that could be used
include
human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C 127 cells, Cos 1, Cos
7 and CV 1, quail
QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the gene can be expressed in stable cell lines that contain the
gene integrated
into a chromosome. The co-transfection with a selectable marker such as dhfr,
gpt, neomycin, or
hygromycin allows the identification and isolation of the transfected cells.
2 0 The transfected gene can also be amplified to express large amounts of the
encoded CHl-
deleted mimetibody or specified portion or variant. The DHFR (dihydrofolate
reductase) marker is
useful to develop cell lines that carry several hundred or even several
thousand copies of the gene of
interest. Another useful selection marker is the enzyme glutamine synthase
(GS) (Murphy, et al.,
Biochem. J. 227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175
(1992)). Using these
2 5 markers, the mammalian cells are grown in selective medium and the cells
with the highest resistance
are selected. These cell lines contain the amplified genes) integrated into a
chromosome. Chinese
hamster ovary (CHO) and NSO cells are often used for the production of CHl-
deleted mimetibody or
specified portion or variants.
The expression vectors pCl and pC4 contain the strong promoter (LTR) of the
Rous Sarcoma
3 0 Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a
fragment of the CMV-enhancer
(Boshart, et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with
the restriction enzyme
cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors
contain in addition the 3' intron, the polyadenylation and termination signal
of the rat preproinsulin
gene.
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Cloning and Expression in CHO Cells
The vector pC4 is used for the expression of CHl-deleted mimetibody or
specified portion or
variant. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession
No. 37146). The
plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
Chinese hamster
ovary- or other cells lacking dihydrofolate activity that are transfected with
these plasmids can be
selected by growing the cells in a selective medium (e.g., alpha minus MEM,
Life Technologies,
Gaithersburg, MD) supplemented with the chemotherapeutic agent methotrexate.
The amplification of
the DHFR genes in cells resistant to methotrexate (MTX) has been well
documented (see, e.g., F. W.
Alt, et al., J. Biol. Chem. 253:1357-1370 (19?8); J. L. Hamlin and C. Ma,
Biochem. et Biophys. Acta
1097:107-143 (1990); and M. J. Page and M. A. Sydenham, Biotechnology 9:64-68
(1991)). Cells
grown in increasing concentrations of MTX develop resistance to the drug by
overproducing the target
enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene
is linked to the DHFR
gene, it is usually co-amplified and over-expressed. It is known in the art
that this approach can be
used to develop cell lines carrying more than 1,000 copies of the amplified
gene(s). Subsequently,
when the methotrexate is withdrawn, cell lines are obtained that contain the
amplified gene integrated
into one or more chromosomes) of the host cell.
Plasmid pC4 contains for expressing the gene of interest the strong promoter
of the long
terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell.
Biol. 5:438-447 (1985))
plus a fragment isolated from the enhancer of the immediate early gene of
human cytomegalovirus
2 0 (CMV) (Boshart, et al., Cell 41:521-530 (1985)). Downstream of the
promoter are BamHI, XbaI, and
Asp718 restriction enzyme cleavage sites that allow integration of the genes.
Behind these cloning
sites the plasmid contains the 3' intron and polyadenylation site of the rat
preproinsulin gene. Other
high efficiency promoters can also be used for the expression, e.g., the human
b-actin promoter, the
SV40 early or late promoters or the long terminal repeats from other
retroviruses, e.g., HIV and
2 5 HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar
systems can be used to
express the EPO in a regulated way in mammalian cells (M. Gossen, and H.
Bujard, Proc. Natl. Acad.
Sci. USA 89: 5547-5551 (1992)). For the polyadenylation of the mRNA other
signals, e.g., from the
human growth hormone or globin genes can be used as well. Stable cell lines
carrying a gene of
interest integrated into the chromosomes can also be selected upon co-
transfection with a selectable
3 0 marker such as gpt, 6418 or hygromycin. It is advantageous to use more
than one selectable marker in
the beginning, e.g., G418 plus methotrexate.
The plasmid pC4 is digested with restriction enzymes and then dephosphorylated
using calf
intestinal phosphatase by procedures known in the art. The vector is then
isolated from a 1 % agarose
gel.
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The DNA sequence encoding the complete CH1-deleted mimetibody or specified
portion or
variant is used, e.g., as presented in SEQID NOS: 13, 14, 15, 16, 17, 18,
corresponding to HC and LC
variable regions of a CH1-deleted mimetibody of the present invention,
according to known method
steps. Isolated nucleic acid encoding a suitable human constant region (i.e.,
HC and LC regions) is
also used in this construct (e.g., as provided in vector p1351).
The isolated variable and constant region encoding DNA and the
dephosphorylated vector are
then ligated with T4 DNA ligase. E. coli HB101 or Xh-1 Blue cells are then
transformed and bacteria
are identified that contain the fragment inserted into plasmid pC4 using, for
instance, restriction
enzyme analysis. -
Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for
transfection. 5
p,g of the expression plasmid pC4 is cotransfected with 0.5 pg of the plasmid
pSV2-neo using
lipofectin. The plasmid pSV2neo contains a dominant selectable marker, the neo
gene from Tn5
encoding an enzyme that confers resistance to a group of antibiotics including
6418. The cells are
seeded in alpha minus MEM supplemented with 1 p,g /ml 6418. After 2 days, the
cells are trypsinized
and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM
supplemented with
10, 25, or 50 ng/ml of methotrexate plus 1 pg /ml 6418. After about 10-14 days
single clones are
trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using
different concentrations of
methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the
highest
concentrations of methotrexate are then transferred to new 6-well plates
containing even higher
2 0 concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same
procedure is
repeated until clones are obtained that grow at a concentration of 100 - 200
mM. Expression of the
desired gene product is analyzed, for instance, by SDS-PAGE and Western blot
or by reverse phase
HPLC analysis.
2 5 Additional constructs can be expressed with single or multiple amino acid
changes in order to
avoid undesirable activities. These changes may be expressed alone or multiple
changes combined in a
single construct. The cysteine normally involved in a disulfide bridge between
the HC and LC will be
mutated to an alanine. While this cysteine may be involved in stabilizing the
construct by forming a
third disulfide bridge, it is possible that it may aberrantly form a disulfide
bond with other cyseines
3 0 within the construct, or it could form a disulfide linkage between two
constructs. By removing the
cysteine, proper folding and assembly could be enhanced and the possibility of
self association
diminished.
It has been shown that mutation of two lysine (L) residues, L234 & L235, in
the IgGl lower
hinge region to alanine (A) will abrogate the ability of the immunoglobulin to
mediate complement
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dependent cytotoxicity (CDC) and antibody dependant cellular cytotoxicity
(ADCC) (Hezereh et al.,
2001, J. Virol., vol. 75 (24), 12161-68).
Another modification that would result in a decrease in mediation of immune
effector functions is the
removal of the glycosylation attachment site. This can be accomplished by
mutation of the asparagine
to glutamine (Q). Aglycosylated versions of the IgGI subclass are known to be
poor mediators of
immune effector function (Jefferis et al. 1998, Immol. Rev., vol. 163, 50-76).
An additional modification that is currently being pursued is the replacement
of the IgGl CH2
and CH3 regions with the same regions of the IgG4 subtype while retaining the
Gl hinge region. As
discussed previously, the ability of the IgG4 subclass to mediate immune
effector functions is much
lower than that of the G1 subclass. So this construct is expected to retain
activity of NfusCGl without
the concerns of potential immune effector functions.
Other envisioned modifications are those that would decrease the potential
immunogenicity
of the constructs. One important determinant of immunogenicity is the ability
of peptides derived from
a protein to be efficiently bound and presented by MHC molecules to T cells
and to elicit a cell based
immune response or T cell help for an antibody response. Using publicly
available web based
algorithms for MHC binding (SYFPETHI, Ramensee et al., 1999, Immunogenetics,
vol. 50, 213-19 and
BIMAS) potential MHC binding epitopes within the NfusCGl were analyzed.
Mutations that would
decrease the predicted immunogenicity of one or more peptides are evaluated
for in vivo effect on
immunogenicity.
2 0 Advantages: The novel construct, NfusCGl described above offers an
alternative way of displaying
bioactive peptides. In addition, proposed modifications are expected to, in
combination and irr addition
to the novel features of mimetibodies of the present invention, enhance their
utility.
It will be clear that the invention can be practiced otherwise than as
particularly described in
the foregoing description and examples.
2 5 Numerous modifications and variations of the present invention are
possible in light of the
above teachings and, therefore, are within the scope of the present invention
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