NZ712058B2 - Gla domains as targeting agents - Google Patents
Gla domains as targeting agents Download PDFInfo
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- NZ712058B2 NZ712058B2 NZ712058A NZ71205814A NZ712058B2 NZ 712058 B2 NZ712058 B2 NZ 712058B2 NZ 712058 A NZ712058 A NZ 712058A NZ 71205814 A NZ71205814 A NZ 71205814A NZ 712058 B2 NZ712058 B2 NZ 712058B2
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Abstract
The disclosure relates to the recombinant Gla domain proteins and their use targeting phosphatidylserine (PtdS) moieties on the surface of cells, particularly those expressing elevated levels of PtdS, such as cells undergoing apoptosis. These proteins can be linked to both diagnostic and therapeutic payloads, thereby permitting identification and treatment of cells expression elevated PtdS. payloads, thereby permitting identification and treatment of cells expression elevated PtdS.
Description
DESCRIPTION
GLA DOMAINS AS TARGETING AGENTS
BACKGROUND
This application claims benefit of priority to U.S. Provisional Application Serial No.
61/787,753, filed March 15, 2013, and to U.S. Provisional Application Serial No. 61/791,537
filed March 15, 2013, the entire contents of which are hereby incorporated by reference. We
further direct the reader’s attention to New Zealand divisional application 751491.
1. Field
This disclosure relates to the targeting of phosphatidylserine (PtdS) on cell
membranes using Gla domain peptides and polypeptides. The use of these peptides and
polypeptides as diagnostic and therapeutic agents is disclosed.
2. Related Art
Phosphatidlyserine (PtdS) is a negatively charged phospholipid component usually
localized to the inner-leaflet (the cytoplasmic side) of the cell membrane. However, PtdS can
be transported by scramblase (a member of the flippase family) from the inner-leaflet to the
outer-leaflet and exposed on the cell surface. With very few exceptions, this active
externalization of PtdS is a response to cellular damage (van den Eijnde et al., 2001; Erwig
and Henson, 2008). For example, tissue injury signals platelets, leukocytes, and endothelial
cells to rapidly and reversibly redistribute PtdS which leads to the promotion of coagulation
and complement activation on cell surfaces. Similarly, apoptotic signals result in the
externalization of PtdS however in a more gradual and sustained manner. This external PtdS
provides a key recognition marker that enables macrophages to ingest dying cells from
surrounding tissue (Erwig and Henson, 2008). This removal process is essential for tissue
homeostasis and in a “healthy” environment it is extremely efficient. In fact, despite the loss
of >10 cells per day, the histological detection of apoptotic cells is a rare event in normal
tissues (Elltiot and Ravichandran, 2010; Elltiot et al., 2009). However, there is evidence that
in many pathological conditions the process of apoptotic cell removal is overwhelmed,
delayed or absent (Elltiot and Ravichandran, 2010; Lahorte et al., 2004). For example several
oncology studies suggest that a high apoptotic index is associated with higher grade tumors,
increased rate of metastasis and a poor prognosis for the patient (Naresh et al., 2001; Loose et
al., 2007; Kurihara et al., 2008; Kietselaer et al., 2002). These studies, and others like them,
suggest that apoptosis and external PtdS expression can be a powerful marker of disease
(Elltiot and Ravichandran, 2010).
There are several proteins with a high affinity for anionic phospholipid surfaces with
Annexin-V being the most widely utilized as a PtdS targeting probe (Lahorte et al., 2004).
With a high affinity for PtdS containing vesicles (K = 0.5-7 nM) and a molecular weight (37
kDa) that falls below the threshold for kidney filtration (approx. 60 kDa) Annexin-V has
shown promise in the clinic as an apoptosis-probe (Lin et al., 2010; Tait and Gibson, 1992).
Moreover, it has been utilized for a wide range of indications including those in oncology,
neurology and cardiology (Lahorte et al., 2004; Boersma et al., 2005; Blankenberg, 2009;
Reutelingsperger et al., 2002). The use of biologic probes which target PtdS cell-surface
expression has been shown both in vitro and in vivo. While their utility in the clinic is
promising, they have, for the most part, not yet been exploited.
SUMMARY
The invention provides a polypeptide suitable for targeting phosphatidylserine cell-surface
expression both in vitro and in vivo, said polypeptide comprising:
a protein S gamma-carboxyglutamic-acid (Gla) domain, lacking a protease or hormone-
binding domain, and
an EGF domain,
wherein the polypeptide:
i) lacks a protease domain and also lacks a hormone-binding domain,
ii) is linked to therapeutic agent.
BRIEF DESCRIPTION
Thus, in accordance with the present disclosure, described herein is method of
targeting cell membrane phosphatidylserine (PtdS) comprising (a) providing an isolated
polypeptide comprising a gamma-carboxyglutamic-acid (Gla) domain and lacking a protease
or hormone-binding domain; and (b) contacting the peptide with a cell surface, wherein the
polypeptide binds to PtdS on the cell membrane. The cell membrane may be a cardiac cell
membrane, a neuronal cell membrane, an endothelial cell membrane, a virus-infected cell
membrane, an apoptotic cell membrane, a platelet membrane or a cancer cell membrane. The
polypeptide may further comprise an EGF binding domain, a Kringle domain, and/or an
aromatic amino acid stack domain. The Gla domain may be from Factor II, Factor VII,
Factor IX, Factor X, protein S or protein C.
The polypeptide may further comprise a detectable label, such as a fluorescent label, a
chemilluminescent label, a radiolabel, an enzyme, a dye or a ligand. The polypeptide may
further comprise a therapeutic agent, such as an anti-cancer agent, including a
chemotherapeutic, a radiotherapeutic, a cytokine, a hormone, an antibody or antibody
fragment or a toxin, or an anti-viral agent. The therapeutic agent may be an enzyme, such as
a prodrug converting enzyme, a cytokine, growth factor, clotting factor, or anti-coagulant.
The polypeptide may be 300 residues or less, 200 residues or less, or 100 residues or less,
including ragnes of 100-200 and 100-300 residues.
The polypeptide may comprise 5-15 Gla residues, 9-13 Gla residues, including 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, or 15 Gla residues. The the polypeptide may comprise more than 13
Gla residues, but less than 30% total Gla residues. The polypeptide may be between about
4.5 and 30 kD in size. The polypeptide may comprise at least one disulfide bond, or 2-5
disulfide bonds. The the polypeptide may comprise a protein S Gla domain. The polypeptide
may comprise a protein S Gla domain plus protein S EGF domain, a prothrombin Gla
domain, a prothrombin Gla domain plus prothrombin Kringle domain, a protein Z Gla
domain, a protein Z Gla domain plus prothrombin Kringle domain, a Factor VII Gla domain,
or a Factor VII Gla domain plus prothrombin Kringle domain. The polypeptide may further
comprise an antibody Fc region. Any of the foregoing may contain conservative substitutions
of the native sequences for the foregoing proteins, and/or exhibit a percentage homology to
the native domains set forth.
In another embodiment, described herein is a method of treating cancer in a subject
comprising administering to the subject an isolated polypeptide comprising a gamma-
carboxyglutamic-acid (Gla) domain and lacking a protease or hormone-binding domain,
wherein the polypeptide is linked to a therapeutic payload. The therapeutic payload may be a
chemotherapeutic, a radiotheraputic or a toxin. The cancer may be breast cancer, brain
cancer, stomach cancer, lung cancer, prostate cancer, ovarian cancer, testicular cancer, colon
cancer, skin cancer, rectal cancer, cervical cancer, uterine cancer, liver cancer, pancreatic
cancer, head & neck cancer or esophageal cancer.
In yet another embodiment, described herein is a method of treating a viral diease in a
subject comprising administering to the subject an isolated polypeptide comprising a gamma-
carboxyglutamic-acid (Gla) domain and lacking a protease or hormone-binding domain,
wherein the polypeptide is linked to an anti-viral agent. The viral disease may be influenza,
human immunodeficiency virus, dengue virus, West Nile virus, smallpox virus, respiratory
syncytial virus, Korean hemorrhagic fever virus, chickenpox, varicella zoster virus, herpes
simplex virus 1 or 2, Epstein-Barr virus, Marburg virus, hantavirus, yellow fever virus,
hepatitis A, B, C or E, Ebola virus, human papilloma virus, rhinovirus, Coxsackie virus, polio
virus, measles virus, rubella virus, rabies virus, Newcastle disease virus, rotavirus, HTLV-1
and -2.
It is contemplated that any method or composition described herein can be
implemented with respect to any other method or composition described herein.
Other objects, features and advantages of the present disclosure will become apparent
from the following detailed description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific embodiments of the
disclosure, are given by way of illustration only, since various changes and modifications
within the spirit and scope of the disclosure will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
The following drawings form part of the present specification and are included to
further demonstrate certain embodiments of the present disclosure. The disclosure may be
better understood by reference to one or more of these drawings in combination with the
detailed.
– Construction of a panel of Gla and Gla-EGF/Kringle domain proteins.
– Testing of Gla domain protein constructs for expression. Transient
transfection into 293 cells using 293cellFectin. 10% gels with reduced samples, 23.3 µl
of media loaded.
– Testing of Gla domain protein constructs for expression. Transient
transfection in BHK21 cells. 10% gels with reduced samples, 20 µl (1/100 total cell
pellet) loaded.
– Changing signal sequence alter secretion. Transient transfection in BHK21
cells. 10% gels with reduced samples, 13.3 µl loaded.
– Protein S Gla + EGF sequence.
– Purification of Protein S Gla + EGF. F1-F4 are column chromatography
fractions. 10% gels, non-reducing conditions.
– Apoptosis Assays for Protein S Gla + EGF. Top and bottom panels
represent identical duplicate procedures except that amounts of Protein S Gla + EGF was
reduced, and the amount of anti-His domain antibody was reduced.
– Apoptosis Assays for Protein S Gla + EGF. Top and bottom panels
represent identical duplicate procedures except for amounts of Annexin V used, which are
double in the bottom panels.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Like annexin, gamma-carboxyglutamic-acid (Gla)-domain proteins such as Factors II,
VII, IX, X, protein C, and protein S bind anionic membranes. In fact, the Gla-domain has
been used as a model for a small molecule that was rationally designed to be an apoptosis-
specific probe (Cohen et al., 2009). Here, the inventors propose the utilization of the
membrane targeting portions of these Gla-domain proteins as a novel class of biological
probes specific for apoptosis and disease. The use of these naturally-occurring and targeted
proteins may lead to enhanced specificity relative to current probes with the added advantage
of a smaller size (<30 kDa). Even in larger embodiments, which would include EGF and/or
Kringle domains, these proteins can still be smaller than Annexin V (37 kDa), and potentially
as small as <5 kDa. These biologic probes can target PtdS cell-surface expression both in
vitro and in vivo. Thus, it is possible to develop an apoptosis/disease targeting probe that is
superior to Annexin V in affinity, specificity and size with the added potential for use as a
therapeutic. These and other embodiments of the disclosure are described in greater detail
below.
Whenever appropriate, terms used in the singular will also include the plural and vice
versa. In the event that any definition set forth below conflicts with the usage of that word in
any other document, including any document incorporated herein by reference, the definition
set forth below shall always control for purposes of interpreting this specification and its
associated claims unless a contrary meaning is clearly intended (for example in the document
where the term is originally used). The use of "or" means "and/or" unless stated otherwise.
The use of “a” herein means “one or more” unless stated otherwise or where the use of “one
or more” is clearly inappropriate. The use of “comprise,” “comprises,” “comprising,”
“include,” “includes,” and “including” are interchangeable and are not limiting. For example,
the term “including” shall mean “including, but not limited to.” The word “about” means
plus or minus 5% of the stated number.
An “isolated peptide or polypeptide,” as used herein, is intended to refer to a peptide
or polypeptide which is substantially free of other biological molecules, including peptides or
polypeptides having distinct sequences. In some embodiments, the isolated peptide or
polypeptide is at least about 75%, about 80%, about 90%, about 95%, about 97%, about 99%,
about 99.9% or about 100% pure by dry weight. In some embodiments, purity can be
measured by a method such as column chromatography, polyacrylamide gel electrophoresis,
or HPLC analysis.
As used herein, “conservative substitutions” refers to modifications of a polypeptide
that involve the substitution of one or more amino acids for amino acids having similar
biochemical properties that do not result in loss of a biological or biochemical function of the
polypeptide. A “conservative amino acid substitution” is one in which the amino acid residue
is replaced with an amino acid residue having a similar side chain. Families of amino acid
residues having similar side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains
(e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). Antibodies of the present disclosure can have one or more
conservative amino acid substitutions yet retain antigen binding activity.
For nucleic acids and polypeptides, the term “substantial homology” indicates that
two nucleic acids or two polypeptides, or designated sequences thereof, when optimally
aligned and compared, are identical, with appropriate nucleotide or amino acid insertions or
deletions, in at least about 80% of the nucleotides or amino acids, usually at least about 85%,
in some embodiments about 90%, 91%, 92%, 93%, 94%, or 95%, in at least one embodiment
at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of the
nucleotides or amino acids. Alternatively, substantial homology for nucleic acids exists when
the segments will hybridize under selective hybridization conditions to the complement of the
strand. Also included are nucleic acid sequences and polypeptide sequences having
substantial homology to the specific nucleic acid sequences and amino acid sequences recited
herein.
The percent identity between two sequences is a function of the number of identical
positions shared by the sequences (i.e., % homology = # of identical positions / total # of
positions x 100), taking into account the number of gaps, and the length of each gap, which
need to be introduced for optimal alignment of the two sequences. Comparison of sequences
and determination of percent identity between two sequences can be accomplished using a
mathematical algorithm, such as without limitation the AlignX™ module of VectorNTI™
(Invitrogen Corp., Carlsbad, CA). For AlignX™, the default parameters of multiple
alignment are: gap opening penalty: 10; gap extension penalty: 0.05; gap separation penalty
range: 8; % identity for alignment delay: 40. (further details at world-wide-web at
invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEA-Communities/Vector-
NTI-Community/Sequence-analysis-and-data-management-software-for-PCs/AlignX-
Module-for-Vector-NTI-Advance.reg.us.html).
Another method for determining the best overall match between a query sequence (a
sequence of the present disclosure) and a subject sequence, also referred to as a global
sequence alignment, can be determined using the CLUSTALW computer program
(Thompson et al., Nucleic Acids Res, 1994, 2(22): 4673-4680), which is based on the
algorithm of Higgins et al., Computer Applications in the Biosciences (CABIOS), 1992, 8(2):
189-191). In a sequence alignment the query and subject sequences are both DNA
sequences. The result of the global sequence alignment is in percent identity. Parameters
that can be used in a CLUSTALW alignment of DNA sequences to calculate percent identity
via pairwise alignments are: Matrix = IUB, k-tuple = 1, Number of Top Diagonals = 5, Gap
Penalty = 3, Gap Open Penalty = 10, Gap Extension Penalty = 0.1. For multiple alignments,
the following CLUSTALW parameters can be used: Gap Opening Penalty = 10, Gap
Extension Parameter = 0.05; Gap Separation Penalty Range = 8; % Identity for Alignment
Delay = 40.
The nucleic acids can be present in whole cells, in a cell lysate, or in a partially
purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially
pure” when purified away from other cellular components with which it is normally
associated in the natural environment. To isolate a nucleic acid, standard techniques such as
the following can be used: alkaline/SDS treatment, CsCl banding, column chromatography,
agarose gel electrophoresis and others well known in the art.
I. Phosphatidylserine (PtdS)
A. Structure and Synthesis
Phosphatidylserine (abbreviated PtdS, Ptd-L-Ser or PS) is a phospholipid component,
usually kept on the inner-leaflet (the cytosolic side) of cell membranes by an enzyme called
flippase. When a cell undergoes apoptosis, phosphatidylserine is no longer restricted to the
cytosolic part of the membrane, but becomes exposed on the surface of the cell. The
chemical formula of PtdS is C H NO P and has a molecular mass of 385.304. The
13 24 10
structure is shown below:
Phosphatidylserine is biosynthesized in bacteria by condensing the amino acid serine
with CDP (cytidine diphosphate)-activated phosphatidic acid. In mammals,
phosphatidylserine is produced by base-exchange reactions with phosphatidylcholine and
phosphatidylethanolamine. Conversely, phosphatidylserine can also give rise to
phosphatidylethanolamine and phosphatidylcholine, although in animals the pathway to
generate phosphatidylcholine from phosphatidylserine only operates in the liver.
B. Function
Early studies of phosphatidylserine distilled the chemical from bovine brain. Modern
studies and commercially available products are made from soybeans, because of concerns
about mad cow disease. The fatty acids attached to the serine in the soy product are not
identical to those in the bovine product and is also impure. Preliminary studies in rats indicate
that the soy product is at least as potent as that of bovine origin.
The U.S. FDA has given “qualified health claim” status to phosphatidylserine, stating
that, “Consumption of phosphatidylserine may reduce the risk of dementia in the elderly” and
"Consumption of phosphatidylserine may reduce the risk of cognitive dysfunction in the
elderly.”
Phosphatidylserine has been demonstrated to speed up recovery, prevent muscle
soreness, improve well-being, and might possess ergogenic properties in athletes involved in
cycling, weight training and endurance running. Soy-PtdS, in a dose dependent manner (400
mg), has been reported to be an effective supplement for combating exercise-induced stress
by blunting the exercise-induced increase in cortisol levels. PtdS supplementation promotes a
desirable hormonal balance for athletes and might attenuate the physiological deterioration
that accompanies overtraining and/or overstretching. In recent studies, PtdS has been shown
to enhance mood in a cohort of young people during mental stress and to improve accuracy
during tee-off by increasing the stress resistance of golfers. First pilot studies indicate that
PtdS supplementation might be beneficial for children with attention-deficit hyperactivity
disorder.
Traditionally, PtdS supplements were derived from bovine cortex (BC-PS); however,
due to the potential transfer of infectious diseases, soy-derived PS (S-PS) has been
established as a potential safe alternative. Soy-derived PS is Generally Recognized As Safe
(GRAS) and is a safe nutritional supplement for older persons if taken up to a dosage of 200
mg three times daily. Phosphatidylserine has been shown to reduce specific immune response
in mice.
PtdS can be found in meat, but is most abundant in the brain and in innards such as
liver and kidney. Only small amounts of PS can be found in dairy products or in vegetables,
with the exception of white beans.
Annexin-A5 is a naturally-occurring protein with avid binding affinity for PtdS.
Labeled-annexin-A5 enables visualization of cells in the early- to mid-apoptotic state in vitro
or in vivo. Another PtdS binding protein is Mfge8. Technetium-labeled annexin-A5 enables
distinction between malignant and benign tumors whose pathology includes a high rate of
cell division and apoptosis in malignant compared with a low rate of apoptosis in benign
tumors.
II. Gla Domain Proteins
A. Gla Domains
The general structure for the Gla-domain proteins is that of a Gla domain followed by
EGF domains and then a C terminal serine protease domain. The exceptions are prothrombin,
which contains Kringle domains in place of EGF domains, and protein S, which does not
have a serine protease domain but rather sex hormone-binding globulin-like (SHBG) domains
(Hansson and Stenflo, 2005). The affinities of Gla-domain proteins to anionic membranes
vary. Roughly, they fall into 3 categories 1) high affinity binders with a Kd of 30-50 nM, 2)
mid-affinity binders with a K of 100-200 nM and 3) low affinity binders with a Kd of 1000-
2000 nM. The high affinity Gla domain proteins have been shown to bind anionic membranes
with Protein S specifically demonstrating binding to apoptotic cells via its interaction with
PtdS (Webb et al., 2002). The low affinity Gla domain proteins use a secondary receptor to
bind to the cell membrane. For example, FVII utilizes Tissue Factor (TF). The Gla
domain/1 EGF domain is believed to constitute the high affinity TF binding domain of FVII.
Importantly for this approach, there are many studies that have shown TF up-regulation on
the surface of cancer cells including colorectal cancer, NSCL carcinoma, and breast cancer
and these high TF levels have been associated with a poor prognosis (Yu et al., 2004).
Although the affinity for anionic membranes is relatively low for FVII, the addition of the
high affinity TF interaction along with the documented up-regulation of TF in cancer makes
it a potentially interesting cancer specific probe.
B. Gla Domain Containing Proteins
1. Factor II
Prothrombin, also known as coagulation factor II, is proteolytically cleaved to form
thrombin in the coagulation cascade, which ultimately results in the stemming of blood loss.
Thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble
strands of fibrin, as well as catalyzing many other coagulation-related reactions. It is
primarily expressed in the liver.
The gene encoding prothrombin is located on chromosome 11 in the region of the
centromere. It is composed of 14 exons and contains 24 kilobases of DNA. The gene encodes
a signal region, a propeptide region, a glutamic acid domain, 2 Kringle regions, and a
catalytic domain. The enzyme gamma-glutamyl carboxylase, in the presence of vitamin K,
converts the N- terminal glutamic acid residues to gamma-carboxyglutamic acid residues.
These gamma-carboxyglutamic acid residues are necessary for the binding of prothrombin to
phospholipids on platelet membranes.
Inherited factor II deficiency is an autosomal recessive disorder that can manifest as
hypoprothrombinemia, a decrease in the overall synthesis of prothrombin, or as
dysprothrombinemia, the synthesis of dysfunctional prothrombin. Homozygous individuals
are generally asymptomatic and have functional prothrombin levels of 2-25%. However,
symptomatic individuals may experience easy bruising, epistaxis, soft-tissue hemorrhage,
excessive postoperative bleeding, and/or menorrhagia.
Prothrombin plays a role in a role in chronic urticaria, an autoimmune disease, and
various vascular disorders. Livedo vasculopathy is associated with immunoglobulin (Ig)M
antiphosphatidylserine-prothrombin complex antibody. The presence of
antiphosphatidylserine-prothrombin complex antibodies and histopathological necrotizing
vasculitis in the upper-to-middle dermis indicates cutaneous leukocytoclastic angiitis rather
than cutaneous polyarteritis nodosa.
Aside from the prothrombin deficiencies, another disorder of prothrombin is the
prothrombin 20210a mutation. A familial cause of venous thromboembolism, the
prothrombin 20210a mutation results in increased levels of plasma prothrombin and a
concurrent increased risk for the development of thrombosis. Although the exact mechanism
of this disorder has not been elucidated, the prothrombin 20210a mutation involves the
substitution of an adenine for a guanine at position 20210 within the 3' untranslated region of
the prothrombin gene. This mutation alters the polyadenylation site of the gene and results in
increased mRNA synthesis, with a subsequent increase in protein expression.
2. Factor VII
Factor VII (formerly known as proconvertin) is one of the proteins that causes blood
to clot in the coagulation cascade. The gene for factor VII is located on chromosome 13
(13q34). It is an enzyme of the serine protease class, and recombinant form of human factor
VIIa (NovoSeven) has U.S. Food and Drug Administration approval for uncontrolled
bleeding in hemophilia patients. It is sometimes used unlicensed in severe uncontrollable
bleeding, although there have been safety concerns. A Biosimilar form of recombinant
activated factor VII (AryoSeven) is manufactured by AryoGen Biopharma.
The main role of factor VII (FVII) is to initiate the process of coagulation in
conjunction with tissue factor (TF/factor III). Tissue factor is found on the outside of blood
vessels - normally not exposed to the bloodstream. Upon vessel injury, tissue factor is
exposed to the blood and circulating factor VII. Once bound to TF, FVII is activated to FVIIa
by different proteases, among which are thrombin (factor IIa), factor Xa, IXa, XIIa, and the
FVIIa-TF complex itself. The most important substrates for FVIIa-TF are Factor X and
Factor IX. Factor VII has been shown to interact with Tissue factor (TF).
The action of the factor is impeded by tissue factor pathway inhibitor (TFPI), which is
released almost immediately after initiation of coagulation. Factor VII is vitamin K
dependent; it is produced in the liver. Use of warfarin or similar anticoagulants decreases
hepatic synthesis of FVII.
Deficiency is rare (congenital proconvertin deficiency) and inherits recessively.
Factor VII deficiency presents as a hemophilia-like bleeding disorder. It is treated with
recombinant factor VIIa (NovoSeven or AryoSeven). Recombinant factor VIIa is also used
for people with hemophilia (with Factor VIII or IX deficiency) who have developed
inhibitors against replacement coagulation factor. It has also been used in the setting of
uncontrollable hemorrhage, but its role in this setting is controversial with insufficient
evidence to support its use outside of clinical trials. The first report of its use in hemorrhage
was in an Israeli soldier with uncontrollable bleeding in 1999. Risks of its use include an
increase in arterial thrombosis.
3. Factor IX
Factor IX (or Christmas factor) is one of the serine proteases of the coagulation
system; it belongs to peptidase family S1. The gene for factor IX is located on the X
chromosome (Xq27.1-q27.2) and is therefore X-linked recessive: mutations in this gene
affect males much more frequently than females. Deficiency of this protein causes
hemophilia B. Factor IX is produced as a zymogen, an inactive precursor. It is processed to
remove the signal peptide, glycosylated and then cleaved by factor XIa (of the contact
pathway) or factor VIIa (of the tissue factor pathway) to produce a two-chain form where the
chains are linked by a disulfide bridge. When activated into factor IXa, in the presence of
Ca , membrane phospholipids, and a Factor VIII cofactor, it hydrolyses one arginine-
isoleucine bond in factor X to form factor Xa. Factor IX is inhibited by antithrombin.
Factors VII, IX, and X all play key roles in blood coagulation and also share a
common domain architecture. The factor IX protein is composed of four protein domains.
These are the Gla domain, two tandem copies of the EGF domain and a C-terminal trypsin-
like peptidase domain which carries out the catalytic cleavage. The N-terminal EGF domain
has been shown to at least in part be responsible for binding Tissue factor. Wilkinson et al.
conclude that residues 88 to 109 of the second EGF domain mediate binding to platelets and
assembly of the Factor X activating complex. The structures of all four domains have been
solved. A structure of the two EGF domains and trypsin like domain was determined for the
pig protein. The structure of the Gla domain, which is responsible for Ca(II)-dependent
phospholipid binding, was also determined by NMR. Several structures of “super active”
mutants have been solved which reveal the nature of Factor IX activation by other proteins in
the clotting cascade.
Deficiency of factor IX causes Christmas disease (hemophilia B). Over 100 mutations
of factor IX have been described; some cause no symptoms, but many lead to a significant
bleeding disorder. Recombinant factor IX is used to treat Christmas disease, and is
commercially available as BeneFIX. Some rare mutations of factor IX result in elevated
clotting activity, and can result in clotting diseases, such as deep vein thrombosis.
4. Factor X
Factor X (Stuart-Prower factor; prothrombinase) is an enzyme of the coagulation
cascade. The human factor X gene is located on the thirteenth chromosome (13q34). It is a
serine endopeptidase (protease group S1). Factor X is synthesized in the liver and requires
vitamin K for its synthesis. Factor X is activated into factor Xa by both factor IX (with its
cofactor, factor VIII in a complex known as intrinsic Xase) and factor VII with its cofactor,
tissue factor (a complex known as extrinsic Xase). The half life of factor X is 40–45 hours. It
is therefore the first member of the final common pathway or thrombin pathway. It acts by
cleaving prothrombin in two places (an arg-thr and then an arg-ile bond), which yields the
active thrombin. This process is optimized when factor Xa is complexed with activated co-
factor V in the prothrombinase complex. Factor X is part of fresh frozen plasma and the
prothrombinase complex. The only commercially available concentrate is “Factor X P
Behring” manufactured by CSL Behring.
Factor Xa is inactivated by protein Z-dependent protease inhibitor (ZPI), a serine
protease inhibitor (serpin). The affinity of this protein for factor Xa is increased 1000-fold by
the presence of protein Z, while it does not require protein Z for inactivation of factor XI.
Defects in protein Z lead to increased factor Xa activity and a propensity for thrombosis.
Inborn deficiency of factor X is very rare (1:500,000), and may present with epistaxis
(nosebleeds), hemarthrosis (bleeding into joints) and gastrointestinal blood loss. Apart from
congenital deficiency, low factor X levels may occur occasionally in a number of disease
states. For example, factor X deficiency may be seen in amyloidosis, where factor X is
adsorbed to the amyloid fibrils in the vasculature. Also, deficiency of vitamin K or
antagonism by warfarin (or similar medication) leads to the production of an inactive factor
X. In warfarin therapy, this is desirable to prevent thrombosis. As of late 2007, four out of
five emerging anti-coagulation therapeutics targeted this enzyme. Direct Xa inhibitors are
popular anticoagulants.
Traditional models of coagulation developed in the 1960s envisaged two separate
cascades, the extrinsic (tissue factor (TF)) pathway and the intrinsic pathway. These
pathways converge to a common point, the formation of the Factor Xa/Va complex which
together with calcium and bound on a phospholipids surface generate thrombin (Factor IIa)
from prothrombin (Factor II). A new model, the cell-based model of anticoagulation appears
to explain more fully the steps in coagulation. This model has three stages: 1) initiation of
coagulation on TF-bearing cells, 2) amplification of the procoagulant signal by thrombin
generated on the TF-bearing cell and 3) propagation of thrombin generation on the platelet
surface. Factor Xa plays a key role in all three of these stages.
In stage 1, Factor VII binds to the transmembrane protein TF on the surface of cells
and is converted to Factor VIIa. The result is a Factor VIIa/TF complex which catalyzes the
activation of Factor X and Factor IX. Factor Xa formed on the surface of the TF-bearing cell
interacts with Factor Va to form the prothrombinase complex which generates small amounts
of thrombin on the surface of TF-bearing cells. In stage 2, the amplification stage, if enough
thrombin has been generated, then activation of platelets and platelet associated cofactors
occurs. In stage 3, thrombin generation, Factor XIa activates free Factor IX on the surface of
activated platelets. The activated Factor IXa with Factor VIIIa forms the “tenase” complex.
This complex activates more Factor X, which in turn forms new prothrombinase complexes
with Factor Va. Factor Xa is the prime component of the prothrombinase complex which
converts large amounts of prothrombin—the “thrombin burst.” Each molecule of Factor Xa
can generate 1000 molecules of thrombin. This large burst of thrombin is responsible for
fibrin polymerization to form a thrombus.
Inhibition of the synthesis or activity of Factor X is the mechanism of action for many
anticoagulants in use today. Warfarin, a synthetic derivative of coumarin, is the most widely
used oral anticoagulant in the U.S. In some European countries, other coumarin derivatives
(phenprocoumon and acenocoumarol) are used. These agents are vitamin K antagonists
(VKA). Vitamin K is essential for the hepatic synthesis of Factors II (prothrombin), VII, IX
and X. Heparin (unfractionated heparin) and its derivatives low molecular weight heparin
(LMWH) bind to a plasma cofactor, antithrombin (AT) to inactivate several coagulation
factors IIa, Xa, XIa and XIIa.
Recently a new series of specific, direct acting inhibitors of Factor Xa has been
developed. These include the drugs rivaroxaban, apixaban, betrixaban, LY517717, darexaban
(YM150), edoxaban and 813893. These agents have several theoretical advantages over
current therapy. They may be given orally. They have rapid onset of action. And they may be
more effective against Factor Xa in that they inhibit both free Factor Xa and Factor Xa in the
prothrombinase complex.
5. Protein S
Protein S is a vitamin K-dependent plasma glycoprotein synthesized in the
endothelium. In the circulation, Protein S exists in two forms: a free form and a complex
form bound to complement protein C4b-binding protein (C4BP). In humans, Protein S is
encoded by the PROS1 gene. The best characterized function of Protein S is its role in the
anti coagulation pathway, where it functions as a cofactor to Protein C in the inactivation of
Factors Va and VIIIa. Only the free form has cofactor activity.
Protein S can bind to negatively charged phospholipids via the carboxylated GLA
domain. This property allows Protein S to function in the removal of cells which are
undergoing apoptosis. Apoptosis is a form of cell death that is used by the body to remove
unwanted or damaged cells from tissues. Cells which are apoptotic (i.e., in the process of
apoptosis) no longer actively manage the distribution of phospholipids in their outer
membrane and hence begin to display negatively-charged phospholipids, such as
phosphatidyl serine, on the cell surface. In healthy cells, an ATP (Adenosine triphosphate)-
dependent enzyme removes these from the outer leaflet of the cell membrane. These
negatively-charged phospholipids are recognized by phagocytes such as macrophages.
Protein S can bind to the negatively-charged phospholipids and function as a bridging
molecule between the apoptotic cell and the phagocyte. The bridging property of Protein S
enhances the phagocytosis of the apoptotic cell, allowing it to be removed 'cleanly' without
any symptoms of tissue damage such as inflammation occurring.
Mutations in the PROS1 gene can lead to Protein S deficiency which is a rare blood
disorder which can lead to an increased risk of thrombosis. Protein S has been shown to
interact with Factor V.
6. Protein C
Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV, is a
zymogenic (inactive) protein, the activated form of which plays an important role in
regulating blood clotting, inflammation, cell death, and maintaining the permeability of blood
vessel walls in humans and other animals. Activated protein C (APC) performs these
operations primarily by proteolytically inactivating proteins Factor V and Factor VIII . APC
is classified as a serine protease as it contains a residue of serine in its active site. In humans,
protein C is encoded by the PROC gene, which is found on chromosome 2.
The zymogenic form of protein C is a vitamin K-dependent glycoprotein that
circulates in blood plasma. Its structure is that of a two-chain polypeptide consisting of a light
chain and a heavy chain connected by a disulfide bond. The protein C zymogen is activated
when it binds to thrombin, another protein heavily involved in coagulation, and protein C's
activation is greatly promoted by the presence of thrombomodulin and endothelial protein C
receptors (EPCRs). Because of EPCR's role, activated protein C is found primarily near
endothelial cells (i.e., those that make up the walls of blood vessels), and it is these cells and
leukocytes (white blood cells) that APC affects. Because of the crucial role that protein C
plays as an anticoagulant, those with deficiencies in protein C, or some kind of resistance to
APC, suffer from a significantly increased risk of forming dangerous blood clots
(thrombosis).
Research into the clinical use of activated protein C also known as drotrecogin alfa-
activated (branded Xigris) has been surrounded by controversy. The manufacturer Eli Lilly
and Company ran an aggressive marketing campaign to promote its use in people with severe
sepsis and septic shock including the sponsoring of the 2004 Surviving Sepsis Campaign
Guidelines. A 2011 Cochrane review however found that its use cannot be recommended as it
does not improve survival (and increases bleeding risk).
Human protein C is a vitamin K-dependent glycoprotein structurally similar to other
vitamin K-dependent proteins affecting blood clotting, such as prothrombin, Factor VII,
Factor IX and Factor X. Protein C synthesis occurs in the liver and begins with a single-chain
precursor molecule: a 32 amino acid N-terminus signal peptide preceding a propeptide.
198 199
Protein C is formed when a dipeptide of Lys and Arg is removed; this causes the
transformation into a heterodimer with N-linked carbohydrates on each chain. The protein has
one light chain (21 kDa) and one heavy chain (41 kDa) connected by a disulfide bond
183 319
between Cys and Cys .
Inactive protein C comprises 419 amino acids in multiple domains: one Gla domain
(residues 43–88); a helical aromatic segment (89–96); two epidermal growth factor (EGF)-
like domains (97–132 and 136–176); an activation peptide (200–211); and a trypsin-like
serine protease domain (212–450). The light chain contains the Gla- and EGF-like domains
and the aromatic segment. The heavy chain contains the protease domain and the activation
petide. It is in this form that 85–90% of protein C circulates in the plasma as a zymogen,
waiting to be activated. The remaining protein C zymogen comprises slightly modified forms
of the protein. Activation of the enzyme occurs when a thrombin molecule cleaves away the
activation peptide from the N-terminus of the heavy chain. The active site contains a catalytic
253 299 402
triad typical of serine proteases (His , Asp and Ser ).
The activation of protein C is strongly promoted by thrombomodulin and endothelial
protein C receptor (EPCR), the latter of which is found primarily on endothelial cells (cells
on the inside of blood vessels). The presence of thrombomodulin accelerates activation by
several orders of magnitude, and EPCR speeds up activation by a factor of 20. If either of
these two proteins is absent in murine specimens, the mouse dies from excessive blood-
clotting while still in an embryonic state. On the endothelium, APC performs a major role in
regulating blood clotting, inflammation, and cell death (apoptosis). Because of the
accelerating effect of thrombomodulin on the activation of protein C, the protein may be said
to be activated not by thrombin but the thrombin-thrombomodulin (or even thrombin-
thrombomodulin-EPCR) complex. Once in active form, APC may or may not remain bound
to EPCR, to which it has approximately the same affinity as the protein zymogen.
The Gla domain is particularly useful for binding to negatively-charged phospholipids
for anticoagulation and to EPCR for cytoprotection. One particular exosite augments protein
C’s ability to inactivate Factor V efficiently. Another is necessary for interacting with
thrombomodulin.
Protein C in zymogen form is present in normal adult human blood plasma at
concentrations between 65–135 IU/dL. Activated protein C is found at levels approximately
2000 times lower than this. Mild protein C deficiency corresponds to plasma levels above 20
IU/dL, but below the normal range. Moderately severe deficiencies describe blood
concentrations between 1 and 20 IU/dL; severe deficiencies yield levels of protein C that are
below 1 IU/dL or are undetectable. Protein C levels in a healthy term infant average 40
IU/dL. The concentration of protein C increases until six months, when the mean level is 60
IU/dL; the level stays low through childhood until it reaches adult levels after adolescence.
The half-life of activated protein C is around 15 minutes.
The protein C pathways are the specific chemical reactions that control the level of
expression of APC and its activity in the body. Protein C is pleiotropic, with two main classes
of functions: anticoagulation and cytoprotection (its direct effect on cells). Which function
protein C performs depends on whether or not APC remains bound to EPCR after it is
activated; the anticoagulative effects of APC occur when it does not. In this case, protein C
functions as an anticoagulant by irreversibly proteolytically inactivating Factor V and Factor
VIII , turning them into Factor V and Factor VIII respectively. When still bound to EPCR,
a i i
activated protein C performs its cytoprotective effects, acting on the effector substrate PAR-
1, protease-activated receptor-1. To a degree, APC's anticoagulant properties are independent
of its cytoprotective ones, in that expression of one pathway is not affected by the existence
of the other.
The activity of protein C may be down-regulated by reducing the amount either of
available thrombomodulin or of EPCR. This may be done by inflammatory cytokines, such as
interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Activated leukocytes release
these inflammatory mediators during inflammation, inhibiting the creation of both
thrombomodulin and EPCR, and inducing their shedding from the endothelial surface. Both
of these actions down-regulate protein C activation. Thrombin itself may also have an effect
on the levels of EPCR. In addition, proteins released from cells can impede protein C
activation, for example eosinophil, which may explain thrombosis in hypereosinophilic heart
disease. Protein C may be up-regulated by platelet factor 4. This cytokine is conjectured to
improve activation of protein C by forming an electrostatic bridge from protein C's Gla
domain to the glycosaminoglycan (GAG) domain of thrombomodulin, reducing the Michaelis
constant (K ) for their reaction. In addition, Protein C is inhibited by protein C inhibitor.
A genetic protein C deficiency, in its mild form associated with simple
heterozygosity, causes a significantly increased risk of venous thrombosis in adults. If a fetus
is homozygous or compound heterozygous for the deficiency, there may be a presentation of
purpura fulminans, severe disseminated intravascular coagulation and simultaneous venous
thromboembolism in the womb; this is very severe and usually fatal. Deletion of the protein
C gene in mice causes fetal death around the time of birth. Fetal mice with no protein C
develop normally at first, but experience severe bleeding, coagulopathy, deposition of fibrin
and necrosis of the liver. The frequency of protein C deficiency among asymptomatic
individuals is between 1 in 200 and 1 in 500. In contrast, significant symptoms of the
deficiency are detectable in 1 in 20,000 individuals. No racial nor ethnic biases have been
detected.
Activated protein C resistance occurs when APC is unable to perform its functions.
This disease has similar symptoms to protein C deficiency. The most common mutation
leading to activated protein C resistance among Caucasians is at the cleavage site in Factor V
for APC. There, Arg is replaced with Gln, producing Factor V Leiden. This mutation is
also called a R506Q. The mutation leading to the loss of this cleavage site actually stops APC
from effectively inactivating both Factor V and Factor VIII . Thus, the person's blood clots
too readily, and he is perpetually at an increased risk for thrombosis. Individuals
heterozygous for the Factor V mutation carry a risk of venous thrombosis 5–7 times
Leiden
higher than in the general population. Homozygous subjects have a risk 80 times higher. This
mutation is also the most common hereditary risk for venous thrombosis among Caucasians.
Around 5% of APC resistance is not associated with the above mutation and Factor
V . Other genetic mutations cause APC resistance, but none to the extent that Factor
Leiden
V does. These mutations include various other versions of Factor V, spontaneous
Leiden
generation of autoantibodies targeting Factor V, and dysfunction of any of APC's cofactors.
Also, some acquired conditions may reduce the efficacy of APC in performing its
anticoagulative functions. Studies suggest that between 20% and 60% of thrombophilic
patients suffer from some form of APC resistance.
C. Gla Domain Peptides and Polypeptide
The present disclosure contemplates the design, production and use of various Gla
domain-containing peptides and polypeptides. The structural features of these molecules are
as follows. First, the peptides or polypeptides have a Gla domain containing about 30-45
consecutive residues comprising a Gla domain. Thus, the term “a peptide having no more
than “X” consecutive residues,” even when including the term “comprising,” cannot be
understood to comprise a greater number of consecutive residues. Second, the peptides and
polypeptides may contain additional non-Gla domain residues, such as EGF domains, Kringle
domains, Fc domains, etc.
In general, the peptides and polypeptides will be 300 residues or less, again,
comprising 30-45 consecutive residues of Gla domain. The overall length may be 30, 40, 50,
60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275 and up to 300 residues. Ranges of
peptide length of 50-300 residues, 100-300 residues, 150-300 residues 200-300, residues, 50-
200 residues, 100-200 residues, and 150-300 residues, and 150-200 residues are
contemplated. The number of consecutive Gla residues may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14 or 15.
The present disclosure may utilize L-configuration amino acids, D-configuration
amino acids, or a mixture thereof. While L-amino acids represent the vast majority of amino
acids found in proteins, D-amino acids are found in some proteins produced by exotic sea-
dwelling organisms, such as cone snails. They are also abundant components of the
peptidoglycan cell walls of bacteria. D-serine may act as a neurotransmitter in the brain. The
L and D convention for amino acid configuration refers not to the optical activity of the
amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which
that amino acid can theoretically be synthesized (D-glyceraldehyde is dextrorotary; L-
glyceraldehyde is levorotary).
One form of an “all-D” peptide is a retro-inverso peptide. Retro-inverso modification
of naturally occurring polypeptides involves the synthetic assemblage of amino acids with α-
carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D-amino
acids in reverse order with respect to the native peptide sequence. A retro-inverso analogue
thus has reversed termini and reversed direction of peptide bonds (NH-CO rather than CO-
NH) while approximately maintaining the topology of the side chains as in the native peptide
sequence. See U.S. Patent 6,261,569, incorporated herein by reference.
D. Synthesis
It will be advantageous to produce peptides and polypeptides using the solid-phase
synthetic techniques (Merrifield, 1963). Other peptide synthesis techniques are well known
to those of skill in the art (Bodanszky et al., 1976; Peptide Synthesis, 1985; Solid Phase
Peptide Synthelia, 1984). Appropriate protective groups for use in such syntheses will be
found in the above texts, as well as in Protective Groups in Organic Chemistry (1973). These
synthetic methods involve the sequential addition of one or more amino acid residues or
suitable protected amino acid residues to a growing peptide chain. Normally, either the
amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively
removable protecting group. A different, selectively removable protecting group is utilized
for amino acids containing a reactive side group, such as lysine.
Using solid phase synthesis as an example, the protected or derivatized amino acid is
attached to an inert solid support through its unprotected carboxyl or amino group. The
protecting group of the amino or carboxyl group is then selectively removed and the next
amino acid in the sequence having the complementary (amino or carboxyl) group suitably
protected is admixed and reacted with the residue already attached to the solid support. The
protecting group of the amino or carboxyl group is then removed from this newly added
amino acid residue, and the next amino acid (suitably protected) is then added, and so forth.
After all the desired amino acids have been linked in the proper sequence, any remaining
terminal and side group protecting groups (and solid support) are removed sequentially or
concurrently, to provide the final peptide. The peptides and polypeptides of the disclosure
are preferably devoid of benzylated or methylbenzylated amino acids. Such protecting group
moieties may be used in the course of synthesis, but they are removed before the peptides and
polypeptides are used. Additional reactions may be necessary, as described elsewhere, to
form intramolecular linkages to restrain conformation.
Aside from the twenty standard amino acids can can be used, there are a vast number
of “non-standard” amino acids. Two of these can be specified by the genetic code, but are
rather rare in proteins. Selenocysteine is incorporated into some proteins at a UGA codon,
which is normally a stop codon. Pyrrolysine is used by some methanogenic archaea in
enzymes that they use to produce methane. It is coded for with the codon UAG. Examples of
non-standard amino acids that are not found in proteins include lanthionine, 2-
aminoisobutyric acid, dehydroalanine and the neurotransmitter gamma-aminobutyric acid.
Non-standard amino acids often occur as intermediates in the metabolic pathways for
standard amino acids - for example ornithine and citrulline occur in the urea cycle, part of
amino acid catabolism. Non-standard amino acids are usually formed through modifications
to standard amino acids. For example, homocysteine is formed through the transsulfuration
pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl
methionine, while hydroxyproline is made by a posttranslational modification of proline.
E. Linkers
Linkers or cross-linking agents may be used to fuse Gla domain peptides or
polypeptides to other proteinaceous sequences (e.g., antibody Fc domains). Bifunctional
cross-linking reagents have been extensively used for a variety of purposes including
preparation of affinity matrices, modification and stabilization of diverse structures,
identification of ligand and receptor binding sites, and structural studies. Homobifunctional
reagents that carry two identical functional groups proved to be highly efficient in inducing
cross-linking between identical and different macromolecules or subunits of a
macromolecule, and linking of polypeptide ligands to their specific binding sites.
Heterobifunctional reagents contain two different functional groups. By taking advantage of
the differential reactivities of the two different functional groups, cross-linking can be
controlled both selectively and sequentially. The bifunctional cross-linking reagents can be
divided according to the specificity of their functional groups, e.g., amino-, sulfhydryl-,
guanidino-, indole-, or carboxyl-specific groups. Of these, reagents directed to free amino
groups have become especially popular because of their commercial availability, ease of
synthesis and the mild reaction conditions under which they can be applied. A majority of
heterobifunctional cross-linking reagents contains a primary amine-reactive group and a
thiol-reactive group.
In another example, heterobifunctional cross-linking reagents and methods of using
the cross-linking reagents are described in U.S. Patent 5,889,155, specifically incorporated
herein by reference in its entirety. The cross-linking reagents combine a nucleophilic
hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example,
of aldehydes to free thiols. The cross-linking reagent can be modified to cross-link various
functional groups and is thus useful for cross-linking polypeptides. In instances where a
particular peptide does not contain a residue amenable for a given cross-linking reagent in its
native sequence, conservative genetic or synthetic amino acid changes in the primary
sequence can be utilized.
F. Additional Peptide/Polypeptide Sequences
One factor drug development is to achieve adequate circulating half-lives, which
impact dosing, drug administration and efficacy, and this has particular important to
biotherapeutics. Small proteins below 60 kD are cleared rapidly by the kidney and therefore
do not reach their target. This means that high doses are needed to reach efficacy. The
modifications currently used to increase the half-life of proteins in circulation include:
PEGylation; conjugation or genetic fusion with proteins, e.g., transferrin (WO06096515A2),
albumin, growth hormone (U.S. Patent Publication 2003104578AA); conjugation with
cellulose (Levy and Shoseyov, 2002); conjugation or fusion with Fc fragments; glycosylation
and mutagenesis approaches (Carter, 2006).
In the case of PEGylation, polyethylene glycol (PEG) is conjugated to the protein,
which can be for example a plasma protein, antibody or antibody fragment. The first studies
regarding the effect of PEGylation of antibodies were performed in the 1980s. The
conjugation can be done either enzymatically or chemically and is well established in the art
(Chapman, 2002; Veronese and Pasut, 2005). With PEGylation the total size can be
increased, which reduces the chance of renal filtration. PEGylation further protects from
proteolytic degradation and slows the clearance from the blood. Further, it has been reported
that PEGylation can reduce immunogenicity and increase solubility. The improved
pharmacokinetics by the addition of PEG is due to several different mechanisms: increase in
size of the molecule, protection from proteolysis, reduced antigenicity, and the masking of
specific sequences from cellular receptors. In the case of antibody fragments (Fab), a 20-fold
increase in plasma half-life has been achieved by PEGylation (Chapman, 2002).
To date there are several approved PEGylated drugs, e.g., PEG-interferon alpha2b
(PEG-INTRON) marketed in 2000 and alpha2a (Pegasys) marketed in 2002. A PEGylated
antibody fragment against TNF alpha, called Cimzia or Certolizumab Pegol, was filed for
FDA approval for the treatment of Crohn's disease in 2007 and has been approved on Apr.
22, 2008. A limitation of PEGylation is the difficulty in synthesizing long monodisperse
species, especially when PEG chains over 1000 kD are needed. For many applications,
polydisperse PEG with a chain length over 10000 kD is used, resulting in a population of
conjugates having different length PEG chains, which need extensive analytics to ensure
equivalent batches between productions. The different length of the PEG chains may result in
different biological activities and therefore different pharmacokinetics. Another limitation of
PEGylation is a decrease in affinity or activity as it has been observed with alpha-interferon
Pegasys, which has only 7% of the antiviral activity of the native protein, but has improved
pharmacokinetics due to the enhanced plasma half-life.
Another approach is to conjugate the drug with a long lived protein, e.g., albumin,
which is 67 kD and has plasma half-life of 19 days in human. Albumin is the most abundant
protein in plasma and is involved in plasma pH regulation, but also serves as a carrier of
substances in plasma. In the case of CD4, increased plasma half-life has been achieved after
fusing it to human serum albumin (Yeh et al., 1992). Other examples for fusion proteins are
insulin, human growth hormone, transferrin and cytokines (Duttaroy et al., 2005; Melder et
al., 2005; Osborn et al., 2002a; Osborn et al., 2002b; Sung et al., 2003) and see (U.S. Patent
Publication 2003104578A1, WO06096515A2, and WO07047504A2, herein incorporated in
entirety by reference).
The effect of glycosylation on plasma half-life and protein activity has also been
extensively studied. In the case of tissue plasminogen activator (tPA), the addition of new
glycosylation sites decreased the plasma clearance, and improved the potency (Keyt et al.,
1994). Glycoengineering has been successfully applied for a number of recombinant proteins
and immunoglobulins (Elliott et al., 2003; Raju and Scallon, 2007; Sinclair and Elliott, 2005;
Umana et al., 1999). Further, glycosylation influences the stability of immunoglobulins
(Mimura et al., 2000; Raju and Scallon, 2006).
Another molecule used for fusion proteins is the Fc fragment of an IgG (Ashkenazi
and Chamow, 1997). The Fc fusion approach has been utilized, for example in the Trap
Technology developed by Regeneron (e.g., IL1 trap and VEGF trap). The use of albumin to
extend the half-life of peptides has been described in U.S. Patent Publication 2004001827A1,
as well as for Fab fragments and scFv-HSA fusion protein. It has been demonstrated that the
prolonged serum half-life of albumin is due to a recycling process mediated by the FcRn
(Anderson et al., 2006; Chaudhury et al., 2003).
Another strategy is to use directed mutagenesis techniques targeting the interaction of
immunoglobulins to their receptor to improve binding properties, i.e., affinity maturation in
the Fc region. With an increased affinity to FcRn a prolonged half-life can be achieved in
vivo (Ghetie et al., 1997; Hinton et al., 2006; Jain et al., 2007; Petkova et al., 2006a; Vaccaro
et al., 2005). However, affinity maturation strategies require several rounds of mutagenesis
and testing. This takes time, is costly and is limited by the number of amino acids that when
mutated result in prolonged half-lives. Therefore, simple alternative approaches are needed to
improve the in vivo half-life of biotherapeutics. Therapeutics with extended half-lives in vivo
are especially important for the treatment of chronic diseases, autoimmune disorders,
inflammatory, metabolic, infectious, and eye diseases, and cancer, especially when therapy is
required over a long time period. Accordingly, a need still exists for the development of
therapeutic agents (e.g., antibodies and Fc fusion proteins) with enhanced persistence and
half-lives in circulation, in order to reduce the dosage and/or the frequency of injections of a
variety of therapeutic agents.
G. Labels
The peptides and polypeptides of the present disclosure may be conjugated to labels
for diagnostic purposes, such as to identify cancer cells or virally-infected cells, including
their use in histochemistry. A label in accordance with the present disclosure is defined as
any moiety which may be detected using an assay. Non-limiting examples of reporter
molecules include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent
molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored
particles or ligands, such as biotin.
Label conjugates are generally preferred for use as diagnostic agents. Diagnostic
agents generally fall within two classes, those for use in in vitro diagnostics, and those for use
in vivo diagnostic protocols, generally known as “directed imaging.” Many appropriate
imaging agents are known in the art, as are methods for their attachment to peptides and
polypeptides (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging
moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-
detectable substances, and X-ray imaging agents.
In the case of paramagnetic ions, one might mention by way of example ions such as
chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II),
neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium
(III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly
preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to
lanthanum (III), gold (III), lead (II), and especially bismuth (III).
In the case of radioactive isotopes for therapeutic and/or diagnostic application, one
211 14 51 36 57 58 67
might mention astatine , carbon, chromium, chlorine, cobalt, cobalt, copper ,
152 67 3 123 125 131 111 59 32
Eu, gallium , hydrogen, iodine , iodine , iodine , indium , iron, phosphorus,
186 188 75 35 99m 90 125
rhenium , rhenium , selenium, sulphur, technicium and/or yttrium . I is often
99m 111
being preferred for use in certain embodiments, and technicium and/or indium are also
often preferred due to their low energy and suitability for long range detection. Radioactively
labeled peptides and polypeptides may be produced according to well-known methods in the
art. For instance, peptides and polypeptides can be iodinated by contact with sodium and/or
potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an
enzymatic oxidizing agent, such as lactoperoxidase. Petides may be labeled with
technetium by ligand exchange process, for example, by reducing pertechnate with
stannous solution, chelating the reduced technetium onto a Sephadex column and applying
the peptide to this column. Alternatively, direct labeling techniques may be used, e.g., by
incubating pertechnate, a reducing agent such as SNCl , a buffer solution such as sodium-
potassium phthalate solution, and the peptide. Intermediary functional groups which are often
used to bind radioisotopes which exist as metallic ions to peptide are
diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).
Among the fluorescent labels contemplated for use as conjugates include Alexa 350,
Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,
BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein
Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514,
Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET,
Tetramethylrhodamine, and/or Texas Red.
Another type of conjugate contemplated is that intended primarily for use in vitro,
where the peptide is linked to a secondary binding ligand and/or to an enzyme (an enzyme
tag) that will generate a colored product upon contact with a chromogenic substrate.
Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen
peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and
streptavidin compounds. The use of such labels is well known to those of skill in the art and
is described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345,
4,277,437, 4,275,149 and 4,366,241.
Other methods are known in the art for the attachment or conjugation of a peptide to
its conjugate moiety. Some attachment methods involve the use of a metal chelate complex
employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid
anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or
tetrachloro-3 α-6 α-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and
4,938,948). Peptides or polypeptides may also be reacted with an enzyme in the presence of a
coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are
prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
IV. Diagnostics and Therapies
A. Pharmaceutical Formulations and Routes of Administration
Where clinical applications are contemplated, it will be necessary to prepare
pharmaceutical compositions in a form appropriate for the intended application. Generally,
this will entail preparing compositions that are essentially free of pyrogens, as well as other
impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render delivery
vectors stable and allow for uptake by target cells. Buffers also will be employed when
recombinant cells are introduced into a patient. Aqueous compositions of the present
disclosure comprise an effective amount of the vector to cells, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred
to as inocula. The phrase “pharmaceutically or pharmacologically acceptable” refer to
molecular entities and compositions that do not produce adverse, allergic, or other untoward
reactions when administered to an animal or a human. As used herein, “pharmaceutically
acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents and the like. The use of such
media and agents for pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the vectors or cells of the
present disclosure, its use in therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the compositions.
The active compositions of the present disclosure may include classic pharmaceutical
preparations. Administration of these compositions according to the present disclosure will
be via any common route so long as the target tissue is available via that route. Such routes
include oral, nasal, buccal, rectal, vaginal or topical route. Alternatively, administration may
be by orthotopic, intradermal, subcutaneous, intramuscular, intratumoral, intraperitoneal, or
intravenous injection. Such compositions would normally be administered as
pharmaceutically acceptable compositions, described supra.
The active compounds may also be administered parenterally or intraperitoneally.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be
prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use, these preparations contain
a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions
or dispersions and sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent
that easy syringability exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of microorganisms, such as
bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying absorption, for example,
aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the
required amount in the appropriate solvent with various other ingredients as enumerated
above, as required, followed by filtered sterilization. Generally, dispersions are prepared by
incorporating the various sterilized active ingredients into a sterile vehicle which contains the
basic dispersion medium and the required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum-drying and freeze-drying techniques which yield a
powder of the active ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be incorporated into the
compositions.
For oral administration the peptides and polypeptides of the present disclosure may be
incorporated with excipients and used in the form of non-ingestible mouthwashes and
dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required
amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
Alternatively, the active ingredient may be incorporated into an antiseptic wash containing
sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be
dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient
may be added in a therapeutically effective amount to a paste dentifrice that may include
water, binders, abrasives, flavoring agents, foaming agents, and humectants.
The compositions of the present disclosure may be formulated in a neutral or salt
form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free
amino groups of the protein) and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic,
and the like. Salts formed with the free carboxyl groups can also be derived from inorganic
bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,
and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically effective. The formulations are
easily administered in a variety of dosage forms such as injectable solutions, drug release
capsules and the like. For parenteral administration in an aqueous solution, for example, the
solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic
with sufficient saline or glucose. These particular aqueous solutions are especially suitable
for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this
connection, sterile aqueous media which can be employed will be known to those of skill in
the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml
of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at
the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences,” 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose for the individual subject.
Moreover, for human administration, preparations should meet sterility, pyrogenicity, general
safety and purity standards as required by FDA Office of Biologics standards.
B. Disease States and Conditions
1. Cancer
Cancer results from the outgrowth of a clonal population of cells from tissue. The
development of cancer, referred to as carcinogenesis, can be modeled and characterized in a
number of ways. An association between the development of cancer and inflammation has
long-been appreciated. The inflammatory response is involved in the host defense against
microbial infection, and also drives tissue repair and regeneration. Considerable evidence
points to a connection between inflammation and a risk of developing cancer, i.e., chronic
inflammation can lead to dysplasia. There are hundreds of different forms of human cancers,
and with an increasing understanding of the underlying genetics and biology of cancer, these
forms are being further subdivided and reclassifed.
Determining what causes cancer is complex. Many things are known to increase the
risk of cancer, including tobacco use, certain infections, radiation, lack of physical activity,
obesity, and environmental pollutants. These can directly damage genes or combine with
existing genetic faults within cells to cause the disease. Approximately five to ten percent of
cancers are entirely hereditary.
Cancer can be detected in a number of ways, including the presence of certain signs
and symptoms, screening tests, or medical imaging. Once a possible cancer is detected it is
diagnosed by microscopic examination of a tissue sample. Cancer is usually treated with
chemotherapy, radiation therapy and surgery. The chances of surviving the disease vary
greatly by the type and location of the cancer and the extent of disease at the start of
treatment. While cancer can affect people of all ages, and a few types of cancer are more
common in children, the risk of developing cancer generally increases with age. In 2007,
cancer caused about 13% of all human deaths worldwide (7.9 million). Rates are rising as
more people live to an old age and as mass lifestyle changes occur in the developing world.
Treatments fall in to five general categories: surgery, chemotherapy, radiation,
alternative medicine and palliative care. Surgery is the primary method of treatment of most
isolated solid cancers and may play a role in palliation and prolongation of survival. It is
typically an important part of making the definitive diagnosis and staging the tumor as
biopsies are usually required. In localized cancer surgery typically attempts to remove the
entire mass along with, in certain cases, the lymph nodes in the area. For some types of
cancer this is all that is needed to eliminate the cancer.
Chemotherapy in addition to surgery has proven useful in a number of different
cancer types including: breast cancer, colorectal cancer, pancreatic cancer, osteogenic
sarcoma, testicular cancer, ovarian cancer, and certain lung cancers. The effectiveness of
chemotherapy is often limited by toxicity to other tissues in the body.
Radiation therapy involves the use of ionizing radiation in an attempt to either cure or
improve the symptoms of cancer. It is used in about half of all cases and the radiation can be
from either internal sources in the form of brachytherapy or external sources. Radiation is
typically used in addition to surgery and or chemotherapy but for certain types of cancer such
as early head and neck cancer may be used alone. For painful bone metastasis it has been
found to be effective in about 70% of people.
Alternative and complementary treatments include a diverse group of health care
systems, practices, and products that are not part of conventional medicine “Complementary
medicine” refers to methods and substances used along with conventional medicine, while
“alternative medicine” refers to compounds used instead of conventional medicine. Most
complementary and alternative medicines for cancer have not been rigorously studied or
tested. Some alternative treatments have been investigated and shown to be ineffective but
still continue to be marketed and promoted.
Finally, palliative care refers to treatment which attempts to make the patient feel
better and may or may not be combined with an attempt to attack the cancer. Palliative care
includes action to reduce the physical, emotional, spiritual, and psycho-social distress
experienced by people with cancer. Unlike treatment that is aimed at directly killing cancer
cells, the primary goal of palliative care is to improve the patient’s quality of life.
2. Viral Infection
A virus is a small infectious agent that can replicate only inside the living cells of an
organism. Viruses can infect all types of organisms, from animals and plants to bacteria and
archaea. About 5,000 viruses have been described in detail, although there are millions of
different types. Viruses are found in almost every ecosystem on Earth and are the most
abundant type of biological entity.
Virus particles (known as virions) consist of two or three parts: i) the genetic material
made from either DNA or RNA, long molecules that carry genetic information; ii) a protein
coat that protects these genes; and in some cases iii) an envelope of lipids that surrounds the
protein coat when they are outside a cell. The shapes of viruses range from simple helical and
icosahedral forms to more complex structures. The average virus is about one one-hundredth
the size of the average bacterium. Most viruses are too small to be seen directly with an
optical microscope.
Viruses spread in many ways; viruses in plants are often transmitted from plant to
plant by insects that feed on plant sap, such as aphids; viruses in animals can be carried by
blood-sucking insects. These disease-bearing organisms are known as vectors. Influenza
viruses are spread by coughing and sneezing. Norovirus and rotavirus, common causes of
viral gastroenteritis, are transmitted by the faecal–oral route and are passed from person to
person by contact, entering the body in food or water. HIV is one of several viruses
transmitted through sexual contact and by exposure to infected blood. The range of host cells
that a virus can infect is called its "host range". This can be narrow or, as when a virus is
capable of infecting many species, broad.
Viral infections in animals provoke an immune response that usually eliminates the
infecting virus. Immune responses can also be produced by vaccines, which confer an
artificially acquired immunity to the specific viral infection. However, some viruses
including those that cause AIDS and viral hepatitis evade these immune responses and result
in chronic infections. Antibiotics have no effect on viruses, but several antiviral drugs have
been developed.
A variety of diseases are fostered by virus infections, including influenza, human
immunodeficiency virus, dengue virus, West Nile virus, smallpox virus, respiratory syncytial
virus, Korean hemorrhagic fever virus, chickenpox, varicella zoster virus, herpes simplex
virus 1 or 2, Epstein-Barr virus, Marburg virus, hantavirus, yellow fever virus, hepatitis A, B,
C or E, Ebola virus, human papilloma virus, rhinovirus, Coxsackie virus, polio virus, measles
virus, rubella virus, rabies virus, Newcastle disease virus, rotavirus, HTLV-1 and -2.
C. Treatment Methods
Peptides and polypeptides can be administered to mammalian subjects (e.g., human
patients) alone or in conjunction with other drugs that treat the diseases set forth above. The
dosage required depends on the choice of the route of administration; the nature of the
formulation, including additional agents attached to the polypeptide; the nature of the
patient’s illness; the subject’s size, weight, surface area, age, and sex; further combination
therapies; and the judgment of the attending physician. Suitable dosages are in the range of
0.0001–100 mg/kg. Wide variations in the needed dosage are to be expected in view of the
variety of compounds available and the differing efficiencies of various routes of
administration. For example, oral administration would be expected to require higher
dosages than administration by intravenous injection. Variations in these dosage levels can
be adjusted using standard empirical routines for optimization as is well understood in the art.
Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-,100-, 150-, or
more times). Encapsulation of the polypeptide in a suitable delivery vehicle (e.g., polymeric
microparticles or implantable devices) may increase the efficiency of delivery, particularly
for oral delivery.
Engineered Gla domain proteins may be used as targeting agents to deliver
therapeutic payloads to cancer cells, such as radionuclides, chemotherapeutic agents or
toxins. Specific chemotherapeutics include temozolomide, epothilones, melphalan,
carmustine, busulfan, lomustine, cyclophosphamide, dacarbazine, polifeprosan, ifosfamide,
chlorambucil, mechlorethamine, busulfan, cyclophosphamide, carboplatin, cisplatin, thiotepa,
capecitabine, streptozocin, bicalutamide, flutamide, nilutamide, leuprolide acetate,
doxorubicin hydrochloride, bleomycin sulfate, daunorubicin hydrochloride, dactinomycin,
liposomal daunorubicin citrate, liposomal doxorubicin hydrochloride, epirubicin
hydrochloride, idarubicin hydrochloride, mitomycin, doxorubicin, valrubicin, anastrozole,
toremifene citrate, cytarabine, fluorouracil, fludarabine, floxuridine, interferon α-2b,
plicamycin, mercaptopurine, methotrexate, interferon α-2a, medroxyprogersterone acetate,
estramustine phosphate sodium, estradiol, leuprolide acetate, megestrol acetate, octreotide
acetate, deithylstilbestrol diphosphate, testolactone, goserelin acetate, etoposide phosphate,
vincristine sulfate, etoposide, vinblastine, etoposide, vincristine sulfate, teniposide,
trastuzumab, gemtuzumab ozogamicin, rituximab, exemestane, irinotecan hydrocholride,
asparaginase, gemcitabine hydrochloride, altretamine, topotecan hydrochloride, hydroxyurea,
cladribine, mitotane, procarbazine hydrochloride, vinorelbine tartrate, pentrostatin sodium,
mitoxantrone, pegaspargase, denileukin diftitix, altretinoin, porfimer, bexarotene, paclitaxel,
docetaxel, arsenic trioxide, or tretinoin. Toxins include Pseudomonas exotoxin (PE38), ricin
A chain, diphtheria toxin, Besides PE and RT, Pokeweed antiviral protein (PAP), saporin and
gelonin. Radionuclides for cancer therapy include Y-90, P-32, I-131, In-111, Sr-89, Re-186,
Sm-153, and Sn-117m.
Agents or factors suitable for therapy against a viral infections include Abacavir,
Aciclovir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir,
Atripla, Boceprevirertet, Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine,
Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Entry inhibitors,
Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Ganciclovir, Ibacitabine,
Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Integrase inhibitor, Interferon type III,
Interferon type II, Interferon type I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc,
Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nucleoside analogues,
Oseltamivir, Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin,
Protease inhibitor, Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine,
Ritonavir, Pyramidine, Saquinavir, Stavudine, Synergistic enhancer (antiretroviral), Tea tree
oil, Telaprevir, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir,
Tromantadine, Truvada, Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Viramidine,
Zalcitabine, Zanamivir and Zidovudine.
The skilled artisan is directed to “Remington's Pharmaceutical Sciences” 15th Edition,
chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose for the individual subject.
Moreover, for human administration, preparations should meet sterility, pyrogenicity, general
safety and purity standards as required by FDA Office of Biologics standards.
V. Examples
The following examples are included to demonstrate particular embodiments of the
disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in
the examples which follow represent techniques discovered by the inventor to function well
in the practice of the disclosure, and thus can be considered to constitute preferred modes for
its practice. However, those of skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific embodiments which are disclosed
and still obtain a like or similar result without departing from the spirit and scope of the
disclosure.
EXAMPLE 1
The affinities of Gla-domain proteins for cell membranes have been determined in
vitro by using prepared phospholipid vesicles (Shah et al., 1998; Nelsestuen, 1999). How
these in vitro values translate to an in vivo context, however, has not been fully elucidated.
The interaction of FVII with TF, for example, underscores the fact that although the Gla
domains of these proteins are very homologous, additional differences in their cell membrane
binding specificity and affinity may be mediated through their EGF and/or Kringle domains.
Unfortunately, these interactions cannot be recapitulated by studies based solely on
phospholipid vesicles and may remain unidentified.
Therefore, inventors proposed making and testing the Gla+EGF/Kringle domains as
well as the Gla domain alone from the following panel of proteins: hS (high affinity binder),
hZ(mid affinity binder), hPT (mid affinity-kringle containing), hFVII (low affinity-utilizes
secondary “receptor” that is also up-regulated in cancer), and B0178 (hFVII with increased
phospholipid affinity). These proteins potentially have varying in vivo binding characteristics
that may be beneficial to their use as probes (and, if validated and selective, potentially as
therapeutics) and that to date have gone unrecognized.
The general approach was to construct recombinant proteins and test them for
expression. Assays would then be developed to assess binding. Then, expression and
purification methods would be optimized, followed by quality control of gamma-
carboxylation.
shows sequences from a variety of Gla domain proteins including
carboxylation sites. shows the expression of a variety of different Gla domain
proteins that were engineered and transiently expressed in 293 cells. shows a similar
study in BHK21 cells. Given that one of the best expressing constructs was a Protein S +
EGF construct, the signal sequence from Protein S was utilized with Prothrombin Gla +
Kringle and Protein Z + EGF. However, expression was only observed intracellularly (FIG.
Protein S Gla + EGF was selected for further study. The sequence is shown in
Protein was produced in BHK21 cells using RF286 medium. 600 ml was harvested and
concentrated 4X. Purification utilized three steps:
1. Ni-NTA column, 10 ml, fresh packed. The medium are loaded to column and
eluted with Imidazole gradient. All the fractions are subject to Gla western blot to
identify the His tagged Gla protein S G+E.
2. Hitrap Q with CaCl step elution. The Gla positive fractions are pooled and subject
to 1 ml Hitrap Q with 10 mM CaCl elution.
3. Hitrap Q with CaCl gradient (0-10 mM shadow gradient). The step purified Gla
proteins were applied to Q and eluted with gradient CaCl (up to 10 mM). A total of 0.9 mg
of protein at a 95% purity level was produced. shows the purification fractions under
both reducing and non-reducing conditions. FIGS. 7 and 8 show different FACs-based
apoptosis assays. Both show that the Protein S Gla + EGF construct is specific for cells
undergoing apoptosis just like Annexin V (, and that Annexin V can compete off the
Protein S Gla + EGF binding.
In summary, Protein S Gla+EGF was expressed and purified. Analysis on the purified
material suggested that it was highly gamma-carboxylated. FACs-based Apoptosis Assays
demonstrated that Protein S G+E (11 Gla) could bind to “apoptotic” cells, and that this
binding was to cells was via targeting of phosphatidylserine, as demonstrated by Annexin V
competition assays.
* * * * * * * * * * * * *
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that
such documents, or such sources of information, in any jurisdiction, are prior art, or form part
of the common general knowledge in the art.
All of the compositions and/or methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present disclosure. While the
compositions and methods of this disclosure have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that variations may be applied to
the compositions and/or methods and in the steps or in the sequence of steps of the method
described herein without departing from the concept, spirit and scope of the disclosure. More
specifically, it will be apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and modifications apparent to
those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure
as defined by the appended claims.
Certain statements that appear herein are broader than what appears in the statements
of the invention. These statements are provided in the interests of providing the reader with a
better understanding of the invention and its practice. The reader is directed to the
accompanying claim set which defines the scope of the invention.
VI. References
The following references, to the extent that they provide exemplary procedural or
other details supplementary to those set forth herein, are specifically incorporated herein by
reference:
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U.S. Patent 5,446,128
U.S. Patent 5,475,085
U.S. Patent 5,597,457
U.S. Patent 5,618,914
U.S. Patent 5,670,155
U.S. Patent 5,672,681
U.S. Patent 5,674,976
U.S. Patent 5,710,245
U.S. Patent 5,790,421
U.S. Patent 5,840,833
U.S. Patent 5,859,184
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Claims (13)
1. A polypeptide suitable for targeting phosphatidylserine cell-surface expression both in vitro and in vivo, said polypeptide comprising: 5 a protein S gamma-carboxyglutamic-acid (Gla) domain, lacking a protease or hormone-binding domain, and an EGF domain, wherein the polypeptide: i) lacks a protease domain and also lacks a hormone-binding domain, 10 ii) is linked to therapeutic agent.
2. The polypeptide according to claim 1 comprising an EGF domain from protein S.
3. The polypeptide according to claim 2, wherein the GLA domain has the sequence 15 shown in SEQ ID NO: 1.
4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide is 300 amino acids or less. 20
5. The polypeptide according to any one of claims 1 to 4, comprising SEQ ID NO: 6.
6. The polypeptide according to any one of claims 1 to 5, wherein the polypeptide comprises an antibody Fc region. 25
7. The polypeptide according to any one of claims 1 to 6, wherein the therapeutic agent is selected from the group consisting of an anti-cancer agent, a chemotherapeutic, a radiotherapeutic, a cytokine, a hormone, an antibody, an antibody binding fragment, a prodrug converting enzyme, a growth factor, a clotting factor and an anti-coagulant. 30
8. The polypeptide according to any one of claims 1 to 7, wherein the polypeptide is linked to a therapeutic agent suitable for treating cancer.
9. The polypeptide according to claim 7 or 8, wherein said therapeutic agent is a chemotherapeutic, a radiotherapeutic or a toxin.
10. The polypeptide according to claim 9, wherein the chemotherapeutic is selected from 5 temozolomide, epothilones, melphalan, carmustine, busulfan, lomustine, cyclophosphamide, dacarbazine, polifeprosan, ifosfamide, chlorambucil, mechlorethamine, busulfan, cyclophosphamide, carboplatin, cisplatin, thiotepa, capecitabine, streptozocin, bicalutamide, flutamide, nilutamide, leuprolide acetate, doxorubicin hydrochloride, bleomycin sulfate, daunorubicin hydrochloride, 10 dactinomycin, liposomal daunorubicin citrate, liposomal doxorubicin hydrochloride, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin, doxorubicin, valrubicin, anastrozole, toremifene citrate, cytarabine, fluorouracil, fludarabine, floxuridine, interferon α-2b, plicamycin, mercaptopurine, methotrexate, interferon α- 2a, medroxyprogersterone acetate, estramustine phosphate sodium, estradiol, 15 leuprolide acetate, megestrol acetate, octreotide acetate, deithylstilbestrol diphosphate, testolactone, goserelin acetate, etoposide phosphate, vincristine sulfate, etoposide, vinblastine, etoposide, vincristine sulfate, teniposide, trastuzumab, gemtuzumab ozogamicin, rituximab, exemestane, irinotecan hydrocholride, asparaginase, gemcitabine hydrochloride, altretamine, topotecan hydrochloride, hydroxyurea, 20 cladribine, mitotane, procarbazine hydrochloride, vinorelbine tartrate, pentrostatin sodium, mitoxantrone, pegaspargase, denileukin diftitix, altretinoin, porfimer, bexarotene, paclitaxel, docetaxel, arsenic trioxide, and tretinoin.
11. The polypeptide according to claim 9, wherein the toxin is selected from 25 Pseudomonas exotoxin (PE38), ricin A chain, diphtheria toxin, Pokeweed antiviral protein (PAP), saporin and gelonin.
12. The polypeptide according to claim 9, wherein the radionuclide is selected from include Y-90, P-32, I-131, In-111, Sr-89, Re-186, Sm-153, and Sn-117m.
13. The polypeptide according to any one of claims 1 to 12, comprising a detectable label.
Priority Applications (1)
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NZ751491A NZ751491B2 (en) | 2013-03-15 | 2014-03-13 | Gla domains as targeting agents |
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US201361791537P | 2013-03-15 | 2013-03-15 | |
US201361787753P | 2013-03-15 | 2013-03-15 | |
US61/787,753 | 2013-03-15 | ||
US61/791,537 | 2013-03-15 | ||
PCT/US2014/025940 WO2014151535A1 (en) | 2013-03-15 | 2014-03-13 | Gla domains as targeting agents |
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