U.S. patent application number 15/411559 was filed with the patent office on 2017-07-20 for lipoprotein lipase for treatment of hypertriglyceridemic-related conditions including acute pancreatitis.
The applicant listed for this patent is Shire Human Genetic Therapies, Inc.. Invention is credited to Michael F. Concino, Tracy Dowie, Omar L. Francone, Lin Guey, Kevin Holmes, Lieh Yoon Low, Dianna Lundberg, Muthuraman Meiyappan, Angela Norton, Bettina Strack-Logue, Bruce Tangarone, Matthew Traylor, Lenore von Krusenstiern, Bohong Zhang.
Application Number | 20170202929 15/411559 |
Document ID | / |
Family ID | 54035309 |
Filed Date | 2017-07-20 |
United States Patent
Application |
20170202929 |
Kind Code |
A1 |
Francone; Omar L. ; et
al. |
July 20, 2017 |
LIPOPROTEIN LIPASE FOR TREATMENT OF HYPERTRIGLYCERIDEMIC-RELATED
CONDITIONS INCLUDING ACUTE PANCREATITIS
Abstract
A lipoprotein lipase (LPL) protein for treating and/or
preventing HTG and its associated diseases, including but not
limited to acute pancreatitis (AP), and in particular, acute
pancreatitis secondary to or exacerbated by hypertriglyceridemia,
and hypertriglyceridemia and its associated diseases in general,
including cardiovascular and metabolic diseases, endocrine
disorders, and fat embolism syndrome.
Inventors: |
Francone; Omar L.; (East
Lyme, CT) ; Guey; Lin; (Watertown, MA) ;
Holmes; Kevin; (Belmont, MA) ; Tangarone; Bruce;
(Hampstead, NH) ; Traylor; Matthew; (Cambridge,
MA) ; von Krusenstiern; Lenore; (Brookline, MA)
; Dowie; Tracy; (Lexington, MA) ; Low; Lieh
Yoon; (Lexington, MA) ; Zhang; Bohong;
(Lexington, MA) ; Meiyappan; Muthuraman;
(Lexington, MA) ; Norton; Angela; (Reading,
MA) ; Strack-Logue; Bettina; (Somerville, MA)
; Lundberg; Dianna; (Brentwood, NH) ; Concino;
Michael F.; (Bolton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shire Human Genetic Therapies, Inc. |
Lexington |
MA |
US |
|
|
Family ID: |
54035309 |
Appl. No.: |
15/411559 |
Filed: |
January 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14830481 |
Aug 19, 2015 |
9597376 |
|
|
15411559 |
|
|
|
|
62039362 |
Aug 19, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61K 9/08 20130101; C12Y 301/01034 20130101; A61K 38/465 20130101;
A61K 9/19 20130101; A61P 3/06 20180101; C12N 9/20 20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; C12N 9/20 20060101 C12N009/20; A61K 9/08 20060101
A61K009/08; A61K 9/00 20060101 A61K009/00; A61K 9/19 20060101
A61K009/19 |
Claims
1. A lipoprotein lipase (LPL) polypeptide for use in the treatment
or prevention of a hypertriglyceridemia-related condition, in a
subject.
2. The LPL polypeptide of claim 1, wherein the LPL polypeptide is
substantially resistant to proteolytic cleavage by proprotein
convertase.
3. The LPL polypeptide of claim 1, wherein the
hypertriglyceridemia-related condition is secondary to or
exacerbated by hypertriglyceridemia.
4. The LPL polypeptide of claim 1, wherein the
hypertriglyceridemia-related condition is selected from the group
consisting of hypertriglyceridemia, acute pancreatitis,
cardiovascular disease, a metabolic disorder, an endocrine
disorder, and fat embolism syndrome.
5. (canceled)
6. The LPL polypeptide of claim 4, wherein the acute pancreatitis
is secondary to or exacerbated by hypertriglyceridemia.
7. The LPL polypeptide of claim 1, wherein the serum or plasma
triglyceride level in the subject exceeds about 150 mg/dl.
8. The LPL polypeptide of claim 1, wherein the LPL polypeptide
comprises or consists of an amino acid sequence having 80%, 90%,
95%, 99%, 99.5% or 99.8% or more identity to SEQ ID NO: 1.
9. The LPL polypeptide of claim 1, wherein the LPL polypeptide
comprises or consists of the amino acid sequence of SEQ ID NO:
2.
10. The LPL polypeptide of claim 1, wherein the LPL polypeptide
exhibits a V.sub.max of about 0.01-50 mmoles FA/hr/mg in a
[.sup.3H]-triolein liposome activity assay and/or a K.sub.m value
of about 0.01-1 .mu.M.
11. The LPL polypeptide of claim 1, wherein the LPL polypeptide is
administered by oral, intravenous, intramuscular, intravenous,
intranasal, intradermal, subcutaneous, or suppository route.
12. The LPL polypeptide of claim 1, wherein the LPL polypeptide is
administered by continuous infusion or by bolus injection.
13. The LPL polypeptide of claim 1, wherein the LPL polypeptide is
administered as a single dose or multiple doses.
14. The LPL polypeptide of claim 1, wherein the LPL polypeptide is
glycosylated.
15. The LPL polypeptide of claim 1, wherein the LPL polypeptide is
non-glycosylated.
16. The LPL polypeptide of claim 1, wherein the LPL polypeptide is
in aqueous form.
17. The LPL polypeptide of claim 1, wherein the LPL polypeptide is
in lyophilized form.
18. A pharmaceutical composition comprising the LPL polypeptide of
claim 1 and a pharmaceutically acceptable carrier.
19. (canceled)
20. A method of treating or preventing a
hypertriglyceridemia-related condition in a subject, comprising the
step of administering to the subject an effective amount of the LPL
polypeptide of claim 1 or a pharmaceutical composition comprising
the LPL polypeptide of claim 1.
21. Use of the LPL polypeptide of claim 1 or a pharmaceutical
composition comprising the LPL polypeptide of claim 1 in the
preparation of a medicament for the treatment or prevention of a
hypertriglyceridemia-related condition in a subject.
22. A method of preparing the LPL polypeptide of, claim 1
comprising performing the recombinant expression of the LPL
polypeptide and LMF1 in a cell.
23. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 14/830,481, filed on Aug. 19, 2015,
which claims priority to U.S. Provisional Application 62/039,362
filed Aug. 19, 2014, which is incorporated by reference in its
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically and is hereby incorporated by
reference in its entirety. The file named "SHR-1166US2_ST25.txt"
was created on Jan. 19, 2017 and is 20,613 bytes in size.
TECHNICAL FIELD
[0003] This invention relates to compositions, uses, and methods
for treating and/or preventing conditions associated with elevated
triglyceride levels, such as hypertriglyceridemia (HTG) and its
associated diseases, including but not limited to acute
pancreatitis (AP) (in particular, acute pancreatitis secondary to
or exacerbated by hypertriglyceridemia), cardiovascular and
metabolic diseases, endocrine disorders such as hypothyroidism, and
fat embolism syndrome.
BACKGROUND
Hypertriglyceridemia
[0004] Hypertriglyceridemia (HTG) is a condition in which
triglyceride (TG) levels are elevated in the blood. The National
Cholesterol Education Program Adult Treatment Panel (NCEP ATP III)
guidelines indicate that normal TG levels are <150 mg/dl. HTG
can be classified into primary or secondary types. Primary HTG is
caused by genetic defects resulting in abnormal triglyceride
metabolism. Secondary HTG is the result of acquired causes such as
diabetes, obesity, hypothyroidism, and certain medications. HTG is
frequently associated with other lipid abnormalities and metabolic
syndrome, and is a risk factor for atherosclerosis and
cardiovascular disease.
Hypertriglyceridemia-Induced Acute Pancreatitis
[0005] HTG is a rare but significant cause of AP.
Hypertriglyceridemic acute pancreatitis (HTGAP) accounts for
approximately 1-11% of all cases of AP. These numbers may represent
somewhat of an underestimation; some investigators have suggested
that HTGAP is under diagnosed, as not all patients diagnosed with
AP have TG levels checked upon their initial presentation to the
hospital, and some patients with severe HTG and abdominal pain are
not correctly diagnosed with pancreatitis. Additionally, it has
been suggested that moderately elevated levels of TG may exacerbate
AP associated with other causative factors.
[0006] The mechanism by which HTG causes AP is currently being
investigated. Without wishing to be bound by theory, one hypothesis
suggests that hydrolysis of excess TGs by pancreatic lipases leads
to the liberation of large amounts of free fatty acids (FFA) within
the pancreas, which initiates the process of pancreatic injury, and
pancreatic enzyme activation and release. Another hypothesis
suggests that the pathologic process is initiated by poor
pancreatic perfusion due to hyperviscosity of the hyperlipidemic
blood, which leads to ischemia, hydrolysis of TG by pancreatic
lipase, and a continuous cycle of pancreatic injury. Genetic
studies in animal models also support a mechanistic link between
HTG and AP. For example, it has been found that LPL-deficient minks
developed spontaneous pancreatitis. LPL-deficient mice showed an
enhanced susceptibility to pancreatitis. Furthermore, high plasma
TG has been shown to exacerbate AP in rats.
[0007] While a value at which patients with elevated TGs develop AP
is not precisely defined, the risk of developing HTGAP increases
significantly when TG levels are above 1000 mg/dl.
[0008] AP is an inflammatory disease of the pancreas. Patients
typically present with acute upper abdominal pain, nausea and
vomiting. The diagnosis is based on patient symptoms, specifically
type and location of abdominal pain, laboratory tests, including
serum amylase and lipase levels, and radiologic evaluations,
including ultrasound, Computed Tomography (CT) or Magnetic
Resonance Imaging (MRI).
[0009] AP leads to the release and activation of digestive enzymes,
such as trypsinogen, from the exocrine pancreas gland causing
inflammation, injury, and autolysis of the pancreas. AP is a
disease with significant morbidity and mortality. Patients with AP
may develop multi-organ failure, pancreatic necrosis, pancreatic
pseudocysts, infection, and shock. Prolonged ICU stays are common,
and various invasive procedures may be required. The overall
mortality rate for all levels of severity is approximately 5%,
however for the sickest patients, those with infected pancreatic
necrosis, mortality has been reported to be as high as 62%. Long
term consequences of AP include glucose intolerance, pancreatic
insufficiency, and recurrent pancreatitis.
[0010] Gallstones and alcohol abuse are by far the two most common
causes of AP, with multiple less frequently occurring etiologies,
including hypertriglyceridemia, undergoing ERCP, drugs, hereditary
AP, among others.
[0011] Pancreatitis can be categorized into different categories
based on level of pancreatic injury, and clinical presentation.
Based on radiologic examinations, AP can be described as either
interstitial edematous pancreatitis, or necrotizing pancreatitis,
with necrotizing pancreatitis being the more severe. In addition,
AP can be described as mild, moderate, or severe, using the revised
Atlanta Criteria, which defines categories based on the patient's
clinical condition, such as the presence or absence of organ
failure: e.g., no organ failure=mild, transient organ failure
(<48 hours)=moderate, and persistent organ failure=severe.
Patients can also be evaluated for severity of illness based on a
large variety of clinical assessment tools (e.g., APACHE II,
Balthazar criteria, SIRS criteria etc.).
[0012] No specific pharmacological interventions have been
demonstrated to improve the clinical course of AP. Current
treatment is limited to supportive therapy: IV hydration, pain
control, nutritional support, intensive care; antibiotics and
surgical interventions are implemented as needed. While
approximately 80% of cases may resolve with supportive therapy,
with hospital stays typically lasting 5-7 days, 20% of patients
have severe disease, which frequently require prolonged ICU stays,
and may require invasive procedures. The mortality rate for all
patients with AP is approximately 5%, while for the sickest
patients the mortality rate has been reported to be upwards of 60%.
In patients with HTGAP, one exemplary goal of therapy is to lower
TG levels as quickly as possible, in an attempt to intervene in the
process of continued pancreatic injury. Currently this can be
achieved through aggressive hydration and nutritional management.
With supportive care, TG levels have been reported to decrease to
.ltoreq.500 mg/dl by 72 hours in most but not all patients. Various
therapeutic interventions to quickly reduce TG levels have been
attempted, however there are currently no approaches that have
proven to be reproducibly effective and safe.
[0013] It has been suggested that HTGAP may be a more aggressive
form of pancreatitis. Due to the small number of patients
accurately diagnosed with HTGAP, there have been few studies large
enough to adequately address this issue. Two studies that examined
larger numbers of patients with HTGAP found that compared to
patients with biliary pancreatitis, patients with HTGAP tend to be
younger, and more likely to progress to severe disease.
Fat Embolism
[0014] Fat embolism syndrome is a potentially fatal complication of
trauma to fat-containing bones and soft tissue and is characterized
by deposits of fat globules in the circulation. Fat embolism
syndrome is more likely to develop after lower limb and pelvic
fractures in contrast to fractures of upper limbs. The primary
symptoms and signs of fat embolism syndrome are respiratory
distress, neurological signs including confusion, drowsiness,
convulsions, coma, and petechial rash. Fat embolism can lead to
severe complications such as pulmonary dysfunction and
microinfarctions. Treatment is limited to supportive care, and
focuses primarily on minimizing pulmonary distress and hypovolemia.
The invention aims to provide improved methods of preventing and/or
treating HTG and its associated diseases, including AP,
cardiovascular disease, metabolic disorders, endocrine disorders,
and fat embolism syndrome.
SUMMARY
[0015] The present invention provides, among other things,
lipoprotein lipase (LPL) polypeptides, related compositions, and
their use in treatment or prevention of various diseases,
disorders, or conditions described herein. In some cases, the LPL
polypeptides may be for use in the treatment or prevention of a
hyperlipidemia and hyperlipidemia-related conditions, including
hypertriglyceridemia (HTG) and hypertriglyceridemia-related
conditions, in a subject. In some cases, the LPL polypeptide is
substantially resistant to proteolytic cleavage by proprotein
convertase. Methods for the treatment and/or prevention of
hyperlipidemia and hyperlipidemia-related conditions (e.g.,
hypertriglyceridemia-related conditions) are also provided. The
method may involve administering any of the LPL polypeptides
disclosed herein to a subject.
[0016] In some cases, the hypertriglyceridemia-related condition is
secondary to or exacerbated by hypertriglyceridemia. The
hypertriglyceridemia-related condition may be, for example,
hypertriglyceridemia, acute pancreatitis, cardiovascular disease, a
metabolic disorder, an endocrine disorder, or fat embolism
syndrome. In some embodiments, the hypertriglyceridemia-related
condition is acute pancreatitis. In some embodiments, the acute
pancreatitis is secondary to or exacerbated by
hypertriglyceridemia. In some embodiments, the serum or plasma
triglyceride level in the subject exceeds about 150 mg/dl.
[0017] In any of the foregoing embodiments, the LPL polypeptide may
comprise or consist of an amino acid sequence having 80%, 90%, 95%,
99%, 99.5% or 99.8% or more identity to SEQ ID NO: 1. In some
embodiments, the LPL polypeptide comprises or consists of the amino
acid sequence of SEQ ID NO: 2. In some embodiments, the LPL
polypeptide exhibits a V.sub.max of about 0.01-50 mmoles FA/hr/mg
in a [.sup.3H]-triolein liposome activity assay and/or a K.sub.m
value of about 0.01-1 .mu.M.
[0018] In any of the foregoing embodiments, the LPL polypeptide may
be administered by oral, intravenous, intramuscular, intravenous,
intranasal, intradermal, subcutaneous, or suppository route. In any
of the foregoing embodiments, the LPL polypeptide may be
administered by continuous infusion or by bolus injection. In any
of the foregoing embodiments, the LPL polypeptide may be
administered as a single dose or multiple doses.
[0019] In any of the foregoing embodiments, the LPL polypeptide may
be glycosylated. In any of the foregoing embodiments, the LPL
polypeptide may be non-glycosylated.
[0020] In some cases, the LPL polypeptide is in aqueous form. In
some cases, the LPL polypeptide is in lyophilized form.
[0021] Methods of preparing the LPL polypeptide disclosed herein
are also provided. In some embodiments, the method may involve
reconstituting an LPL polypeptide in lyophilized form with aqueous
material. In some embodiments, the method may comprise performing
recombinant expression of an LPL polypeptide and LMF1 in a cell.
The cell may be a mammalian cell, a plant cell, an insect cell, or
another type of cell. In some cases, the cell is a HT1080 cell,
HEK293 cell, CHO cell, or a variant of a CHO cell.
[0022] Pharmaceutical compositions are also provided. The
pharmaceutical composition may comprise any of the LPL polypeptides
disclosed herein, and a pharmaceutically acceptable carrier.
[0023] Methods of treating or preventing a
hypertriglyceridemia-related condition in a subject are also
provided comprising the step of administering to the subject an
effective amount of an LPL polypeptide as disclosed herein or a
pharmaceutical composition as disclosed herein.
[0024] Use of LPL polypeptides disclosed herein, or pharmaceutical
compositions disclosed herein, in the preparation of a medicament
for the treatment or prevention of a hypertriglyceridemia-related
condition in a subject are also provided.
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIG. 1 shows LPL titer estimates of LPL expressed in HT1080
cells ("R324A") and in HT1080 cells co-transfected with LMF1
("R324A/LMF1") by ELISA.
[0026] FIGS. 2A and 2B show the enzymatic properties of the LPL
using Michaelis-Menten kinetics (FIG. 2A) and Eadie-Hofstee
kinetics (FIG. 2B).
[0027] FIGS. 3A and 3B show representative time-courses in
triglyceride (TG) and free fatty acid (FFA) concentrations in
plasma samples from patients who have HTG (FIG. 3A) and HTGAP (FIG.
3B) following treatment with LPL. Baseline TG levels are 1250 mg/dl
and 6202 mg/dl in FIGS. 3A and 3B respectively.
[0028] FIGS. 4A and 4B show changes in TG and FFA levels in plasma
obtained in 16 HTG individuals (denoted by open squares) and 2
individuals with HTGAP (denoted by asterisk).
[0029] FIG. 5 shows the change in FFA concentration following
incubation of increasing amounts of LPL in human HTG plasma
demonstrating a dose-dependent hydrolysis of TGs.
[0030] FIG. 6 shows changes in NEFA concentration in plasma
obtained from 11 HTG NHPs following incubation with LPL (1 .mu.g
LPL in 125 .mu.L plasma). Baseline plasma TGs were 1566.+-.352
mg/dL (range=352-5360 mg/dL).
[0031] Table 1 shows serum TG levels following a single IV
administration of recombinant LPL (1 mg/kg) or vehicle to ob/ob
mice at 5 and 20 minutes post-injection. Data shown as
mean.+-.standard deviation.
[0032] Table 2 shows serum TG levels in ob/ob mice following a
single IV administration of recombinant LPL at 0 (vehicle control),
0.1 mg/kg, 0.5 mg/kg and 1 mg/kg. Data shown as mean.+-.standard
deviation.
DISCLOSURE OF THE INVENTION
[0033] The invention provides a lipoprotein lipase (LPL)
polypeptide for use in the treatment and/or prevention of HTG and
its associated diseases, including but not limited to AP,
cardiovascular disease, metabolic disorders, endocrine disorders,
and fat embolism syndrome in a subject. The subject may have HTG.
The serum triglyceride level in the subject may exceed
approximately 150 mg/dl. The AP may be secondary to or exacerbated
by hyperlipidemia, preferably HTG.
[0034] LPL can play a major role in the metabolism and transport of
lipids as the enzyme responsible for the hydrolysis of
triglycerides in chylomicrons and very low-density lipoproteins
(VLDLs). LPL is primarily anchored to the luminal surface of
capillary endothelial cells and can interact with lipoproteins and
facilitate lipoprotein particle uptake. LPL can also promote the
exchange of lipids between lipoproteins, and consequently has an
important role in the kinetics of many lipoprotein particles. Thus,
when used with the invention, LPL is capable of reducing
plasma/serum triglyceride (TG) level in the subject, thereby
ameliorating the symptoms of the diseases.
[0035] The invention uses LPL polypeptides, which include both
wild-type LPL and its sequence variants. The invention preferably
uses a human LPL polypeptide, e.g. the LPL polypeptide comprises or
consists of the amino acid sequence recited in SEQ ID NO: 1. A LPL
polypeptide used with the invention preferably comprises or
consists of an amino acid sequence having 80%, 90%, 95%, 99%, 99.5%
or 99.8% or more identity to a LPL polypeptide, e.g. the mature
human lipoprotein lipase (LPL) (SEQ ID NO: 1). LPL polypeptides may
exhibit certain properties (e.g., enzymatic properties), including
a V.sub.max of about 0.01-50 mmoles/h/mg in a [.sup.3H]-triolein
liposome activity assay and/or a K.sub.m value of about 0.01-1
.mu.M. In some cases, the LPL polypeptide may be substantially
resistant to proteolytic cleavage, for example, by proprotein
convertase (PC). For example, the LPL polypeptide may be a variant
of LPL containing at least one point mutation that replaces,
modifies or deletes any of the amino acids at the RAKR sequence
located at amino acid positions 294-297 of SEQ ID NO: 1 (which
corresponds to the amino acid positions 320-324 of the wild-type
LPL precursor, SEQ ID NO: 3). In some cases, the LPL polypeptide
may have an amino acid substitution at position 297 of SEQ ID NO:
1. Preferably, the LPL variant comprises or consists of the amino
acid sequence recited in SEQ ID NO: 2 (referred to as LPL R324A
herein). In some cases, the LPL polypeptide is not an S447X mutant
of LPL (SEQ ID NO: 6).
[0036] The LPL polypeptide may be, for example, 448 amino acids in
length, e.g. having the amino acid sequence of SEQ ID NO: 1 or 2.
In other instances, the LPL polypeptide may be 446 amino acids in
length, e.g. having the amino acid sequence of SEQ ID NO: 6.
[0037] In vivo, endogenous LPL is secreted as head-to-tail
homodimers following synthesis. The LPL homodimers are translocated
from the interstitial space to endothelial cells. The LPL
homodimers are anchored to endothelial cells by ion interaction
with heparin sulfate proteoglycans (HSPG) and/or by glycosyl
phosphatidylinositol (e.g. GPI-HBP1). LPL homodimers are active in
the capillary lumen, and the activity requires interaction with the
cofactor ApoCII. The inventors report herein that in plasma samples
obtained from HTG patients with and without AP, when LPL
polypeptide was added to the samples, LPL was capable of rapidly
hydrolyzing TGs. This demonstrates that exogenously added LPL
polypeptide remains enzymatically active in plasma, and is
functional to reduce TG levels in a disease context.
[0038] In fact, LPL polypeptide is capable of rapidly hydrolyzing
triglycerides in human plasma of HTG individuals and HTG
individuals undergoing an attack of AP. This was demonstrated by
incubating an isolated LPL polypeptide variant R324A in plasma
samples obtained from 16 HTG individuals (baseline TG
levels=1182.+-.873 mg/dL, range 342-3449 mg/dL) and two individuals
with HTGAP (baseline TG levels=1255 and 6202 mg/dL) for up to 24
hours and assessing changes in TG and non-esterified fatty acid or
free fatty acid (FFA) levels. FIGS. 3A and 3B show representative
time-courses of TG and FFA concentrations following incubation of
LPL for an individual with HTG and HTGAP respectively (baseline TG
levels=1250 mg/dl and 6202 mg/dl respectively). In both samples,
LPL led to a rapid and precipitous decline of TGs along with a
concomitant increase in FFAs (FIG. 3). This was observed across all
HTG samples (FIG. 4, asterisk denotes HTGAP samples). Specifically,
plasma TG levels decreased 42.+-.11%, 54.+-.8%, 67.+-.9% and
79.+-.11% at 15 minutes, 1 hour, 6 hours and 24 hours respectively
across all HTG samples (all p<0.001) (FIG. 4A). Additionally a
corresponding increase in FFAs consistent with the reduction in TG
levels was observed in each of the samples (FIG. 4B). Treatment
with LPL also resulted in a dose-dependent hydrolysis of TGs in HTG
plasma as demonstrated by the increased release of FFAs following
incubation with increasing amounts of LPL (FIG. 5).
[0039] Embodiments disclosed herein provide for the use of LPL
polypeptides in various therapeutic applications. Given that LPL
had previously been known to have a very short half-life in humans
(only 6-30 min), it was surprising that exogenous LPL exhibited
sufficient activity in plasma to reduce TG levels in vivo, in a
therapeutically-relevant way. Building upon the ex vivo experiments
described herein, the inventors conducted various experiments in
vivo, using the ob/ob mouse model. In these experiments, LPL R324A
was administered intravenously (IV) at doses of 0.1 mg/kg, 0.5
mg/kg and 1 mg/kg. Table 1 shows a >85% drop in serum TG levels
following a single IV administration of 1 mg/kg of recombinant LPL
in ob/ob mice compared to vehicle controls (p<0.001). Despite
the large drop in serum TGs, serum NEFA concentration remained
unchanged. Fatty acids are rapidly transported to target tissues by
albumin and thus our findings are consistent with the rapid
clearance of fatty acids as reported in the literature.
Furthermore, LPL elicited a dose-dependent hydrolysis of TGs
consistent with ex vivo experiments (Table 2). This provides
further evidence that an exogenously added LPL polypeptide has in
vivo functional activity when administered.
[0040] Before commencing this work, an uncertainty faced the
inventors, in that the AP condition causes the release of
proteolytic enzymes from the gut into the plasma. The work reported
herein is the first to show that LPL remains active in the presence
of these proteolytic enzymes, to the extent that a real reduction
in TG levels can be generated in a therapeutic context. This
therefore paves the way for the observation that LPL and variants
thereof could be effective in lowering TG levels, e.g. by
hydrolyzing triglycerides in lipoproteins, such as those found in
chylomicrons and very low-density lipoproteins (VLDL), in AP
patients.
[0041] The LPL polypeptides used with the invention may be
administered by oral, intravenous, intramuscular, intravenous,
intranasal, intradermal, subcutaneous, or suppository routes. The
LPL polypeptides of the invention may also be administered
parenterally or intraperitoneally. In some cases, the LPL
polypeptides may be administered to a subject via intravenous,
subcutaneous or intra muscular injection, by continuous infusion,
or by bolus injection. The LPL polypeptides used with the invention
may be administered as a single dose or multiple doses.
[0042] The LPL polypeptides used with the invention are preferably
glycosylated. A glycosylated polypeptide used in accordance with
the invention preferably has a molecular weight of about 50-60 kDa.
In some cases, the LPL polypeptides may be non-glycosylated, as
some, or all of the N-linked consensus sites.
[0043] The LPL polypeptide preferably has a V.sub.max of about
0.01-50 mmoles/h/mg in a [.sup.3H]-triolein liposome activity
assay. Preferably, the LPL polypeptide of the invention has a
K.sub.m value of about 0.01-1 .mu.M.
[0044] LPL is preferably administered in the form of protein into a
subject. The LPL polypeptide used with the invention may be in
aqueous form or a formulation compatible with the hydrophobic
nature of the molecule, including but not limited to lipid or
polymeric nanoparticles.
[0045] The invention may use a lyophilizate of the LPL polypeptides
of the invention. This lyophilizate can be reconstituted with
aqueous material to provide an aqueous composition comprising the
LPL polypeptide of the invention. For administration, the
lyophilizate is thus reconstituted with a suitable liquid diluent
(e.g. a buffer, saline solution, water for injection (wfi)).
[0046] The invention also provides a pharmaceutical composition
comprising a LPL polypeptide of the invention (e.g., wild-type LPL
or a variant thereof) and a pharmaceutically acceptable
carrier.
[0047] The invention also provides a method of preparing a LPL
polypeptide of the invention, comprising performing the recombinant
expression of the LPL polypeptide together with the LMF1 protein in
a cell. Suitable expression systems may be stable or transient
expression systems. The cell may be a mammalian cell, a plant cell,
an insect cell, or other types of cells. Preferably, the cell is a
HT1080, HEK293, COS or CHO cell (including all CHO variants).
[0048] The invention preferably uses LPL polypeptides in enzyme
therapy, i.e. the LPL is administered in the form of protein. This
is in contrast to a gene therapy, which involves administering the
subject with a viral nucleic acid encoding a protein such as LPL.
LPL enzyme therapy provides the subject with a supply of LPL
polypeptides, and this sudden increase in LPL polypeptide
concentration is capable of allowing a rapid reduction in
plasma/serum TG level and/or maintaining plasma/serum TG at a
certain concentration to prevent conditions related to HTG.
LPL and Variants
[0049] The lipoprotein lipase protein is herein annotated as `LPL`
(EC 3.1.1.34). LPL was originally called `heparin-releasable
clearing factor`, and has also been referred to as `clearing factor
lipase`, `diacylglycerol lipase`, `diglyceride lipase`, `HDLCQ11`
and `LIPD` in the literature.
[0050] In humans, a wild-type LPL has the amino acid sequence SEQ
ID NO: 3 (NP_000228, GI: 4557727). LPL is initially translated as a
precursor protein, having 475 amino acids (SEQ ID NO: 3). In
action, the signal peptide at amino acid positions 1-27 of SEQ ID
NO: 3 is then cleaved, leaving the mature form having roughly 448
amino acids (e.g. SEQ ID NO: 1).
[0051] The invention preferably uses a human LPL polypeptide, e.g.
the LPL polypeptide comprises or consists of the amino acid
sequence recited in SEQ ID NO: 1 or 3. Preferably, the LPL
polypeptide comprises or consists of the amino acid sequence
recited in SEQ ID NO: 1.
[0052] A LPL polypeptide used in accordance with the invention may
comprise or consist of an amino acid sequence having 80% or more
identity (e.g. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99.5%, 99.8% or more) to wild-type LPL, e.g., the LPL
polypeptide used with the invention may comprise or consist of any
of the amino acid sequences recited in SEQ ID NOs: 1-6.
[0053] Preferably, a LPL polypeptide used with the invention may
retain the activity of the wild-type LPL. For example, the LPL
polypeptide may exhibit activity that is not worse (e.g., <5%
activity) than the wild-type human LPL (SEQ ID NO: 3). For example,
the LPL polypeptide may exhibit activity that is similar to (e.g.,
within .+-.5% activity) or better (e.g., >5% activity) than the
wild-type human LPL (SEQ ID NO: 3). Preferably, the LPL polypeptide
may exhibit a V.sub.max of about 0.01-50 mmoles/h/mg in a
[.sup.3H]-triolein liposome activity assay and/or a K.sub.m value
of about 0.01-1 .mu.M.
[0054] LPL polypeptides disclosed herein may be substantially
resistant to proteolytic cleavage (in particular, by proprotein
convertase), e.g. LPL R324A (SEQ ID NO: 2). The LPL variants
disclosed herein may have the advantage of increased stability when
administered, especially in a subject having AP, e.g., where AP
causes the release of a large amount of proteolytic enzymes from
the gut into the plasma.
[0055] LPL polypeptide can be inactivated by proteolytic cleavage
at the RAKR sequence located at amino acid positions 320-324 of SEQ
ID NO: 3 by a proprotein convertase (PC). Thus, a useful LPL
polypeptide for use in accordance with the present invention is one
that is resistant to proteolytic cleavage by PC. For example, such
a LPL polypeptide may comprise at least one point mutation that
replaces, modifies or deletes any of the amino acids at the RAKR
sequence located at amino acid positions 294-297 of SEQ ID NO: 1.
The LPL polypeptide may have an amino acid substitution at position
297 of SEQ ID NO: 1, e.g., replacement with an alanine residue
resulting in SEQ ID NO: 2 (referred to as LPL R324A herein). The
LPL polypeptide may comprise an amino acid sequence having 80% or
more identity (e.g. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.5%, 99.8% or more) to SEQ ID NO: 2. Preferably, the
LPL polypeptide comprises or consists of the amino acid sequence
recited in SEQ ID NO: 2.
[0056] In some cases, the LPL polypeptide does not comprise or
consist of the amino acid sequence recited in SEQ ID NO: 6, which
is a S447X variant of wild-type human LPL.
[0057] The invention also encompasses the use of LPL polypeptides
having single nucleotide polymorphisms (SNPs). For example, the
human LPL gene locus is highly polymorphic and contains many SNPs
in both coding and non-coding regions. For example, two SNPs in the
coding DNA (cSNPs) occur at high frequencies in the general human
population, and they concern point mutations in exon 2 and 6,
causing the substitution of an aspartic acid to an asparagine
residue at position 9 (D9N), and an asparagine to a serine residue
at position 291 (N291S), respectively.
[0058] The invention is not limited to human LPL sequences, but
rather encompasses such variants and homologs from other species
(e.g. guinea pig, mouse, rat, chicken, baboon, ox, sheep, pig and
fish), as well as non-natural variants. Standard search and
alignment techniques can be used to identify the homolog of any
particular LPL sequence. For example, cDNA clones for LPL have been
isolated and sequenced from a number of species, including human,
guinea pig, mouse, rat, chicken, baboon, ox, sheep, pig and fish.
Homology between the primary protein sequences of all the mammalian
species is in excess of 90% except in the case of the guinea pig,
where homology is in the region of 80% compared to other mammals.
Moreover, the available sequences from the LPL proteins can be used
to design primers for amplification of homologous sequences from
other species.
[0059] In general, suitable variants of a particular SEQ ID NO
include its allelic variants, its polymorphic forms, its homologs,
its orthologs, its paralogs, its mutants, etc.
[0060] Standard search and alignment techniques can be used to
identify the homolog of any particular sequence from LPL in public
sequence databases. Thus, for instance, LPL polypeptides used with
the invention may, compared to the SEQ ID NOs herein, include one
or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc) amino acid
substitutions, such as conservative substitutions (i.e.,
substitutions of one amino acid with another which has a related
side chain). Genetically-encoded amino acids are generally divided
into four families: (1) acidic i.e. aspartate, glutamate; (2) basic
i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar i.e. glycine, asparagine,
glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified jointly as
aromatic amino acids. In general, substitution of single amino
acids within these families does not have a major effect on the
biological activity. The LPL polypeptides may also include one or
more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) single amino acid
deletions relative to the SEQ ID NO sequences. The LPL polypeptides
may also include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
etc.) insertions (e.g., each of 1, 2, 3, 4 or 5 amino acids)
relative to the SEQ ID NO sequences.
[0061] Similarly, a LPL polypeptide used with the invention may
comprise an amino acid sequence that: [0062] (a) is identical (i.e.
100% identical) to a sequence disclosed in the sequence listing;
[0063] (b) shares sequence identity (e.g. 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a sequence
disclosed in the sequence listing; [0064] (c) has 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 (or more) single amino acid alterations (deletions,
insertions, substitutions), which may be at separate locations or
may be contiguous, as compared to the sequences of (a) or (b); and
[0065] (d) when aligned with a particular sequence from the
sequence listing using a pairwise alignment algorithm, each moving
window of x amino acids from N-terminus to C-terminus (such that
for an alignment that extends top amino acids, where p>x, there
are p-x+1 such windows) has at least xy identical aligned amino
acids, where: x is selected from 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 150, 200; y is selected from 0.50, 0.60, 0.70,
0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97,
0.98, 0.99; and if xy is not an integer then it is rounded up to
the nearest integer. The preferred pairwise alignment algorithm is
the Needleman-Wunsch global alignment algorithm, using default
parameters (e.g., with Gap opening penalty=10.0, and with Gap
extension penalty=0.5, using the EBLOSUM62 scoring matrix). This
algorithm is conveniently implemented in the needle tool in the
EMBOSS package. Within group (c), deletions or substitutions may be
at the N-terminus and/or C-terminus, or may be between the two
termini. Thus a truncation is an example of a deletion. Truncations
may involve deletion of up to 40 (or more) amino acids at the
N-terminus and/or C-terminus. N-terminus truncation can remove
leader peptides e.g., to facilitate recombinant expression in a
heterologous host. C-terminus truncation can remove anchor
sequences e.g., to facilitate recombinant expression in a
heterologous host. Preferably, the LPL polypeptide contains
alterations which substantially retain desirable properties (e.g.,
V.sub.max of about 0.01-50 mmoles/h/mg in a [.sup.3H]-triolein
liposome activity assay and/or a K.sub.m value of about 0.01-1
.mu.M).
[0066] In general, when a LPL polypeptide of the invention
comprises a sequence that is not identical to a complete sequence
from the sequence listing (e.g., when it comprises a sequence
listing with <100% sequence identity thereto, or when it
comprises a fragment thereof) it is preferred in each individual
instance that the LPL polypeptide is at least capable of
hydrolyzing plasma or serum TGs into a diacylglycerol and a
carboxylate (e.g., a free fatty acid and monoglyceride/glycerol).
Preferably, the LPL polypeptide is capable of hydrolyzing
triglycerides in lipoproteins, such as those found in chylomicrons
and very low-density lipoproteins (VLDL), into free fatty acids and
monoglyceride/glycerol molecules. Preferably, the LPL polypeptide
has similar enzyme kinetics as the known wild-type LPL. For
example, the LPL polypeptide may have a V.sub.max of about 0.01-50
mmoles/h/mg (e.g. when triolein liposome is used as a substrate)
and/or a K.sub.m value of about 0.01-1 .mu.M (e.g. when triolein
liposome is used as a substrate).
LPL Fragments
[0067] LPL is organized into two structurally distinct regions,
consisting of a larger amino-terminal domain (SEQ ID NO: 4, which
corresponds to residues 1-312 of SEQ ID NO: 1) and a smaller
carboxy-terminal end (SEQ ID NO: 5, which corresponds to residues
313-448 of SEQ ID NO: 1) connected by a flexible peptide. The
C-terminus is required for the binding to the lipoprotein
substrate, whereas the N-terminal domain is the region responsible
for catalysis.
[0068] Polypeptides used with the invention may omit either one of
the domains. Thus, a useful polypeptide may comprise an amino acid
sequence comprising a fragment of at least `n` consecutive amino
acids of SEQ ID NO: 1 (or variants thereof described herein),
wherein `n` is 50 or more (e.g. 55, 60, 70, 80, 90 or more), whilst
retaining at least the lipoprotein substrate-binding function or
the catalysis activity of LPL.
[0069] Preferred fragments comprise the catalysis active site in
the N-terminal domain of SEQ ID NO: 1. Preferably, the LPL
polypeptide comprises an amino acid sequence having 80% or more
identity (e.g. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99.5%, 99.8% or more) to the amino acid sequence recited in
SEQ ID NO: 4. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the N-terminus, while retaining at least the catalysis activity of
LPL.
[0070] Preferred fragments comprise the lipoprotein
substrate-binding site in the C-terminal domain of SEQ ID NO: 1.
Preferably, the LPL polypeptide comprises an amino acid sequence
having 80% or more identity (e.g. 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or more) to the amino acid
sequence recited in SEQ ID NO: 5. Other preferred fragments lack
one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25 or more) from the C-terminus, while retaining at least the
lipoprotein substrate-binding function of LPL.
Hybrid Polypeptides
[0071] The invention may use a `hybrid` polypeptide, which includes
at least two LPL polypeptides of the invention (i.e. a dimer)
expressed as a single polypeptide chain. An advantage of using a
hybrid polypeptide is that a LPL polypeptide of the invention may
be unstable or poorly expressed on its own, and this can be
assisted by adding a suitable hybrid partner that overcomes the
problem. For example, a hybrid polypeptide of the invention may
consist of two monomers, each monomer being a LPL polypeptide
comprising an amino acid sequence having 80% or more identity (e.g.
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%
or more) to any of the amino acid sequences recited in SEQ ID NOs:
1-6. The two monomers may or may not be the same. The dimer may
have a head-to-tail arrangement of the monomers. Alternatively, the
dimer may have a head-to-head arrangement of monomers.
[0072] A hybrid polypeptide may also consist of monomers and/or
oligomers of the fragments of LPL described above.
[0073] Hybrids consisting of amino acid sequences from different
organisms can be useful. For example, a hybrid polypeptide may
consist of the N-terminal domain of LPL from one species (e.g.
human) and amino acid sequences comprising the C-terminal domain of
LPL from another species (e.g. bovine).
[0074] Hybrid polypeptides can be represented by the formula
NH.sub.2-A-{-X-L-}.sub.n-B--COOH, wherein: X is an amino acid
sequence of a LPL polypeptide of the invention, as described above;
L is an optional linker amino acid sequence; A is an optional
N-terminal amino acid sequence; B is an optional C-terminal amino
acid sequence; n is an integer of 2 or more (e.g., 2, 3, 4, 5, 6,
etc.). Usually n is 2. The linker amino acid sequence L will
typically be short (e.g., 20 or fewer amino acids i.e. 20, 19, 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1).
Examples comprise short peptide sequences which facilitate cloning,
or poly-glycine linkers. Other suitable linker amino acid sequences
will be apparent to those skilled in the art.
[0075] The individual monomers within the hybrid (i.e. individual
--X-- moieties) may be from one or more species. Where n=2, for
instance, X.sub.2 may be from the same species as X.sub.1 or from a
different species. Where n=3, the species might be (i)
X.sub.1=X.sub.2=X.sub.3 (ii) X.sub.1=X.sub.2.noteq.X.sub.3 (iii)
X.sub.1.noteq.X.sub.2=X.sub.3 (iv)
X.sub.1.noteq.X.sub.2.noteq.X.sub.3 or (v)
X.sub.1=X.sub.3.noteq.X.sub.2, etc.
[0076] Different hybrid polypeptides may be mixed together in a
single formulation. The hybrid polypeptides can also be combined
with polypeptides of the invention as described above. Usefully,
these hybrid polypeptides are at least capable of hydrolyzing
plasma or serum TGs into a diacylglycerol and a carboxylate (e.g.,
a free fatty acid and monoglyceride/glycerol). Preferably, the
hybrid is capable of hydrolyzing triglycerides in lipoproteins,
such as those found in chylomicrons and very low-density
lipoproteins (VLDL), into free fatty acids and
monoglyceride/glycerol molecules. Preferably, the hybrid has
similar enzyme kinetics as the known wild-type LPL. For example,
the LPL polypeptide may have a V.sub.max of about 0.01-50
mmoles/h/mg (e.g. when triolein liposome is used as a substrate)
and/or a K.sub.m value of about 0.01-1 .mu.M (e.g. when triolein
liposome is used as a substrate).
LPL Polypeptides Used with the Invention
[0077] LPL polypeptides used with the invention can take various
forms (e.g. native, fusions, glycosylated, non-glycosylated,
lipidated, non-lipidated, phosphorylated, non-phosphorylated,
myristoylated, non-myristoylated, monomeric, multimeric,
particulate, denatured, etc.).
[0078] Preferably the invention uses LPL polypeptides that are
glycosylated. The mature LPL may contain approximately 0-15%
carbohydrate, and may have a molecular weight of about 50-60 kDa.
The human wild-type LPL sequence contains three consensus sites for
N-linked glycosylation (Asn-X-Ser, where X can be any amino acid)
that are located at Asn-43, Asn-257 and Asn-359 of SEQ ID NO: 1.
Experimental evidence suggests that N-linked glycosylation of LPL
is important for its catalytic activity. Thus, a LPL polypeptide of
the invention is preferably glycosylated. An exemplary glycosylated
polypeptide may have approximately 9% carbohydrate, and/or a
molecular weight of about 55 kDa.
[0079] In some embodiments, the LPL polypeptides used with the
invention may be non-glycosylated.
[0080] Polypeptides used with the invention are preferably human
polypeptides.
[0081] The LPL polypeptides used with the invention may be in the
form of an oligomer. For example, the LPL polypeptide may be in the
form of a dimer (e.g., homodimer), preferably a non-covalent dimer.
In some embodiments, the LPL polypeptide may be in the form of a
tetramer.
[0082] The term "polypeptide" refers to amino acid polymers of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified naturally or by intervention; for example, disulphide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labelling component. Also included are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), as well as other
modifications known in the art. Polypeptides can occur as single
chains or associated chains.
The invention provides polypeptides comprising a sequence --P-Q- or
-Q-P--, wherein: --P-- is an amino acid sequence as defined above
and -Q- is not a sequence as defined above i.e. the invention
provides fusion proteins. Where the N-terminus codon of --P-- is
not ATG, but this codon is not present at the N-terminus of a
polypeptide, it will be translated as the standard amino acid for
that codon rather than as a Met. Where this codon is at the
N-terminus of a polypeptide, however, it will be translated as Met.
Examples of -Q- moieties include, but are not limited to, histidine
tags (i.e. His.sub.n where n=3, 4, 5, 6, 7, 8, 9, 10 or more), one
or more myc tag (i.e. SEQ ID NO: 7: EQKLISEEDL), maltose-binding
protein, glutathione-S-transferase (GST).
[0083] In some embodiments, the present invention provides LPL
polypeptides in a fusion protein configuration. For example, a
suitable LPL polypeptide may be a fusion protein between an LPL
domain and another domain or moiety that typically can facilitate a
therapeutic effect of LPL by, for example, enhancing or increasing
stability, potency and/or delivery of LPL, or reducing or
eliminating immunogenicity, clearance, or toxicity. Such suitable
domains or moieties for an LPL fusion protein include but are not
limited to Fc domain, XTEN domain. In some embodiments, a suitable
recombinant LPL protein contains an Fc domain or a portion thereof
that binds to the FcRn receptor. As a non-limiting example, a
suitable Fc domain may be derived from an immunoglobulin subclass
such as IgG. In some embodiments, a suitable Fc domain is derived
from IgG1, IgG2, IgG3, or IgG4. In some embodiments, a suitable Fc
domain is derived from IgM, IgA, IgD, or IgE. Particularly suitable
Fc domains include those derived from human or humanized
antibodies. In some embodiments, a suitable Fc domain is a modified
Fc portion, such as a modified human Fc portion with improved
binding between Fc domain and the FcRn receptor resulting in
prolonged serum half-life.
Exemplary Amino Acid Sequences for LPL Polypeptides
TABLE-US-00001 [0084] SEQ ID NO: 1
ADQRRDFIDIESKFALRTPEDTAEDTCHLIPGVAESVATCHFNHSSKTFMVIHGWTVTGM
YESWVPKLVAALYKREPDSNVIVVDWLSRAQEHYPVSAGYTKLVGQDVARFINWMEE
EFNYPLDNVHLLGYSLGAHAAGIAGSLTNKKVNRITGLDPAGPNFEYAEAPSRLSPDDA
DFVDVLHTFTRGSPGRSIGIQKPVGHVDIYPNGGTFQPGCNIGEAIRVIAERGLGDVDQL
VKCSHERSIHLFIDSLLNEENPSKAYRCSSKEAFEKGLCLSCRKNRCNNLGYEINKVRAK
RSSKMYLKTRSQMPYKVFHYQVKIHFSGTESETHTNQAFEISLYGTVAESENIPFTLPEVS
TNKTYSFLIYTEVDIGELLMLKLKWKSDSYFSWSDWWSSPGFAIQKIRVKAGETQKKVI
FCSREKVSHLQKGKAPAVFVKCHDKSLNKKSG SEQ ID NO: 2
ADQRRDFIDIESKFALRTPEDTAEDTCHLIPGVAESVATCHFNHSSKTFMVIHGWTVTGM
YESWVPKLVAALYKREPDSNVIVVDWLSRAQEHYPVSAGYTKLVGQDVARFINWMEE
EFNYPLDNVHLLGYSLGAHAAGIAGSLTNKKVNRITGLDPAGPNFEYAEAPSRLSPDDA
DFVDVLHTFTRGSPGRSIGIQKPVGHVDIYPNGGTFQPGCNIGEAIRVIAERGLGDVDQL
VKCSHERSIHLFIDSLLNEENPSKAYRCSSKEAFEKGLCLSCRKNRCNNLGYEINKVRAK
ASSKMYLKTRSQMPYKVFHYQVKIHFSGTESETHTNQAFEISLYGTVAESENIPFTLPEV
STNKTYSFLIYTEVDIGELLMLKLKWKSDSYFSWSDWWSSPGFAIQKIRVKAGETQKKV
IFCSREKVSHLQKGKAPAVFVKCHDKSLNKKSG SEQ ID NO: 3
MESKALLVLTLAVWLQSLTASRGGVAAADQRRDFIDIESKFALRTPEDTAEDTCHLIPG
VAESVATCHFNHSSKTFMVIHGWTVTGMYESWVPKLVAALYKREPDSNVIVVDWLSR
AQEHYPVSAGYTKLVGQDVARFINWMEEEFNYPLDNVHLLGYSLGAHAAGIAGSLTNK
KVNRITGLDPAGPNFEYAEAPSRLSPDDADFVDVLHTFTRGSPGRSIGIQKPVGHVDIYP
NGGTFQPGCNIGEAIRVIAERGLGDVDQLVKCSHERSIHLFIDSLLNEENPSKAYRCSSKE
AFEKGLCLSCRKNRCNNLGYEINKVRAKRSSKMYLKTRSQMPYKVFHYQVKIHFSGTE
SETHTNQAFEISLYGTVAESENIPFTLPEVSTNKTYSFLIYTEVDIGELLMLKLKWKSDSY
FSWSDWWSSPGFAIQKIRVKAGETQKKVIFCSREKVSHLQKGKAPAVFVKCHDKSLNK KSG SEQ
ID NO: 4
ADQRRDFIDIESKFALRTPEDTAEDTCHLIPGVAESVATCHFNHSSKTFMVIHGWTVTGM
YESWVPKLVAALYKREPDSNVIVVDWLSRAQEHYPVSAGYTKLVGQDVARFINWMEE
EFNYPLDNVHLLGYSLGAHAAGIAGSLTNKKVNRITGLDPAGPNFEYAEAPSRLSPDDA
DFVDVLHTFTRGSPGRSIGIQKPVGHVDIYPNGGTFQPGCNIGEAIRVIAERGLGDVDQL
VKCSHERSIHLFIDSLLNEENPSKAYRCSSKEAFEKGLCLSCRKNRCNNLGYEINKVRAK
RSSKMYLKTRSQMPYK SEQ ID NO: 5
VFHYQVKIHFSGTESETHTNQAFEISLYGTVAESENIPFTLPEVSTNKTYSFLIYTEVDIGE
LLMLKLKWKSDSYFSWSDWWSSPGFAIQKIRVKAGETQKKVIFCSREKVSHLQKGKAP
AVFVKCHDKSLNKKSG SEQ ID NO: 6
ADQRRDFIDIESKFALRTPEDTAEDTCHLIPGVAESVATCHFNHSSKTFMVIHGWTVTGM
YESWVPKLVAALYKREPDSNVIVVDWLSRAQEHYPVSAGYTKLVGQDVARFINWMEE
EFNYPLDNVHLLGYSLGAHAAGIAGSLTNKKVNRITGLDPAGPNFEYAEAPSRLSPDDA
DFVDVLHTFTRGSPGRSIGIQKPVGHVDIYPNGGTFQPGCNIGEAIRVIAERGLGDVDQL
VKCSHERSIHLFIDSLLNEENPSKAYRCSSKEAFEKGLCLSCRKNRCNNLGYEINKVRAK
RSSKMYLKTRSQMPYKVFHYQVKIHFSGTESETHTNQAFEISLYGTVAESENIPFTLPEVS
TNKTYSFLIYTEVDIGELLMLKLKWKSDSYFSWSDWWSSPGFAIQKIRVKAGETQKKVI
FCSREKVSHLQKGKAPAVFVKCHDKSLNKK
Expression and Purification
[0085] LPL polypeptides used with the invention can be prepared by
various means (e.g. recombinant expression, purification from cell
culture, chemical synthesis, etc.). For example, the LPL
polypeptide may be expressed in the presence of dextran and
sulfate.
[0086] The expression of the LPL polypeptides of the invention
usually takes place in a mammalian host. The invention may use a
heterologous host for expression (recombinant expression). The
heterologous host may be prokaryotic (e.g., a bacterium) or
eukaryotic. Preferably, the heterologous host is eukaryotic, for
example, human cells (e.g. HT1080 cells, HEK293 cells), and hamster
cells (e.g. CHO cells and variants thereof). In some cases, the
heterologous host is a plant cell. In some cases, the heterologous
host is an insect cell.
[0087] Generally, LPL polypeptides of the invention are prepared by
recombinant expression. For example, a nucleic acid molecule (e.g.
a vector) encoding a LPL polypeptide of the invention may be stably
or transiently introduced into the cells to stably or transiently
express the LPL polypeptide. Preferably, the nucleic acid molecule
(e.g. a vector) encoding the LPL polypeptide is stably introduced
into the cells for stable expression of the LPL polypeptide.
[0088] The inventors have found that LMF1 plays a role in LPL
folding, dimer formation, and secretion, thereby giving a higher
titer in conditioned medium (see results of ELISA experiments shown
in FIG. 1). The LPL expression level from HT1080 cells increased
20-25 fold following co-expression with LMF1. This may promote
assembly and secretion of the LPL protein, thereby giving a
significantly higher yield. Thus, the invention preferably uses a
cell that expresses (e.g., prepared by recombinant expression) a
polypeptide of the invention and LMF1. The expression can be stable
or transient. Cells suitable for this can be prepared in various
ways.
[0089] For example, a method according to this aspect of the
invention involves transfecting a cell with nucleic acids encoding:
(a) a LPL polypeptide of the invention and (b) LMF1, to express the
LPL polypeptide and LMF1, respectively. The nucleic acids encoding
the LPL polypeptide of the invention and LMF1 may be present in the
same or different expression vectors. The nucleic acids encoding
(a) a LPL polypeptide of the invention and (b) LMF1 may be
transfected simultaneously or in series.
[0090] For example, such a method may involve: (i) transfecting a
cell with a nucleic acid molecule (e.g. a vector) encoding LMF1 to
express LMF1, and (ii) transfecting the cell with a nucleic acid
molecule (e.g. a vector) encoding a LPL polypeptide of the
invention to express that polypeptide. In one embodiment, step (i)
is carried out before (ii). In another embodiment, step (i) is
carried out after step (ii). In another embodiment, steps (i) and
(ii) are carried out simultaneously.
[0091] For example, such a method may involve transfecting a cell
with a nucleic acid molecule (e.g. a vector) encoding a LPL
polypeptide of the invention, wherein the cell stably expresses
LMF1. In this example, the cell may have been previously
transfected with a nucleic acid molecule (e.g. a vector) encoding
LMF1 to express LMF1.
[0092] Another method involves transfecting a cell with a nucleic
acid molecule (e.g. a vector) encoding LMF1, wherein the cell
stably expresses a LPL polypeptide of the invention. In this
example, the cell may have been previously transfected with a
nucleic acid molecule (e.g. a vector) encoding the LPL polypeptide
to express the LPL polypeptide.
[0093] Transient gene expression in cells can be carried out by
various techniques, including the calcium phosphate or calcium
chloride co-precipitation, lipofection, electroporation,
viral-mediated transfection methods etc. Suitable methods for
transfecting host cells can be found in common laboratory
manuals.
[0094] Techniques for stable gene expression in cells are also
known in the art. For example, a nucleic acid molecule encoding the
LPL polypeptide may be stably introduced into the cells (e.g. by
chromosomal integration). For example, the cells can be transfected
with expression vectors which may contain origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells may be allowed to grow for 1-2 days in an
enriched media before they are switched to selective media. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
that successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.
[0095] The invention also encompasses inducible gene expression
systems, e.g. doxycycline-inducible expression system.
[0096] LPL polypeptides of the invention can be chemically
synthesized using standard techniques. Automated peptide
synthesizers are commercially available (e.g. Advanced ChemTech
Model 396; Milligen/Biosearch 9600). Polypeptides may be dimerised
via a disulfide bridge formed by oxidation of the cysteins.
Following HPLC purification dimer formation may be verified, by
mass spectrometry.
[0097] LPL polypeptides used with the invention may be purified by
HPLC. The LPL polypeptides may be purified using a 2-column
purification process (for example, eButyl-Heparin) wherein the
capture column may be any hydrophobic interacting column (HIC)
(e.g., octyl, phenyl, butyl). The purification process may include
additional column for removing cell culture residuals such as host
cell proteins and host cell DNA.
[0098] The purified LPL polypeptides may be analyzed and assayed
for activity in accordance with standard methods. For example, the
purified product may be analyzed by mass spectrometry and
additional analytical methods including those capable of analyzing
glycan heterogeneity (e.g. 2-aminobenzamide hydrophilic-interaction
liquid chromatography (2AB/HILIC)), charge heterogeneity (e.g.
imaged capillary isoelectric focusing (iCE)), variation in the
multimeric state of the molecule (e.g. size exclusion (SE)-HPLC)
and product fragments (e.g. SDS-CE). LPL polypeptides of the
invention are preferably provided in purified or substantially
purified form i.e. substantially free from other polypeptides
(e.g., free from naturally-occurring polypeptides), particularly
from other host cell polypeptides, and are generally at least about
50% pure (by weight), and usually at least about 90% pure,
preferably about >95% pure. Hence, less than about 50%, and more
preferably less than about 10% (e.g., 5%) of a composition is made
up of other expressed polypeptides. Thus the LPL polypeptides of
the invention in the compositions are separated from the whole
organism with which the LPL polypeptide is expressed.
Pharmaceutical Compositions
[0099] Pharmaceutical compositions of the invention should be
pharmaceutically acceptable. Such compositions will usually include
components in addition to the antigens e.g., they typically include
one or more pharmaceutically acceptable carrier(s) and/or
excipient(s). A thorough discussion of such components is available
in reference Gennaro, 2000, Remington: The Science and Practice of
Pharmacy. 20th edition.
[0100] Depending on the route of administration, the peptides of
the invention may be coated in a material to protect said
ingredients from the action of enzymes, acids or other natural
conditions. Compositions will generally be in aqueous form.
Suitable compositions include aqueous solutions or dispersions and
powders for the extemporaneous preparation of solutions or
dispersion. The compositions are stable under the conditions of
manufacture and storage and are preserved against the contaminating
action of microorganisms such as bacteria and fungi. The aqueous
solutions are sterile and fluid to the extent that the solution may
readily be placed in a syringe.
[0101] The carrier may be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
monoglyceride/glycerol, propylene glycol, and polyethylene glycol,
and the like), suitable mixtures thereof, and vegetable oils. The
fluidity may 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
preventions of the action of microorganisms may 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 may be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0102] Solutions are prepared by incorporating the peptides of the
invention in the required amount in the appropriate solvent with
various of the other ingredients enumerated above, as required,
followed by filter sterilization.
[0103] Generally, dispersions are prepared by incorporating the
various polypeptides of the invention into vehicle which contains
the basic dispersion medium and the required other ingredients from
those enumerated above. Dispersions may also be prepared in
monoglyceride/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.
[0104] In the case of powders for the preparation of solutions, the
preferred methods of preparation are vacuum drying and the
freeze-drying (lyophilization) technique which yield a powder of
the peptides plus any additional desired ingredient from previously
sterile-filtered solution thereof. Thus the invention also provides
a lyophilizate of an aqueous composition of the invention. This is
prepared by lyophilizing an aqueous composition of the invention.
It can then be reconstituted with aqueous material to provide an
aqueous immunogenic composition of the invention. If the material
is reconstituted with a smaller volume of material than before
lyophilization then these materials will be present in more
concentrated form.
[0105] Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed.
[0106] A pharmaceutical composition comprising a polypeptide of the
invention preferably contains less than 10 ng (preferably less than
ing, and more preferably less than 100 pg) of residual host cell
DNA per dose, although trace amounts of host cell DNA may be
present.
[0107] The peptides of the invention may be incorporated into
sustained-release preparations and formulations.
[0108] The invention also provides a kit or composition of the
invention for use as a medicament in enzyme therapy.
Methods of Treatment
[0109] LPL polypeptides and compositions of the invention are
suitable for administration to subjects. Such LPL polypeptides and
compositions may be useful in treating hyperlipidemia and
hyperlipidemia-related conditions, including conditions that are
secondary to or exacerbated by hyperlipidemia, e.g.
hypertriglyceridemia (HTG), dyslipidemia, chlymicroemia,
hypercholesterolemia, dysbetalipoproteinemia, mixed
hyperlipoprotienemia and/or combined hyperlipidemia. For example,
the invention provides a LPL polypeptide or a composition for use
in treating HTG and its associated diseases, in a subject. The
HTG-associated diseases include diseases that are secondary to
and/or are exacerbated by HTG, e.g., AP, cardiovascular disease,
metabolic disorders, endocrine disorders, and fat embolism
syndrome.
[0110] Preferably, the invention provides a method of treating AP
in a subject, comprising the step of administering to the subject
an effective amount of a LPL polypeptide or a composition of the
invention. The invention also provides the use of a LPL polypeptide
or a composition of the invention in the preparation of a
medicament for the treatment of AP in a subject. The invention can
be used to treat AP at the time of onset of AP or at any point
during the course of the disease.
[0111] The subject may have hyperlipidemia, e.g.
hypertriglyceridemia (HTG), dyslipidemia, chlymicroemia,
hypercholesterolemia, dysbetalipoproteinemia, mixed
hyperlipoproteinemia and/or combined hyperlipidemia. Preferably,
the subject may have HTG.
[0112] The serum triglyceride level in the subject in need of
treatment may exceed about 150 mg/dl, about 200 mg/dl, about 300
mg/dl, about 400 mg/dl, about 500 mg/dl, about 600 mg/dl, about 600
mg/dl, about 700 mg/dl, about 800 mg/dl, about 900 mg/dl, or about
1000 mg/dl.
[0113] The AP may be secondary to or exacerbated by hyperlipidemia,
e.g. hypertriglyceridemia (HTG), dyslipidemia, chlymicroemia,
hypercholesterolemia, dysbetalipoproteinemia, mixed
hyperlipoprotienemia and/or combined hyperlipidemia. Preferably,
the AP is secondary to HTG. In other cases, the AP is exacerbated
by HTG.
[0114] The subject may have interstitial edematous AP or
necrotizing AP.
[0115] The subject may have mild, moderate or severe AP.
[0116] After administration of the LPL polypeptides of the
invention, the plasma TG level in the subject preferably decreases
below about 2000 mg/dl, about 1000 mg/dl, or about 500 mg/dl, or
about 150 mg/dl. In some cases, the plasma TG level in the subject
decreased by about 15%, about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80% or more, after administration of
the LPL polypeptides disclosed herein. Preferably, the decrease in
TG level may be observed after 1 hour, 6 hour, 12 hours, 1 day, or
1 week. Assessment of triglyceride levels in the plasma and serum
is well known in the art.
[0117] Preferably, administration of the LPL polypeptides of the
invention may lead to an improvement in AP or other diseases
disclosed herein. Potential endpoints that may be affected include
hospital length of stay, ICU length of stay, mortality rate,
incidence of complications, organ dysfunction, need for invasive
treatment, decrease in severity scores and/or markers of
inflammation, time to resolution of pain, cost of hospitalization,
relapses or recurrent AP, pancreatic insufficiency/glucose
intolerance, pain, clinician reported outcomes, quality of life
measures and other markers of clinical improvement.
Prophylactic Treatment
[0118] LPL polypeptides and compositions of the invention are also
useful in prophylactic treatment of hyperlipidemia and
hyperlipidemia-related conditions, including conditions that are
secondary to or exacerbated by hyperlipidemia, e.g.
hypertriglyceridemia (HTG), dyslipidemia, chlymicroemia,
hypercholesterolemia, dysbetalipoproteinemia, mixed
hyperlipoprotienemia and/or combined hyperlipidemia. Prophylactic
treatment refers to treatment before onset or reoccurrence of the
disease to prevent, inhibit or reduce the occurrence or
reoccurrence of the disease.
[0119] For example, the invention provides a LPL polypeptide or a
composition for use in preventing, inhibiting and/or reducing the
occurrence of HTG and its associated diseases, in a subject. The
HTG-associated diseases include diseases that are secondary to
and/or are exacerbated by HTG, e.g., AP, cardiovascular disease,
metabolic disorders, endocrine disorders, and fat embolism
syndrome.
[0120] AP patients following hospital discharge are typically
advised to maintain low TG levels. However, the patients have a
high recurrent AP rate (20-60%) and this suggests that the current
standard of care to maintain TG levels is ineffective. The
invention is also suited for prophylactic treatment of AP.
[0121] Thus, the invention also provides a LPL polypeptide (e.g.,
human wild-type LPL, or its variant) for use in the prevention,
inhibition and/or reduction in the occurrence or reoccurrence of AP
in a subject. The invention also provides a method of preventing,
inhibiting and/or reducing the occurrence or reoccurrence of AP in
a subject, comprising the step of administering to the mammal an
effective amount of the LPL polypeptide of the invention. The
invention also provides the use of the LPL polypeptide of the
invention in the preparation of a medicament for the prevention,
inhibition and/or reduction in the occurrence or reoccurrence of AP
in a subject.
[0122] Typically, the subject's plasma or serum triglyceride level
is maintained below about 150 mg/dl, below about 500 mg/dl, below
about 1000 mg/dl, or below about 2000 mg/dl.
[0123] For example, a subject at risk of AP who previously had an
attack of HTGAP can be prophylactically treated according to the
method of the present invention prior to the onset or recurrence of
AP.
[0124] The peptides of the invention are not suitable solely for
these groups, however, and may be used more generally in a
population.
Administration of LPL
[0125] The invention includes administering an effective amount of
a LPL polypeptide of the invention to the subject. An effective
amount is an amount sufficient to reduce, prevent, or inhibit the
effects of AP, or other diseases disclosed herein, in the
subject.
[0126] Preferably, the LPL is not administered in the form of a
nucleic acid, e.g., in a gene therapy vector (e.g., using an
adeno-associated virus). Preferably, the invention does not include
administering to a subject a nucleic acid (e.g., in a gene therapy
vector, such as using an adeno-associated virus) that encodes the
amino acid sequence recited in SEQ ID NO: 6 (LPL S447X).
[0127] In a preferred embodiment, the therapeutic amount ranges
from about 1 .mu.g to about 100 mg per kg of body weight per day.
In some embodiment, the LPL polypeptide variant, R324A, exhibits a
V.sub.max=153.+-.14 .mu.moles FA/min/mg and K.sub.m=0.28.+-.0.07
.mu.M in a [.sup.3H]-triolein liposome activity assay, which is
within the values reported in the literature for human LPL using
the same activity assay. LPL polypeptides used in accordance with
the invention preferably possess an activity with a V.sub.max of
0.01-50 mmoles FA/hr/mg and a K.sub.m of 0.01-1 uM.
[0128] The invention may use the LPL polypeptides at a
concentration of .ltoreq.8, .ltoreq.9, .ltoreq.10 or .ltoreq.11,
.ltoreq.12 mg/kg, most suitably, at a dose of .ltoreq.100 mg/kg, or
maximum feasible dose (MFD).
[0129] LPL polypeptides or compositions of the invention will be
administered directly to a patient. A practical advantage is that
the peptide may be administered in a convenient manner such as by
the oral, intravenous, intramuscular, intravenous, intranasal,
intradermal, subcutaneous, or suppository routes. The peptides of
the invention may also be administered parenterally or
intraperitoneally, or by other methods disclosed herein.
[0130] The administration route is preferably intravenous.
Preferably, a LPL polypeptide of the invention may be administered
at a dose of .ltoreq.100 mg/kg by intravenous route or MFD.
[0131] The administration is preferably subcutaneous. Preferably, a
LPL polypeptide of the invention may be administered at a
concentration of at a concentration of .ltoreq.100 mg/kg by
subcutaneous route or MFD.
[0132] The LPL polypeptides or compositions of the invention may be
administered by continuous infusion. The LPL polypeptides or
compositions of the invention may be administered by a single
injection (e.g., a bolus injection), or by multiple repeated
doses.
[0133] The dosage regimen may be adjusted to provide the optimum
therapeutic response. Dosage can be by a single dose schedule or a
multiple dose schedule. For example, several divided doses may be
administered daily. Multiple doses may be administered at least 1
week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks,
about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about
16 weeks, etc.). The dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation.
Preferably the subject is a mammal. Preferably the subject is a
human. Suitable examples of mammals other than humans include, for
example, rabbits, rats, mice, horses, minks, goats, or
primates.
General
[0134] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature.
[0135] "GI" numbering is used above. A GI number, or "GenInfo
Identifier", is a series of digits assigned consecutively to each
sequence record processed by NCBI when sequences are added to its
databases. The GI number bears no resemblance to the accession
number of the sequence record. When a sequence is updated (e.g.,
for correction, or to add more annotation or information) then it
receives a new GI number. Thus the sequence associated with a given
GI number is never changed.
[0136] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g., X+Y.
The word "substantially" does not exclude "completely" e.g., a
composition which is "substantially free" from Y may be completely
free from Y. Where necessary, the word "substantially" may be
omitted from the definition of the invention.
[0137] The term "about" in relation to a numerical value x means,
for example, x.+-.10%.
[0138] References to a percentage sequence identity between two
amino acid sequences means that, when aligned, that percentage of
amino acids are the same in comparing the two sequences. This
alignment and the percent homology or sequence identity can be
determined using software programs known in the art. A preferred
alignment is determined by the Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 2, BLOSUM matrix of 62.
[0139] Where a compound is administered to the body as part of a
composition then that compound may alternatively be replaced by a
suitable prodrug.
EXAMPLES AND EMBODIMENTS
LPL Expression and Purification
[0140] This experiment aimed to improve LPL titer. LPL was
expressed in either HT1080 cells or a stable HT1080 cell line that
co-expresses lipase maintaining factor 1 (LMF1) in the presence of
dextran sulfate. The LPL was then purified from the conditioned
medium using a 2-column purification process (Butyl-Heparin). A
human LPL variant, LPL R324A (SEQ ID NO: 2), was used in the
experiment.
[0141] The amount of LPL expressed from HT1080 cells co-expressing
LMF1 was measured by ELISA, and the results are shown in FIG. 1.
The titer of LPL R324A obtained using an HT1080 stable cell line
was approximately 1 mg/L. In contrast, the stable cell line that
co-expresses LMF1 provided >20-fold increase in LPL R324A titer,
giving a titer of about 25 mg/L.
[0142] Such results indicated that LMF1 can play a role in LPL
folding, dimer formation, and secretion, yielding a significantly
higher LPL titer in conditioned medium.
Specific Activity
[0143] The lipolytic activity of the LPL R324A was measured with
3[H]-triolein liposome as a substrate. The results are shown in
FIG. 2. The specific lipolytic activity of the LPL was 4-10
mmoles/hr/mg, which is within the values reported in the literature
for human LPL.
Enzymatic Properties
[0144] The enzymatic properties of LPL R324A LPL were measured and
the results are shown in FIG. 2 using a [.sup.3H]-triolein liposome
substrate. Following Michaelis-Menten kinetics (FIG. 3A), the
recombinant LPL has: Vmax=153.+-.14 .mu.moles FA/min/mg,
Km=0.28.+-.0.07 .mu.M and R.sup.2=0.98. Following Eadie-Hofstee
kinetics (FIG. 3B), the recombinant LPL has: Vmax=158.+-.14
.mu.moles FA/min/mg, Km=0.30.+-.0.04 .mu.M and R.sup.2=0.92.
LPL Activity Ex Vivo
[0145] This experiment aims to investigate whether exogenous LPL is
active in human plasma. This experiment also aims to investigate
whether LPL R324A remains active in plasma obtained from
individuals with HTGAP.
[0146] Also, AP causes the release of proteolytic enzymes from the
gut into the plasma, so it was unclear whether LPL would remain
active in the presence of the proteolytic enzymes. Plasma samples
were obtained from HTG patients with or without AP. Recombinant LPL
R324A was added to the samples, and the concentrations of TGs and
FFAs were measured over time. It was found that LPL R324A rapidly
hydrolyzed TGs with a concomitant increase in FFAs in both HTG and
HTGAP patients (FIGS. 3-4).
[0147] There is a dose-dependent hydrolysis of TGs as demonstrated
by the increase in FFA concentration following treatment with
increasing amounts of LPL (FIG. 5).
[0148] Additionally, LPL polypeptide is also capable of rapidly
hydrolyzing triglycerides in non-human primate (NHP) plasma with
HTG. This was demonstrated by incubating an isolated LPL
polypeptide variant R324A in plasma samples obtained from 11 HTG
NHPs (baseline TGs 1566.+-.352 mg/dL, range 352-5360 mg/dL) and
assessing changes in non-esterified fatty acid (NEFA)
concentrations. FIG. 6 shows time-courses of NEFA concentrations
following incubation of LPL for all 11 HTG NHPs. In all samples,
LPL led to a rapid hydrolysis of triglycerides as demonstrated by a
rapid increase in NEFA concentrations.
[0149] It was surprising that the exogenous LPL was active in human
plasma and that the LPL remained active in plasma from HTG patients
with or without AP. This therefore suggests that LPL could be
effective in lowering TG levels, e.g. by hydrolyzing triglycerides
in lipoproteins, such as those found in chylomicrons and very
low-density lipoproteins (VLDL), in AP patients.
LPL Activity In Vivo
[0150] LPL has been reported to have a short half-life (6-30 min),
so it was unclear whether exogenously administered LPL would be
sufficient to reduce TG levels in vivo.
[0151] The ob/ob mouse model was used for this study. LPL R324A was
administered IV at doses of 0.1 mg/kg, 0.5 mg/kg and 1 mg/kg. As
negative controls, mice were injected with the vehicle only (20 MM
TrisCl, pH8, 5 mM CaCl.sub.2, 30% glycerol, 1.5M NaCl).
[0152] Table 1 shows a >85% drop in serum triglyceride levels
(p<0.001) following IV administration of 1 mg/kg recombinant in
ob/ob mice. Despite the large drop in serum TGs, serum FFA
concentration remained unchanged. FFAs are rapidly transported to
target tissues by albumin and thus our findings are consistent with
the rapid clearance of FFAs as reported in the literature. Table 2
shows that TG hydrolysis in ob/ob mice is dose-dependent. Thus,
recombinant LPL has functional activity when administered in
vivo.
[0153] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
Sequence CWU 1
1
71448PRTArtificial Sequencechemically synthesized polypeptide 1Ala
Asp Gln Arg Arg Asp Phe Ile Asp Ile Glu Ser Lys Phe Ala Leu 1 5 10
15 Arg Thr Pro Glu Asp Thr Ala Glu Asp Thr Cys His Leu Ile Pro Gly
20 25 30 Val Ala Glu Ser Val Ala Thr Cys His Phe Asn His Ser Ser
Lys Thr 35 40 45 Phe Met Val Ile His Gly Trp Thr Val Thr Gly Met
Tyr Glu Ser Trp 50 55 60 Val Pro Lys Leu Val Ala Ala Leu Tyr Lys
Arg Glu Pro Asp Ser Asn 65 70 75 80 Val Ile Val Val Asp Trp Leu Ser
Arg Ala Gln Glu His Tyr Pro Val 85 90 95 Ser Ala Gly Tyr Thr Lys
Leu Val Gly Gln Asp Val Ala Arg Phe Ile 100 105 110 Asn Trp Met Glu
Glu Glu Phe Asn Tyr Pro Leu Asp Asn Val His Leu 115 120 125 Leu Gly
Tyr Ser Leu Gly Ala His Ala Ala Gly Ile Ala Gly Ser Leu 130 135 140
Thr Asn Lys Lys Val Asn Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro 145
150 155 160 Asn Phe Glu Tyr Ala Glu Ala Pro Ser Arg Leu Ser Pro Asp
Asp Ala 165 170 175 Asp Phe Val Asp Val Leu His Thr Phe Thr Arg Gly
Ser Pro Gly Arg 180 185 190 Ser Ile Gly Ile Gln Lys Pro Val Gly His
Val Asp Ile Tyr Pro Asn 195 200 205 Gly Gly Thr Phe Gln Pro Gly Cys
Asn Ile Gly Glu Ala Ile Arg Val 210 215 220 Ile Ala Glu Arg Gly Leu
Gly Asp Val Asp Gln Leu Val Lys Cys Ser 225 230 235 240 His Glu Arg
Ser Ile His Leu Phe Ile Asp Ser Leu Leu Asn Glu Glu 245 250 255 Asn
Pro Ser Lys Ala Tyr Arg Cys Ser Ser Lys Glu Ala Phe Glu Lys 260 265
270 Gly Leu Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Asn Leu Gly Tyr
275 280 285 Glu Ile Asn Lys Val Arg Ala Lys Arg Ser Ser Lys Met Tyr
Leu Lys 290 295 300 Thr Arg Ser Gln Met Pro Tyr Lys Val Phe His Tyr
Gln Val Lys Ile 305 310 315 320 His Phe Ser Gly Thr Glu Ser Glu Thr
His Thr Asn Gln Ala Phe Glu 325 330 335 Ile Ser Leu Tyr Gly Thr Val
Ala Glu Ser Glu Asn Ile Pro Phe Thr 340 345 350 Leu Pro Glu Val Ser
Thr Asn Lys Thr Tyr Ser Phe Leu Ile Tyr Thr 355 360 365 Glu Val Asp
Ile Gly Glu Leu Leu Met Leu Lys Leu Lys Trp Lys Ser 370 375 380 Asp
Ser Tyr Phe Ser Trp Ser Asp Trp Trp Ser Ser Pro Gly Phe Ala 385 390
395 400 Ile Gln Lys Ile Arg Val Lys Ala Gly Glu Thr Gln Lys Lys Val
Ile 405 410 415 Phe Cys Ser Arg Glu Lys Val Ser His Leu Gln Lys Gly
Lys Ala Pro 420 425 430 Ala Val Phe Val Lys Cys His Asp Lys Ser Leu
Asn Lys Lys Ser Gly 435 440 445 2 448PRTArtificial
Sequencechemically syntehsized polypeptide 2Ala Asp Gln Arg Arg Asp
Phe Ile Asp Ile Glu Ser Lys Phe Ala Leu 1 5 10 15 Arg Thr Pro Glu
Asp Thr Ala Glu Asp Thr Cys His Leu Ile Pro Gly 20 25 30 Val Ala
Glu Ser Val Ala Thr Cys His Phe Asn His Ser Ser Lys Thr 35 40 45
Phe Met Val Ile His Gly Trp Thr Val Thr Gly Met Tyr Glu Ser Trp 50
55 60 Val Pro Lys Leu Val Ala Ala Leu Tyr Lys Arg Glu Pro Asp Ser
Asn 65 70 75 80 Val Ile Val Val Asp Trp Leu Ser Arg Ala Gln Glu His
Tyr Pro Val 85 90 95 Ser Ala Gly Tyr Thr Lys Leu Val Gly Gln Asp
Val Ala Arg Phe Ile 100 105 110 Asn Trp Met Glu Glu Glu Phe Asn Tyr
Pro Leu Asp Asn Val His Leu 115 120 125 Leu Gly Tyr Ser Leu Gly Ala
His Ala Ala Gly Ile Ala Gly Ser Leu 130 135 140 Thr Asn Lys Lys Val
Asn Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro 145 150 155 160 Asn Phe
Glu Tyr Ala Glu Ala Pro Ser Arg Leu Ser Pro Asp Asp Ala 165 170 175
Asp Phe Val Asp Val Leu His Thr Phe Thr Arg Gly Ser Pro Gly Arg 180
185 190 Ser Ile Gly Ile Gln Lys Pro Val Gly His Val Asp Ile Tyr Pro
Asn 195 200 205 Gly Gly Thr Phe Gln Pro Gly Cys Asn Ile Gly Glu Ala
Ile Arg Val 210 215 220 Ile Ala Glu Arg Gly Leu Gly Asp Val Asp Gln
Leu Val Lys Cys Ser 225 230 235 240 His Glu Arg Ser Ile His Leu Phe
Ile Asp Ser Leu Leu Asn Glu Glu 245 250 255 Asn Pro Ser Lys Ala Tyr
Arg Cys Ser Ser Lys Glu Ala Phe Glu Lys 260 265 270 Gly Leu Cys Leu
Ser Cys Arg Lys Asn Arg Cys Asn Asn Leu Gly Tyr 275 280 285 Glu Ile
Asn Lys Val Arg Ala Lys Ala Ser Ser Lys Met Tyr Leu Lys 290 295 300
Thr Arg Ser Gln Met Pro Tyr Lys Val Phe His Tyr Gln Val Lys Ile 305
310 315 320 His Phe Ser Gly Thr Glu Ser Glu Thr His Thr Asn Gln Ala
Phe Glu 325 330 335 Ile Ser Leu Tyr Gly Thr Val Ala Glu Ser Glu Asn
Ile Pro Phe Thr 340 345 350 Leu Pro Glu Val Ser Thr Asn Lys Thr Tyr
Ser Phe Leu Ile Tyr Thr 355 360 365 Glu Val Asp Ile Gly Glu Leu Leu
Met Leu Lys Leu Lys Trp Lys Ser 370 375 380 Asp Ser Tyr Phe Ser Trp
Ser Asp Trp Trp Ser Ser Pro Gly Phe Ala 385 390 395 400 Ile Gln Lys
Ile Arg Val Lys Ala Gly Glu Thr Gln Lys Lys Val Ile 405 410 415 Phe
Cys Ser Arg Glu Lys Val Ser His Leu Gln Lys Gly Lys Ala Pro 420 425
430 Ala Val Phe Val Lys Cys His Asp Lys Ser Leu Asn Lys Lys Ser Gly
435 440 445 3 475PRTArtificial Sequencechemically synthesized
polypeptide 3Met Glu Ser Lys Ala Leu Leu Val Leu Thr Leu Ala Val
Trp Leu Gln 1 5 10 15 Ser Leu Thr Ala Ser Arg Gly Gly Val Ala Ala
Ala Asp Gln Arg Arg 20 25 30 Asp Phe Ile Asp Ile Glu Ser Lys Phe
Ala Leu Arg Thr Pro Glu Asp 35 40 45 Thr Ala Glu Asp Thr Cys His
Leu Ile Pro Gly Val Ala Glu Ser Val 50 55 60 Ala Thr Cys His Phe
Asn His Ser Ser Lys Thr Phe Met Val Ile His 65 70 75 80 Gly Trp Thr
Val Thr Gly Met Tyr Glu Ser Trp Val Pro Lys Leu Val 85 90 95 Ala
Ala Leu Tyr Lys Arg Glu Pro Asp Ser Asn Val Ile Val Val Asp 100 105
110 Trp Leu Ser Arg Ala Gln Glu His Tyr Pro Val Ser Ala Gly Tyr Thr
115 120 125 Lys Leu Val Gly Gln Asp Val Ala Arg Phe Ile Asn Trp Met
Glu Glu 130 135 140 Glu Phe Asn Tyr Pro Leu Asp Asn Val His Leu Leu
Gly Tyr Ser Leu 145 150 155 160 Gly Ala His Ala Ala Gly Ile Ala Gly
Ser Leu Thr Asn Lys Lys Val 165 170 175 Asn Arg Ile Thr Gly Leu Asp
Pro Ala Gly Pro Asn Phe Glu Tyr Ala 180 185 190 Glu Ala Pro Ser Arg
Leu Ser Pro Asp Asp Ala Asp Phe Val Asp Val 195 200 205 Leu His Thr
Phe Thr Arg Gly Ser Pro Gly Arg Ser Ile Gly Ile Gln 210 215 220 Lys
Pro Val Gly His Val Asp Ile Tyr Pro Asn Gly Gly Thr Phe Gln 225 230
235 240 Pro Gly Cys Asn Ile Gly Glu Ala Ile Arg Val Ile Ala Glu Arg
Gly 245 250 255 Leu Gly Asp Val Asp Gln Leu Val Lys Cys Ser His Glu
Arg Ser Ile 260 265 270 His Leu Phe Ile Asp Ser Leu Leu Asn Glu Glu
Asn Pro Ser Lys Ala 275 280 285 Tyr Arg Cys Ser Ser Lys Glu Ala Phe
Glu Lys Gly Leu Cys Leu Ser 290 295 300 Cys Arg Lys Asn Arg Cys Asn
Asn Leu Gly Tyr Glu Ile Asn Lys Val 305 310 315 320 Arg Ala Lys Arg
Ser Ser Lys Met Tyr Leu Lys Thr Arg Ser Gln Met 325 330 335 Pro Tyr
Lys Val Phe His Tyr Gln Val Lys Ile His Phe Ser Gly Thr 340 345 350
Glu Ser Glu Thr His Thr Asn Gln Ala Phe Glu Ile Ser Leu Tyr Gly 355
360 365 Thr Val Ala Glu Ser Glu Asn Ile Pro Phe Thr Leu Pro Glu Val
Ser 370 375 380 Thr Asn Lys Thr Tyr Ser Phe Leu Ile Tyr Thr Glu Val
Asp Ile Gly 385 390 395 400 Glu Leu Leu Met Leu Lys Leu Lys Trp Lys
Ser Asp Ser Tyr Phe Ser 405 410 415 Trp Ser Asp Trp Trp Ser Ser Pro
Gly Phe Ala Ile Gln Lys Ile Arg 420 425 430 Val Lys Ala Gly Glu Thr
Gln Lys Lys Val Ile Phe Cys Ser Arg Glu 435 440 445 Lys Val Ser His
Leu Gln Lys Gly Lys Ala Pro Ala Val Phe Val Lys 450 455 460 Cys His
Asp Lys Ser Leu Asn Lys Lys Ser Gly 465 470 475 4312PRTArtificial
Sequencechemically synthesized polypeptide 4Ala Asp Gln Arg Arg Asp
Phe Ile Asp Ile Glu Ser Lys Phe Ala Leu 1 5 10 15 Arg Thr Pro Glu
Asp Thr Ala Glu Asp Thr Cys His Leu Ile Pro Gly 20 25 30 Val Ala
Glu Ser Val Ala Thr Cys His Phe Asn His Ser Ser Lys Thr 35 40 45
Phe Met Val Ile His Gly Trp Thr Val Thr Gly Met Tyr Glu Ser Trp 50
55 60 Val Pro Lys Leu Val Ala Ala Leu Tyr Lys Arg Glu Pro Asp Ser
Asn 65 70 75 80 Val Ile Val Val Asp Trp Leu Ser Arg Ala Gln Glu His
Tyr Pro Val 85 90 95 Ser Ala Gly Tyr Thr Lys Leu Val Gly Gln Asp
Val Ala Arg Phe Ile 100 105 110 Asn Trp Met Glu Glu Glu Phe Asn Tyr
Pro Leu Asp Asn Val His Leu 115 120 125 Leu Gly Tyr Ser Leu Gly Ala
His Ala Ala Gly Ile Ala Gly Ser Leu 130 135 140 Thr Asn Lys Lys Val
Asn Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro 145 150 155 160 Asn Phe
Glu Tyr Ala Glu Ala Pro Ser Arg Leu Ser Pro Asp Asp Ala 165 170 175
Asp Phe Val Asp Val Leu His Thr Phe Thr Arg Gly Ser Pro Gly Arg 180
185 190 Ser Ile Gly Ile Gln Lys Pro Val Gly His Val Asp Ile Tyr Pro
Asn 195 200 205 Gly Gly Thr Phe Gln Pro Gly Cys Asn Ile Gly Glu Ala
Ile Arg Val 210 215 220 Ile Ala Glu Arg Gly Leu Gly Asp Val Asp Gln
Leu Val Lys Cys Ser 225 230 235 240 His Glu Arg Ser Ile His Leu Phe
Ile Asp Ser Leu Leu Asn Glu Glu 245 250 255 Asn Pro Ser Lys Ala Tyr
Arg Cys Ser Ser Lys Glu Ala Phe Glu Lys 260 265 270 Gly Leu Cys Leu
Ser Cys Arg Lys Asn Arg Cys Asn Asn Leu Gly Tyr 275 280 285 Glu Ile
Asn Lys Val Arg Ala Lys Arg Ser Ser Lys Met Tyr Leu Lys 290 295 300
Thr Arg Ser Gln Met Pro Tyr Lys 305 310 5136PRTArtificial
Sequencechemically synthesized polypeptide 5Val Phe His Tyr Gln Val
Lys Ile His Phe Ser Gly Thr Glu Ser Glu 1 5 10 15 Thr His Thr Asn
Gln Ala Phe Glu Ile Ser Leu Tyr Gly Thr Val Ala 20 25 30 Glu Ser
Glu Asn Ile Pro Phe Thr Leu Pro Glu Val Ser Thr Asn Lys 35 40 45
Thr Tyr Ser Phe Leu Ile Tyr Thr Glu Val Asp Ile Gly Glu Leu Leu 50
55 60 Met Leu Lys Leu Lys Trp Lys Ser Asp Ser Tyr Phe Ser Trp Ser
Asp 65 70 75 80 Trp Trp Ser Ser Pro Gly Phe Ala Ile Gln Lys Ile Arg
Val Lys Ala 85 90 95 Gly Glu Thr Gln Lys Lys Val Ile Phe Cys Ser
Arg Glu Lys Val Ser 100 105 110 His Leu Gln Lys Gly Lys Ala Pro Ala
Val Phe Val Lys Cys His Asp 115 120 125 Lys Ser Leu Asn Lys Lys Ser
Gly 130 135 6446PRTArtificial Sequencechemically synthesized
polypeptide 6Ala Asp Gln Arg Arg Asp Phe Ile Asp Ile Glu Ser Lys
Phe Ala Leu 1 5 10 15 Arg Thr Pro Glu Asp Thr Ala Glu Asp Thr Cys
His Leu Ile Pro Gly 20 25 30 Val Ala Glu Ser Val Ala Thr Cys His
Phe Asn His Ser Ser Lys Thr 35 40 45 Phe Met Val Ile His Gly Trp
Thr Val Thr Gly Met Tyr Glu Ser Trp 50 55 60 Val Pro Lys Leu Val
Ala Ala Leu Tyr Lys Arg Glu Pro Asp Ser Asn 65 70 75 80 Val Ile Val
Val Asp Trp Leu Ser Arg Ala Gln Glu His Tyr Pro Val 85 90 95 Ser
Ala Gly Tyr Thr Lys Leu Val Gly Gln Asp Val Ala Arg Phe Ile 100 105
110 Asn Trp Met Glu Glu Glu Phe Asn Tyr Pro Leu Asp Asn Val His Leu
115 120 125 Leu Gly Tyr Ser Leu Gly Ala His Ala Ala Gly Ile Ala Gly
Ser Leu 130 135 140 Thr Asn Lys Lys Val Asn Arg Ile Thr Gly Leu Asp
Pro Ala Gly Pro 145 150 155 160 Asn Phe Glu Tyr Ala Glu Ala Pro Ser
Arg Leu Ser Pro Asp Asp Ala 165 170 175 Asp Phe Val Asp Val Leu His
Thr Phe Thr Arg Gly Ser Pro Gly Arg 180 185 190 Ser Ile Gly Ile Gln
Lys Pro Val Gly His Val Asp Ile Tyr Pro Asn 195 200 205 Gly Gly Thr
Phe Gln Pro Gly Cys Asn Ile Gly Glu Ala Ile Arg Val 210 215 220 Ile
Ala Glu Arg Gly Leu Gly Asp Val Asp Gln Leu Val Lys Cys Ser 225 230
235 240 His Glu Arg Ser Ile His Leu Phe Ile Asp Ser Leu Leu Asn Glu
Glu 245 250 255 Asn Pro Ser Lys Ala Tyr Arg Cys Ser Ser Lys Glu Ala
Phe Glu Lys 260 265 270 Gly Leu Cys Leu Ser Cys Arg Lys Asn Arg Cys
Asn Asn Leu Gly Tyr 275 280 285 Glu Ile Asn Lys Val Arg Ala Lys Arg
Ser Ser Lys Met Tyr Leu Lys 290 295 300 Thr Arg Ser Gln Met Pro Tyr
Lys Val Phe His Tyr Gln Val Lys Ile 305 310 315 320 His Phe Ser Gly
Thr Glu Ser Glu Thr His Thr Asn Gln Ala Phe Glu 325 330 335 Ile Ser
Leu Tyr Gly Thr Val Ala Glu Ser Glu Asn Ile Pro Phe Thr 340 345 350
Leu Pro Glu Val Ser Thr Asn Lys Thr Tyr Ser Phe Leu Ile Tyr Thr 355
360 365 Glu Val Asp Ile Gly Glu Leu Leu Met Leu Lys Leu Lys Trp Lys
Ser 370 375 380 Asp Ser Tyr Phe Ser Trp Ser Asp Trp Trp Ser Ser Pro
Gly Phe Ala 385 390 395 400 Ile Gln Lys Ile Arg Val Lys Ala Gly Glu
Thr Gln Lys Lys Val Ile 405 410 415 Phe Cys Ser Arg Glu Lys Val Ser
His Leu Gln Lys Gly Lys Ala Pro 420 425 430 Ala Val Phe Val Lys Cys
His
Asp Lys Ser Leu Asn Lys Lys 435 440 445 710PRTArtificial
Sequencechemically synthesized polypeptide 7Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu 1 5 10
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