NZ734570B2 - Methods of treating or preventing cholesterol related disorders - Google Patents
Methods of treating or preventing cholesterol related disorders Download PDFInfo
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- NZ734570B2 NZ734570B2 NZ734570A NZ73457012A NZ734570B2 NZ 734570 B2 NZ734570 B2 NZ 734570B2 NZ 734570 A NZ734570 A NZ 734570A NZ 73457012 A NZ73457012 A NZ 73457012A NZ 734570 B2 NZ734570 B2 NZ 734570B2
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- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Abstract
Disclosed and claimed is a method of lowering serum LDL cholesterol in a patient comprising administering at least one anti-PCSK9 antibody to the patient in need thereof at a dose of about 105-280mg no more frequently than once per week, thereby lowering said serum LDL cholesterol level by at least about 15%. Wherein PCSK9 comprises the amino acids of SEQ ID NO 1. about 15%. Wherein PCSK9 comprises the amino acids of SEQ ID NO 1.
Description
METHODS OF TREATING OR PREVENTING CHOLESTEROL RELATED
DISORDERS
The present application is a divisional application of New Zealand Application
No. 717550, which is incorporated in its entirety herein by reference.
This application claims the benefit of U.S. Provisional Application No. 61/642,363
filed May 3, 2012, U.S. Provisional Application No. 61/614,417 filed March 22, 2012, U.S.
Provisional Application No. 61/595,526 filed February 6, 2012, U.S. Provisional Application
No. 61/562,303 filed November 21, 2011, U.S. Provisional Application No. 61/484,610 filed
May 10, 2011, all of which are incorporated by reference herein.
REFERENCE TO THE SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic
format. The Sequence Listing is provided as a file entitled AWO-
PCT_Sequence_Listing.txt created May 10, 2012 which is 315 KB in size. The information
in the electronic format of the Sequence Listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
The present invention relates to methods of treating or preventing cholesterol related
disorders, such as hypercholesterolemia, hyperlipidemia or dyslipidemia, using antigen
binding proteins, including antibodies, against proprotein convertase subtilisin/kexin type 9
(PCSK9). Pharmaceutical formulations and methods of producing said formulations are also
described.
BACKGROUND
Any discussion of the prior art throughout the specification should in no way be
considered as an admission that such prior art is widely known or forms part of common
general knowledge in the field.
“Cholesterol related disorders” (which include “serum cholesterol related disorders”)
include any one or more of the following: hypercholesterolemia, hperlipidemia, heart disease,
metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular diseases,
Alzheimer’s disease and generally dyslipidemias, which can be manifested, for example, by
an elevated total serum cholesterol, elevated LDL, elevated triglycerides, elevated VLDL,
and/or low HDL. Hypercholesterolemia is, in fact, an established risk factor for coronary
heart disease (CHD) in humans. Lowering of low-density lipoprotein cholesterol (LDL-C)
results in a reduction of cardiovascular risk and is a primary goal in pharmacotherapy for
CHD. Statins (hydroxymethylglutaryl coenzyme A [HMG CoA] reductase inhibitors) are
currently the treatment of choice for hypercholesterolemia. However, emerging data indicate
that more aggressive treatment of hypercholesterolemia is associated with lower risk for CHD
events. In addition, a subset of patients are intolerant to, or do not respond adequately to,
statin therapy. Thus, novel therapies that can be used alone or in combination with existing
agents to more effectively reduce LDL-C may be useful.
It is well established that recycling of the hepatic cell surface low-density lipoprotein
receptor (LDLR) plays a critical role in the maintenance of cellular and whole body
cholesterol balance by regulating plasma LDL-C levels. More recently it has been shown
that proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in the
recycling and regulation of LDLR. PCSK9 is a member of the subtilisin family of serine
proteases and is expressed predominantly in the liver. Following secretion, it causes post-
translational down regulation of hepatic cell surface LDLR by a mechanism that involves
direct binding to the LDLR. Down regulation of hepatic LDLR in turn leads to increased
levels of circulating LDL-C. Thus PCSK9 may represent a target for inhibition by novel
therapeutics in the setting of hypercholesterolemia. Strong rationale for such an approach is
available from studies in preclinical models and from findings that humans with PCSK9 loss-
of-function mutations have cholesterol levels lower than normal and reduced incidence of
CHD.
SUMMARY OF VARIOUS EMBODIMENTS
In some aspects of the invention, there is provided a stable formulation comprising at
least one monoclonal antibody that specifically binds to PCSK9, wherein PCSK9 comprises
the amino acids of SEQ ID NO 1, the monoclonal antibody in an amount of about 40 mg/ml
to about 300 mg/ml, and a pharmaceutically acceptable buffer in an amount of about .05 mM
to about 40 mM, and a pharmaceutically acceptable surfactant in an amount that is about
.01% w/v to about 20% w/v, and at least one pharmaceutically acceptable stabilizer of about
0.5% w/v to about 10% w/v, wherein the stable formulation has a pH of between about 4.0 to
about 6.0 is provided. In some embodiments the above stable formulation comprises a
pharmaceutically acceptable buffer chosen from thee group consisting of glutamate,
phosphate, phosphate buffered saline, sodium acetate, sodium citrate, and Tris buffer. In
particular embodiments the pharmaceutically acceptable buffer of the above stable
formulation is present in an amount of 10-20 mM. In a particular embodiment the
pharmaceutically acceptable buffer is sodium acetate in the amount of 10-20 mM. In some
embodiments, the pharmaceutically acceptable surfactant is present in an amount of about
0.004% w/v to about 0.01% w/v. In particular embodiments the pharmaceutically acceptable
surfactant of the above stable formulation is polysorbate 80 or polysorbate 20. In further
embodiments the pharmaceutically acceptable surfactant is polysorbate 80 or polysorbate 20
present in an amount of about 0.004% w/v to about 0.01% w/v.
In some embodiments the pharmaceutically acceptable stabilizer of the above stable
formulation is selected from the group consisting of a polyhydroxy hydrocarbon, a
disaccharide, a polyol, proline, arginine, lysine, methionine, taurine, and benzyl alcohol. In
some embodiments the pharmaceutically acceptable stabilizer is a polyhydroxy hydrocarbon
selected from the group consisting of sorbital, mannitol, and glycerol. In a particular
embodiment, the polyhydroxy hydrocarbon of the above stable formulation is sorbital. In
some embociments the pharmaceutically acceptable stabilizer is a disaccharide selected from
the group consisting of sucrose, maltose, lactose, fructose and trehelose. In some
embodiments disaccharide stabilizer is present in an amount of about 9% w/v. In some
embodiments, said disaccharide is sucrose. In particular embodiments the sucrose is present
in the above stable formulation in an amount of about 9% w/v. In some embodiments
stabilizer is an amino acid selected from the group consistin of proline, arginine, lysine,
methionine, and taurine. In a particular embodiment the stabilizer is proline. In a further
embodiment the proline is present in the above stable formulation in an amount of between
about 2% and 3% w/v. In some embodiments, the pH of the above stable formulation is
between about 5.0 to about 5.5.
In some embodiments the above stable formulation comprises a monoclonal antibody
comprises: a light chain variable region that comprises an amino acid sequence that is at least
90% identical to that of SEQ ID NO: 23 and a heavy chain variable region that comprises and
amino acid sequence that is at least 90% identical to that of SEQ ID NO:49; a light chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO: 12 and a heavy chain variable region that comprises an amino acid sequence
that is at least 90% identical to that of SEQ ID NO:67;a light chain variable region that
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 461
and a heavy chain variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:459;a light chain variable region that comprises an amino
acid sequence that is at least 90% identical to that of SEQ ID NO:465 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO:463, or a light chain variable region that comprises an amino acid sequence that
is at least 90% identical to that of SEQ ID NO: 485 and a heavy chain variable region that
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:483.
In some embodiments the above stable formulation includes a monoclonal antibody
that comprises:a light chain variable region that comprises the amino acid sequence SEQ ID
NO: 23 and a heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:49; a light chain variable region that comprises the amino acid sequence of SEQ ID NO:
12 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID
NO:67;a light chain variable region that comprises the amino acid sequence of SEQ ID NO:
461 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID
NO:459;a light chain variable region that comprises the amino acid sequence of SEQ ID
NO:465 and a heavy chain variable region that comprises the amino acid of SEQ ID
NO:463, or a light chain variable region that comprises the amino acid sequenceof SEQ ID
NO: 485 and a heavy chain variable region that comprises the amino acid sequence of SEQ
ID NO:483.
In some embodiments, the above stable formulation comprises the monoclonal
antibody 21B12, 31H4, 8A3, 11F1, or 8A1.
In some embodiments, the above stable formulation comprises a viscosity of 30 cP or
less at 25°C. In particular embodiments, the above stable formulation the monoclonal
antibody is present at about 70 mg/ml to about 150 mg/ml and the stable formulation
comprises a viscosity of 12 cP or less at 25°C. In some embodiments the above stable
formulation comprises an osmolality of between about 250 mOsmol/kg to about 350
mOsmol/kg. In some embodiments, the above stable formulation remains stable for at least
3, 6, 12 or 24 months.
In particular embodiments, the above stable formulation comprises monoclonal
antibody having a variable region that is at least 90% identical to that of SEQ ID NO:465
and a heavy chain variable region that is at least 90% identical to that of of SEQ ID NO:463.
In some embodiments the above stable formulation comprises a monoclonal antibody having
a light chain variable region comprising the amino acid sequence of SEQ ID NO:465 and a
heavy chain variable region comprising the amino acid sequence of SEQ ID NO:463 and
whererein the amount of the monoclonal antibody is about 150
In some embodiments, the above stable formulation comprises an antibody comprises
a light chain variable region that is at least 90% identical to that of SEQ ID NO:23 and a
heavy chain variable region that is at least 90% identical to that of of SEQ ID NO:49. In
some embodiments, the above stable formulation comprises a monoclonal antibody having a
light chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a
heavy chain variable region comprising the amino acid sequence of SEQ ID NO:49 and
whererein the amount of the monoclonal antibody is about 120 mg/ml or 140 mg/ml..
In particular embociments the above stable formulation, comprises (a) a monoclonal
antibody in an amount of about 70 mg/ml to about 200 mg/ml, said monoclonal antibody
comprising:a light chain variable region that comprises the amino acid sequence SEQ ID NO:
23 and a heavy chain variable region comprises the amino acid sequence of SEQ ID NO:49;
a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 12 and
a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:67;a
light chain variable region that comprises the amino acid sequence of SEQ ID NO: 461 and a
heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:459;a
light chain variable region that comprises the amino acid sequence of SEQ ID NO:465 and a
heavy chain variable region that comprises the amino acid of SEQ ID NO:463, ora light
chain variable region that comprises the amino acid sequenceof SEQ ID NO: 485 and a heavy
chain variable region that comprises the amino acid sequence of SEQ ID NO:483 and about
10 mM sodium acetate; about 9.0% w/v sucrose; about 0.004% to about 0.01% w/v
polysorbate 20 or polysorbate 80, anda pH of about 5.2.
In this aspect the mono clonal antibody may be 21B12, 8A3, 11F1. In particular
embodiments of this aspect, the monoclonal antibody is 21B12 and is present in the above
stabe formulation in an amount of about 140 mg/ml. In further embodiments of this aspect the
stable formulation of claims comprises about .004% polysorbate 20. In further particular
embodiments of this aspect the above stable formulation comprises the monoclonal antibody
is 8A3 which is present in an amount of about 150 mg/ml.
In furher embodiments of this aspect, the above stable formulation comprises the
monoclonal antibody is 11F1 in an amount of about 140, 150, 160. 170, 180, 190, or 200
mg/ml. In particular embodiments, the stable formulation comprising 11F1 also comprises
about 0.01% polysorbate 80.
In a further embodiment, the stable formulation, compries (a) a monoclonal antibody
in an amount of about 70 mg/ml to about 200 mg/ml, said monoclonal antibody comprising:a
light chain variable region that comprises the amino acid sequence SEQ ID NO: 23 and a
heavy chain variable region comprises the amino acid sequence of SEQ ID NO:49; a light
chain variable region that comprises the amino acid sequence of SEQ ID NO: 12 and a heavy
chain variable region that comprises the amino acid sequence of SEQ ID NO:67;a light chain
variable region that comprises the amino acid sequence of SEQ ID NO: 461 and a heavy
chain variable region that comprises the amino acid sequence of SEQ ID NO:459;a light
chain variable region that comprises the amino acid sequence of SEQ ID NO:465 and a heavy
chain variable region that comprises the amino acid of SEQ ID NO:463, or a light chain
variable region that comprises the amino acid sequenceof SEQ ID NO: 485 and a heavy chain
variable region that comprises the amino acid sequence of SEQ ID NO:483, and about 10
mM sodium acetate; between about 2.0% to 3.0% w/v proline; about about 0.01% w/v
polysorbate 20 or polysorbate 80, anda pH of about 5.0. In some embodiments of this aspect,
the stable formulation comprises the monoclonal antibody is 21B12, 8A3 or 11F1.
In another aspect of the invention, a stable formulation, comprising an anti-PCSK9
monoclonal antibody in an amount of about 70 mg/ml to about 200 mg/ml, said monoclonal
antibody comprising: a light chain variable region that comprises the amino acid sequence
having at least 90% identity to the sequence of SEQ ID NO: 577 and a heavy chain variable
region that comprises the amino acid sequence having at least 90% identity to the sequence of
SEQ ID NO: 576; a light chain variable region that comprises the amino acid sequence of
SEQ ID NO: 577 and a heavy chain variable region that comprises the amino acid sequence
of SEQ ID NO:576; a light chain variable region that comprises the amino acid sequence
having at least 90% identity to the sequence of SEQ ID NO: 588 and a heavy chain variable
region that comprises the amino acid sequence having at least 90% identity to the sequence of
SEQ ID NO: 589 or a light chain variable region that comprises the amino acid sequence of
SEQ ID NO: 588 and a heavy chain variable region that comprises the amino acid sequence
of SEQ ID NO:589; and (b) about 10 mM sodium acetate;(c) about 9.0% w/v sucrose; (d)
about 0.004% to about 0.01% w/v polysorbate 20 or polysorbate 80, and(e) a pH of about
.2.
In another aspect of the invention, a stable formulation, comprising an anti-PCSK9
monoclonal antibody in an amount of about 70 mg/ml to about 200 mg/ml, said monoclonal
antibody comprising: a light chain variable region that comprises the amino acid sequence
having at least 90% identity to the sequence of SEQ ID NO: 577 and a heavy chain variable
region that comprises the amino acid sequence having at least 90% identity to the sequence of
SEQ ID NO: 576; a light chain variable region that comprises the amino acid sequence of
SEQ ID NO: 577 and a heavy chain variable region that comprises the amino acid sequence
of SEQ ID NO:576 ; a light chain variable region that comprises the amino acid sequence
having at least 90% identity to the sequence of SEQ ID NO: 588 and a heavy chain variable
region that comprises the amino acid sequence having at least 90% identity to the sequence of
SEQ ID NO: 589 or a light chain variable region that comprises the amino acid sequence of
SEQ ID NO: 588 and a heavy chain variable region that comprises the amino acid sequence
of SEQ ID NO:589; and (b) about 10 mM sodium acetate;(c) between about 2.0% to 3.0%
w/v proline; (d) about 0.01% w/v polysorbate 20 or polysorbate 80, and (e) a pH of about
.0.
In some aspects, the invention provided comprises a method of lowering serum LDL
cholesterol in a patient comprising administering at least one anti-PCSK9 antibody to the
patient in need thereof at a dose of about 10 mg to about 3000 mg, thereby lowering said
serum LDL cholesterol level by at least about 15%, as compared to a predose level of serum
LDL cholesterol in the patient. In some embodiments of this aspect of the invention, the
serum LDL cholesterol level of said patient is lowered by at least about 20%, at least about
%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, or at least about 90% as compared to
a predose level of serum LDL cholesterol in the patient.
In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is
administered to a patient at a dose of about 35 mg to about 3000 mg, of about 35 mg to about
2800 mg, of about 35 mg to about 2500 mg, of about 35 mg to about 2000 mg, of about 35
mg to about 1800 mg, of about 35 mg to about 1400 mg, of about 25 mg to about 1200 mg, of
about 35 mg to about 1000 mg, of about 35 mg to about 700 mg, of about 45 mg to about 700
mg, of about 45 mg to about 600 mg, of about 45 mg to about 450 mg, of about 70 mg to
about 450 mg, of about 105 mg to about 420 mg, of about 120 mg to about 200 mg, of about
140 mg to about 200 mg, of about 140 mg to about 180 mg, or of about 140 mg to about 170
mg, of about 420 mg to about 3000 mg, of about 700 mg to about 3000 mg, of about 1000 mg
to about 3000 mg, of about 1200 to about 3000 mg, of about 1400 mg to about 3000 mg, of
about 1800 mg to about 3000 mg, of about 2000 mg to about 3000 mg, of about 2400 mg to
about 3000 mg, or about 2800 mg to about 3000 mg. In some embodiments of this aspect, the
anti-PCSK9 antibody is administered to a patient at a dose of about 35 mg, of about 45 mg, of
about 70 mg, of about 105 mg, of about 120 mg of about 140 mg, of about 150 mg, of about
160 mg, of about 170 mg, of about 180 mg, of about 190 mg, of about 200 mg, of about 210
mg, of about 280 mg, of about 360 mg, of about 420 mg, of about 450 mg, of about 600 mg,
of about 700 mg, of about 1200 mg, of about 1400 mg, of about 1800 mg, of about 2000 mg,
of about 2500 mg, of about 2800 mg, or about 3000 mg.
In some embodiments of this aspect of the invention the anti-PCSK9 antibody is
administered to a patient on a schedule selected from the group consisting of: (1) once a
week, (2) once every two weeks, (3) once a month, (4) once every other month, (5) once
every three months (6)once every six months and (7) once every twelve months. In some
embodiments of this aspect of the invention the anti-PCSK9 antibody is administered
parenterally. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody
is administered intravenously. In some embodiments of this aspect of the invention, the anti-
PCSK9 antibody is administered subcutaneously.
In some embodiments of this aspect of the invention the anti-PCSK9 antibody
comprises: a light chain variable region that comprises an amino acid sequence that is at least
90% identical to that of SEQ ID NO: 23 and a heavy chain variable region that comprises and
amino acid sequence that is at least 90% identical to that of SEQ ID NO:49; a light chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO: 12 and a heavy chain variable region that comprises an amino acid sequence
that is at least 90% identical to that of SEQ ID NO:67; a light chain variable region that
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 461
and a heavy chain variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:459; a light chain variable region that comprises an amino
acid sequence that is at least 90% identical to that of SEQ ID NO:465 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO:463; a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 485 and a heavy chain variable region that
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:483; or
a light chain variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO: 582 and a heavy chain variable region that comprises and
amino acid sequence that is at least 90% identical to that of SEQ ID NO:583. In some
embodiments of this aspect of the invention the anti-PCSK9 antibody comprises: a light chain
variable region that comprises an amino acid sequence, SEQ ID NO: 23, and a heavy chain
variable region that comprises and amino acid sequence, SEQ ID NO:49; a light chain
variable region that comprises an amino acid sequence, SEQ ID NO: 12, and a heavy chain
variable region that comprises an amino acid sequence, SEQ ID NO:67; a light chain variable
region that comprises amino acid sequence SEQ ID NO: 461 and a heavy chain variable
region that comprises amino acid sequence SEQ ID NO:459; a light chain variable region that
comprises the amino acid sequence of SEQ ID NO:465 and a heavy chain variable region that
comprises the amino acid sequence of SEQ ID NO:463; a light chain variable region that
comprises the amino acid sequence of SEQ ID NO: 485 and a heavy chain variable region
that comprises the amino acid sequence of SEQ ID NO:483; or a light chain variable region
that comprises an amino acid sequence, SEQ ID NO: 582, and a heavy chain variable region
that comprises and amino acid sequence, SEQ ID NO:583. In some embodiments of this
aspect of the invention the anti-PCSK9 antibody is selected from the group consisting of
21B12, 31H4, 8A3, 11F1 and 8A1.
In some aspects, the invention comprises a method of treating or preventing a
cholesterol related disorder in a patient having a serum LDL cholesterol level comprising
administering at least one anti-PCSK9 antibody to the patient in need thereof at thereof at a
dose of about 10 mg to about 3000 mg, thereby treating or preventing the cholesterol related
disorder in the patient. In an aspect of this embodiment, the cholesterol related disorder to be
treated or prevented is familial hypercholesterolemia, including heterozygous familial
hypercholesterolemia and homozygous familial hypercholesterolemia, non-familial
hypercholesterolemia, elevated lipoprotein (a), heart disease, metabolic syndrome, diabetes,
coronary heart disease, stroke, cardiovascular disease, Alzheimer’s disease, peripheral arterial
disease, hyperlipidemia or dyslipidemia. In some embodiments of this aspect, the serum LDL
cholesterol level of said patient is lowered by at least about 15%, at least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about
70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% as
compared to a predose level of serum LDL cholesterol in said patient.
In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is
administered to a patient at a dose of about 35 mg to about 3000 mg, of about 35 mg to about
2800 mg, of about 35 mg to about 2500 mg, of about 35 mg to about 2000 mg, of about 35
mg to about 1800 mg, of about 35 mg to about 1400 mg, of about 25 mg to about 1200 mg, of
about 35 mg to about 1000 mg, of about 35 mg to about 700 mg, of about 45 mg to about 700
mg, of about 45 mg to about 600 mg, of about 45 mg to about 450 mg, of about 70 mg to
about 450 mg, of about 105 mg to about 420 mg, of about 120 mg to about 200 mg, of about
140 mg to about 200 mg, of about 140 mg to about 180 mg, or of about 140 mg to about 170
mg, of about 420 mg to about 3000 mg, of about 700 mg to about 3000 mg, of about 1000 mg
to about 3000 mg, of about 1200 to about 3000 mg, of about 1400 mg to about 3000 mg, of
about 1800 mg to about 3000 mg, of about 2000 mg to about 3000 mg, of about 2400 mg to
about 3000 mg, or about 2800 mg to about 3000 mg. In some embodiments of this aspect,
the anti-PCSK9 antibody is administered to a patient at a dose of about 35 mg, of about 45
mg, of about 70 mg, of about 105 mg, of about 120 mg of about 140 mg, of about 150 mg, of
about 160 mg, of about 170 mg, of about 180 mg, of about 190 mg, of about 200 mg, of
about 210 mg, of about 280 mg, of about 360 mg, of about 420 mg, of about 450 mg, of
about 600 mg, of about 700 mg, of about 1200 mg, of about 1400 mg, of about 1800 mg, of
about 2000 mg, of about 2500 mg, of about 2800 mg, or about 3000 mg.
In some embodiments of this aspect of the invention the anti-PCSK9 antibody is
administered to a patient on a schedule selected from the group consisting of: (1) once a
week, (2) once every two weeks, (3) once a month, (4) once every other month, (5) once
every three months (6)once every six months and (7) once every twelve months. In some
embodiments of this aspect of the invention the anti-PCSK9 antibody is administered
parenterally. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody
is administered intravenously. In some embodiments of this aspect of the invention, the anti-
PCSK9 antibody is administered subcutaneously.
In some embodiments of this aspect of the invention the anti-PCSK9 antibody
comprises: a light chain variable region that comprises an amino acid sequence that is at least
90% identical to that of SEQ ID NO: 23 and a heavy chain variable region that comprises and
amino acid sequence that is at least 90% identical to that of SEQ ID NO:49; a light chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO: 12 and a heavy chain variable region that comprises an amino acid sequence
that is at least 90% identical to that of SEQ ID NO:67; a light chain variable region that
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 461
and a heavy chain variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:459; a light chain variable region that comprises an amino
acid sequence that is at least 90% identical to that of SEQ ID NO:465 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO:463; a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 485 and a heavy chain variable region that
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:483; or
a light chain variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO: 582 and a heavy chain variable region that comprises and
amino acid sequence that is at least 90% identical to that of SEQ ID NO:583. In some
embodiments of this aspect of the invention the anti-PCSK9 antibody comprises: a light chain
variable region that comprises an amino acid sequence, SEQ ID NO: 23, and a heavy chain
variable region that comprises and amino acid sequence, SEQ ID NO:49; a light chain
variable region that comprises an amino acid sequence, SEQ ID NO: 12, and a heavy chain
variable region that comprises an amino acid sequence, SEQ ID NO:67; a light chain variable
region that comprises amino acid sequence SEQ ID NO: 461 and a heavy chain variable
region that comprises amino acid sequence SEQ ID NO:459; a light chain variable region that
comprises the amino acid sequence of SEQ ID NO:465 and a heavy chain variable region that
comprises the amino acid sequence of SEQ ID NO:463; a light chain variable region that
comprises the amino acid sequence of SEQ ID NO: 485 and a heavy chain variable region
that comprises the amino acid sequence of SEQ ID NO:483; or a light chain variable region
that comprises an amino acid sequence, SEQ ID NO: 582, and a heavy chain variable region
that comprises and amino acid sequence, SEQ ID NO:583. In some embodiments of this
aspect of the invention the anti-PCSK9 antibody is selected from the group consisting of
21B12, 31H4, 8A3, 11F1 and 8A1.
In some embodiments of this aspect of the invention the anti-PCSK9 antibody is
administered to a patient on a schedule selected from the group consisting of: (1) once a
week, (2) once every two weeks, (3) once a month, (4) once every other month, (5) once
every three months (6) once every six months and (7) once every twelve months. In some
embodiments of this aspect of the invention the anti-PCSK9 antibody is administered
parenterally. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody
is administered intravenously. In some embodiments of this aspect of the invention, the anti-
PCSK9 antibody is administered subcutaneously.
In particular embodiments of the invention, the anti-PCSK9 antibody is 21B12 and
31H4. In some embodiments the anti-PCSK9 antibody comprises: a light chain variable
region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID
NO:23 and a heavy chain variable region that comprises an amino acid sequence that is at
least 90% identical to that of SEQ ID NO:49. In some embodiments the anti-PCSK9
antibody comprises: a light chain variable region that comprises the amino acid sequence of
SEQ ID NO:23 and a heavy chain variable region that comprises the amino acid sequence of
SEQ ID NO:49. In some embodiments, the anti-PCSK9 antibody is 21B12. In a particular
embodiment wherein the anti-PCSK9 antibody comprises an amino acid sequence that is at
least 90% identical to that of SEQ ID NO:23 and a heavy chain variable region that
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:49, or
comprises a light chain variable region that comprises the amino acid sequence of SEQ ID
NO:23 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID
NO:49, or is antibody is 21B12, the anti-PCSK9 antibody is administered to a patient at a
dose of about 21 mg to about 70 mg subcutaneously once a week, wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 3-10 days; is
administered to a patient at a dose of about 21 mg subcutaneously once a week, wherein the
serum LDL cholesterol level of the patient is lowered at least about 15-50% for about 3-10
days; is administered to a patient at a dose of about 35 mg subcutaneously once a week,
wherein the serum LDL cholesterol level of the patient is lowered at least about 15-50% for
about 3-10 days; is administered to a patient at a dose of about 70 mg subcutaneously once a
week, wherein the serum LDL cholesterol level of the patient is lowered at least about 15-
50% for about 3-10 days; is administered to a patient at a dose of about 70 mg to about 280
mg subcutaneously once every other week, wherein the serum LDL cholesterol level of the
patient is lowered at least about 15-50% for about 7-14 days; is administered to a patient at a
dose of about 70 mg subcutaneously once every other week, wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 7-14 days; is
administered to a patient at a dose of about 105 mg subcutaneously once every other week,
wherein the serum LDL cholesterol level of the patient is lowered at least about 15-50% for
about 7-14 days; is administered to a patient at a dose of about 120 mg subcutaneously once
every other week, wherein the serum LDL cholesterol level of the patient is lowered at least
about 15-50% for about 7-14 days; is administered to a patient at a dose of about 140 mg
subcutaneously once every other week, wherein the serum LDL cholesterol level of the
patient is lowered at least about 15-50% for about 7-14 days; is administered to a patient at a
dose of about 210 mg subcutaneously once every other week, wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 7-14 days; is
administered to a patient at a dose of about 280 mg subcutaneously once every other week,
wherein the serum LDL cholesterol level of the patient is lowered at least about 15-50% for
about 7-14 days; is administered to a patient at a dose of about 280 mg to about 420 mg
subcutaneously once every four weeks, wherein the serum LDL cholesterol level of the patent
is lowered at least about 15-50% for about 21-31 days; is administered to a patient at a dose
of about 280 mg subcutaneously once every four weeks, wherein the serum LDL cholesterol
level of the patient is lowered at least about 15-50% for about 21-31 days; is administered to
a patient at a dose of about 350 mg subcutaneously once every four weeks wherein the serum
LDL cholesterol level of the patient is lowered at least about 15-50% for about 21-31 days; is
administered to a patient at a dose of about 420 mg subcutaneously every four weeks,
wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 21-31
days.
In another particular embodiment, wherein the anti-PCSK9 antibody comprises an
amino acid sequence that is at least 90% identical to that of SEQ ID NO:23 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO:49, or comprises a light chain variable region that comprises the amino acid
sequence of SEQ ID NO:23 and a heavy chain variable region that comprises the amino acid
sequence of SEQ ID NO:49, or is antibody is 21B12, the anti-PCSK9 antibody is
administered to a patient at a dose of about 420 mg to about 3000 mg intraveneously every
week, wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 3-
days, is administered to a patient at a dose of about 700 mg intraveneously every week,
wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 3-10
days; is administered to a patient at a dose of about 1200 mg intraveneously every week,
wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 3-10
days; is administered to a patient at a dose of about greater than 1200 mg to about 3000 mg
intraveneously every week, wherein the serum LDL cholesterol level of the patient is lowered
-50% for about 3-10 days; is administered to a patient at a dose of about 420 mg to about
3000 mg intraveneously other week, wherein the serum LDL cholesterol level of the patient
is lowered 15-50% for about 7-14 days; is administered to a patient at a dose of about 700 mg
intraveneously every other week, wherein the serum LDL cholesterol level of the patient is
lowered 15-50% for about 7-14 days; is administered to a patient at a dose of about 1200 mg
intraveneously every other week, wherein the serum LDL cholesterol level of the patient is
lowered 15-50% for about 21-31 days; is administered to a patient at a dose of about greater
than 1200 mg to about 3000 mg intraveneously every other week, wherein the serum LDL
cholesterol level of the patient is lowered 15-50% for about 7-14 days; is administered to a
patient at a dose of about 420 mg to about 3000 mg intraveneously four weeks, wherein the
serum LDL cholesterol level of the patient is lowered 15-50% for about 21-31 days, is
administered to a patient at a dose of about 700 mg intraveneously every four weeks, wherein
the serum LDL cholesterol level of the patient is lowered 15-50% for about 21-31 days; is
administered to a patient at a dose of about 1200 mg intraveneously every four weeks,
wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 21-31
days; is administered to a patient at a dose of about greater than 1200 mg to about 3000 mg
intraveneously every four weeks, wherein the serum LDL cholesterol level of the patient is
lowered 15-50% for about 21-31 days.
In another particular embodiment wherein the anti-PCSK9 antibody comprises an
amino acid sequence that is at least 90% identical to that of SEQ ID NO:23 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO:49 or comprises a light chain variable region that comprises the amino acid
sequence of SEQ ID NO:23 and a heavy chain variable region that comprises the amino acid
sequence of SEQ ID NO:49 or is antibody is 21B12, the anti-PCSK9 antibody is
administered to a patient at a dose of about 21 mg subcutaneously once a week, wherein the
serum LDL cholesterol level of the patient is lowered at least about 30-50% for about 7-10
days; is administered to a patient at a dose of about 35 mg subcutaneously once a week,
wherein the serum LDL cholesterol level of the patient is lowered at least about 30-50% for
about 7-10 days; is administered to a patient at a dose of about 70 mg subcutaneously once a
week, wherein the serum LDL cholesterol level of the patient is lowered at least about 30-
50% for about 7-10 days; is administered to a patient at a dose of about 70 mg
subcutaneously once every other week wherein the serum LDL cholesterol level of the
patient is lowered at least about 30-50% for about 10-14 days; is administered to a patient at a
dose of about 105 mg subcutaneously once every other week, wherein the serum LDL
cholesterol level of the patient is lowered at least about 30-50% for about 10-14 days; is
administered to a patient at a dose of about 120 mg subcutaneously once every other week,
wherein the serum LDL cholesterol level of the patient is lowered at least about 30-50% for
about 10-14 days; is administered to a patient at a dose of about 140 mg subcutaneously once
every other week, wherein the serum LDL cholesterol level of the patient is lowered at least
about 30-50% for about 10-14 days; is administered to a patient at a dose of about 210 mg
subcutaneously once every other week, wherein the serum LDL cholesterol level of the
patient is lowered at least about 30-50% for about 10-14 days; is administered to a patient at a
dose of about 280 mg subcutaneously once every other week, wherein the serum LDL
cholesterol level of the patient is lowered at least about 30-50% for about 10-14 days; is
administered to a patient at a dose of about 280 mg to about 420 mg subcutaneously once
every four weeks, wherein the serum LDL cholesterol level of the patent is lowered at least
about 30-50% for about 24-28 days; is administered to a patient at a dose of about 280 mg
subcutaneously once every four weeks, wherein the serum LDL cholesterol level of the
patient is lowered at least about 30-50% for about 24-28 days; is administered to a patient at a
dose of about 350 mg subcutaneously once every four weeks wherein the serum LDL
cholesterol level of the patient is lowered at least about 30-50% for about 24-28 days; is
administered to a patient at a dose of about 420 mg subcutaneously every 4 weeks, wherein
the serum LDL cholesterol level of the patient is lowered 30-50% for about 24-28 days.
In another particular embodiment, wherein the anti-PCSK9 antibody comprises an
amino acid sequence that is at least 90% identical to that of SEQ ID NO:23 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90% identical to that of
SEQ ID NO:49, or comprises a light chain variable region that comprises the amino acid
sequence of SEQ ID NO:23 and a heavy chain variable region that comprises the amino acid
sequence of SEQ ID NO:49, or is antibody is 21B12, the anti-PCSK9 antibody is
administered to a patient at a dose of about 420 mg to about 3000 mg intraveneously every
week, wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 7-
days; is administered to a patient at a dose of about 700 mg intraveneously every week,
wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 7-10
days; is administered to a patient at a dose of about 1200 mg intraveneously every week,
wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 7-10
days; is administered to a patient at a dose of about greater than 1200 mg to about 3000 mg
intraveneously every week, wherein the serum LDL cholesterol level of the patient is lowered
30-50% for about 7-10 days; is administered to a patient at a dose of about 420 mg to about
3000 mg intraveneously other week, wherein the serum LDL cholesterol level of the patient
is lowered 30-50% for about 10-14 days; is administered to a patient at a dose of about 700
mg intraveneously every other week, wherein the serum LDL cholesterol level of the patient
is lowered 30-50% for about 10-14 days; is administered to a patient at a dose of about 1200
mg intraveneously every other week, wherein the serum LDL cholesterol level of the patient
is lowered 30-50% for about 10-14 days; is administered to a patient at a dose of about
greater than 1200 mg to about 3000 mg intraveneously every other week, wherein the serum
LDL cholesterol level of the patient is lowered 30-50% for about 10-14 days; is administered
to a patient at a dose of about 420 mg to about 3000 mg intraveneously four weeks, wherein
the serum LDL cholesterol level of the patient is lowered 30-50% for about 24-28 days, is
administered to a patient at a dose of about 700 mg intraveneously every four weeks, wherein
the serum LDL cholesterol level of the patient is lowered 30-50% for about 24-28 days; is
administered to a patient at a dose of about 1200 mg intraveneously every four weeks,
wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 24-28
days; is administered to a patient at a dose of about greater than 1200 mg to about 3000 mg
intraveneously every four weeks, wherein the serum LDL cholesterol level of the patient is
lowered 30-50% for about 24-28 days.
In particular embodiments of the invention, the anti-PCSK9 antibody is 8A3, 11F1 and
8A1. In some embodiments the anti-PCSK9 antibody comprises: a light chain variable
region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID
NO:465 and a heavy chain variable region that comprises an amino acid sequence that is at
least 90% identical to that of SEQ ID NO:463. In some embodiments the anti-PCSK9
antibody comprises: a light chain variable region that comprises the amino acid sequence of
SEQ ID NO:465 and a heavy chain variable region that comprises the amino acid sequence of
SEQ ID NO:463. In some embodiments the anti-PCSK9 antibody is 11F1. In a particular
embodiment, wherein the anti-PCSK9 antibody comprises an amino acid sequence that is at
least 90% identical to that of SEQ ID NO:465 and a heavy chain variable region that
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:463, or
comprises a light chain variable region that comprises the amino acid sequence of SEQ ID
NO:465 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID
NO:463, or is antibody is 11F1, the anti-PCSK9 antibody is administered to a patient at a
dose of about 45 mg subcutaneously once a week wherein the serum LDL cholesterol level of
the patient is lowered at least about 15-50% for about 3-10 days, is administered to a patient
at a dose of about 150 mg subcutaneously once every other week wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 7-14 days; is
administered to a patient at a dose of about 150 mg subcutaneously once every four weeks
wherein the serum LDL cholesterol level of the patent is lowered at least about 15-50% for
about 21-31 days; is administered to a patient at a dose of about greater than 150 mg to about
200 mg subcutaneously once every four weeks, wherein the serum LDL cholesterol level of
the patient is lowered at least about 15-50% for about 21-31 days; is administered to a patient
at a dose of about 170 mg to about 180 mg subcutaneously once every four weeks, wherein
the serum LDL cholesterol level of the patient is lowered at least about 15-50% for about 21-
31 days; is administered to a patient at a dose of about 150 mg to about 170 mg
subcutaneously once every four weeks, wherein the serum LDL cholesterol level of the
patient is lowered at least about 15-50% for about 21-31 days; is administered to a patient at a
dose of about 450 mg subcutaneously once every four weeks, wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 21-31 days; is
administered to a patient at a dose of about 150 mg subcutaneously once every six weeks
wherein the serum LDL cholesterol level of the patent is lowered at least about 15-50% for
about 31-42 days; is administered to a patient at a dose of about greater than 150 mg to about
200 mg subcutaneously once every six weeks, wherein the serum LDL cholesterol level of
the patient is lowered at least about 15-50% for about 31-42 days; is administered to a patient
at a dose of about 170 mg to about 180 mg subcutaneously once every six weeks wherein the
serum LDL cholesterol level of the patient is lowered at least about 15-50% for about 31-42
days; is administered to a patient at a dose of about 150 mg to about 170 mg subcutaneously
once every six weeks wherein the serum LDL cholesterol level of the patient is lowered at
least about 15-50% for about 31-42 days; is administered to a patient at a dose of about 450
mg subcutaneously once every six weeks wherein the serum LDL cholesterol level of the
patient is lowered at least about 15-50% for about 31-42 days; is administered to a patient at a
dose of about 140 mg to about 200 mg subcutaneously every 8 weeks wherein the serum
LDL cholesterol level of the patient is lowered 15-50% for about 45-56 days; is administered
to a patient at a dose of about 170 mg to about 180 mg subcutaneously every 8 weeks
wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 45-56
days; is administered to a patient at a dose of about 150 mg to about 170 mg subcutaneously
every 8 weeks wherein the serum LDL cholesterol level of the patient is lowered 15-50% for
about 45-56 days; is administered to a patient at a dose of about 450 mg subcutaneously
every 8 weeks wherein the serum LDL cholesterol level of the patient is lowered 15-50% for
about 45-56 days; at a dose of about 600 mg subcutaneously once every 8 weeks wherein
the serum LDL cholesterol level of the patient is lowered at least about 15-50% for about 45-
56 days; at a dose of about 700 mg subcutaneously once every 8 weeks wherein the serum
LDL cholesterol level of the patient is lowered at least about 15-50% for about 45-56 days; at
a dose of about 600 mg subcutaneously once every 12 weeks wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 74-84 days; at a
dose of about 700 mg subcutaneously once every 12 weeks wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 74-84 days; at a
dose of about 600 mg subcutaneously once every 16 weeks wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 100-112 days; at a
dose of about 700 mg subcutaneously once every 16 weeks wherein the serum LDL
cholesterol level of the patient is lowered at least about 15-50% for about 100 -112 days.
In particular embodiments of the invention wherein the anti-PCSK9 antibody
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:465
and a heavy chain variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:463 or comprises a light chain variable region that comprises
the amino acid sequence of SEQ ID NO:465 and a heavy chain variable region that comprises
the amino acid sequence of SEQ ID NO:463 or is antibody is 11F1, the anti-PCSK9 antibody
is administered to a patient at a dose of about 45 mg subcutaneously once a week wherein the
serum LDL cholesterol level of the patient is lowered at least about 30-50% for about 7-10
days, is administered to a patient at a dose of about 150 mg subcutaneously once every other
week wherein the serum LDL cholesterol level of the patient is lowered at least about 30-
50% for about 10-14 days; is administered to a patient at a dose of about 150 mg
subcutaneously once every four weeks wherein the serum LDL cholesterol level of the patent
is lowered at least about 30-50% for about 24-28 days; is administered to a patient at a dose
of about greater than 150 mg to about 200 mg subcutaneously once every four weeks,
wherein the serum LDL cholesterol level of the patient is lowered at least about 30-50% for
about 24-28 days; is administered to a patient at a dose of about 170 mg to about 180 mg
subcutaneously once every four weeks, wherein the serum LDL cholesterol level of the
patient is lowered at least about 30-50% for about 24-28 days; is administered to a patient at a
dose of about 150 mg to about 170 mg subcutaneously once every four weeks, wherein the
serum LDL cholesterol level of the patient is lowered at least about 30-50% for about 24-28
days; is administered to a patient at a dose of about 450 mg subcutaneously once every four
weeks, wherein the serum LDL cholesterol level of the patient is lowered at least about 30-
50% for about 24-28 days; is administered to a patient at a dose of about 150 mg
subcutaneously once every six weeks wherein the serum LDL cholesterol level of the patent
is lowered at least about 30-50% for about 40-41 days; is administered to a patient at a dose
of about greater than 150 mg to about 200 mg subcutaneously once every six weeks, wherein
the serum LDL cholesterol level of the patient is lowered at least about 30-50% for about 40-
41 days; is administered to a patient at a dose of about 170 mg to about 180 mg
subcutaneously once every six weeks wherein the serum LDL cholesterol level of the patient
is lowered at least about 30-50% for about 40-41 days; is administered to a patient at a dose
of about 150 mg to about 170 mg subcutaneously once every six weeks wherein the serum
LDL cholesterol level of the patient is lowered at least about 30-50% for about 40-41 days; is
administered to a patient at a dose of about 450 mg subcutaneously once every six weeks
wherein the serum LDL cholesterol level of the patient is lowered at least about 30-50% for
about 40-41 days; is administered to a patient at a dose of about 140 mg to about 200 mg
subcutaneously every 8 weeks wherein the serum LDL cholesterol level of the patient is
lowered 30-50% for about 50-56 days; is administered to a patient at a dose of about 170 mg
to about 180 mg subcutaneously every 8 weeks wherein the serum LDL cholesterol level of
the patient is lowered 30-50% for about 50-56 days; is administered to a patient at a dose of
about 150 mg to about 170 mg subcutaneously every 8 weeks wherein the serum LDL
cholesterol level of the patient is lowered 30-50% for about 50-56 days; is administered to a
patient at a dose of about 450 mg subcutaneously every 8 weeks wherein the serum LDL
cholesterol level of the patient is lowered 30-50% for about 50-56 days; at a dose of about
600 mg subcutaneously once every 8 weeks wherein the serum LDL cholesterol level of the
patient is lowered at least about 30-50% for about 50-56 days; at a dose of about 700 mg
subcutaneously once every 8 weeks wherein the serum LDL cholesterol level of the patient is
lowered at least about 30-50% for about 50-56 days; at a dose of about 600 mg
subcutaneously once every 12 weeks wherein the serum LDL cholesterol level of the patient
is lowered at least about 30-50% for about 80-84 days; at a dose of about 700 mg
subcutaneously once every 12 weeks wherein the serum LDL cholesterol level of the patient
is lowered at least about 30-50% for about 80-84 days; at a dose of about 600 mg
subcutaneously once every 16 weeks wherein the serum LDL cholesterol level of the patient
is lowered at least about 30-50% for about 105-112 days; at a dose of about 700 mg
subcutaneously once every 16 weeks wherein the serum LDL cholesterol level of the patient
is lowered at least about 30-50% for about 105 -112 days.
In particular embodiments of the invention wherein the anti-PCSK9 antibody
comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:465
and a heavy chain variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:463 or comprises a light chain variable region that comprises
the amino acid sequence of SEQ ID NO:465 and a heavy chain variable region that comprises
the amino acid sequence of SEQ ID NO:463 or is antibody is 11F1, the anti-PCSK9 antibody
is administered to a patient the anti-PCSK9 antibody is administered to a patient at a dose of
about 420 mg to about 3000 mg intraveneously every week, wherein the serum LDL
cholesterol level of the patient is lowered 30-50% for about 7-10 days; is administered to a
patient at a dose of about 700 mg intraveneously every week, wherein the serum LDL
cholesterol level of the patient is lowered 30-50% for about 7-10 days; is administered to a
patient at a dose of about 1200 mg intraveneously every week, wherein the serum LDL
cholesterol level of the patient is lowered 30-50% for about 7-10 days; is administered to a
patient at a dose of about greater than 1200 mg to about 3000 mg intraveneously every week,
wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 7-10
days; is administered to a patient at a dose of about 420 mg to about 3000 mg intraveneously
other week, wherein the serum LDL cholesterol level of the patient is lowered 30-50% for
about 10-14 days; is administered to a patient at a dose of about 700 mg intraveneously every
other week, wherein the serum LDL cholesterol level of the patient is lowered 30-50% for
about 10-14 days; is administered to a patient at a dose of about 1200 mg intraveneously
every other week, wherein the serum LDL cholesterol level of the patient is lowered 30-50%
for about 10-14 days; is administered to a patient at a dose of about greater than 1200 mg to
about 3000 mg intraveneously every other week, wherein the serum LDL cholesterol level of
the patient is lowered 30-50% for about 10-14 days; is administered to a patient at a dose of
about 420 mg to about 3000 mg intraveneously four weeks, wherein the serum LDL
cholesterol level of the patient is lowered 30-50% for about 24-28 days, is administered to a
patient at a dose of about 700 mg intraveneously every four weeks, wherein the serum LDL
cholesterol level of the patient is lowered 30-50% for about 24-28 days; is administered to a
patient at a dose of about 1200 mg intraveneously every four weeks, wherein the serum LDL
cholesterol level of the patient is lowered 30-50% for about 24-28 days; is administered to a
patient at a dose of about greater than 1200 mg to about 3000 mg intraveneously every four
weeks, wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about
24-28 days; is administered at a dose of about 1000 mg – 3000 mg intravenously once every
24 weeks wherein the serum LDL cholesterol level of the patient is lowered at least about 15-
50% for about 150 to 168 days; is administered at a dose of about 1000 mg – 3000 mg
intravenously once every 24 weeks wherein the serum LDL cholesterol level of the patient is
lowered at least about 30-50% for about 160 to 168 days; is administered at a dose of about
1000 mg – 3000 mg intravenously once every 52 weeks wherein the serum LDL cholesterol
level of the patient is lowered at least about 15-50% for about 350 to 365 days; is
administered at a dose of about 1000 mg – 3000 mg intravenously once every 52 weeks
wherein the serum LDL cholesterol level of the patient is lowered at least about 30-50% for
about 360 to 365 days.
In another aspect of the invention, the at least one anti-PCSK9 antibody is
administered to the patient before, after or concurrent with at least one other cholesterol-
lowering agent. Cholesterol lowering agents include statins, including, atorvastatin,
cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin,
simvastatin, nicotinic acid (niacin), slow relese niacin (SLO-NIACIN), laropiprant
(CORDAPTIVE), fibric acid (LOPID (Gemfibrozil), TRICOR (fenofibrate)), Bile acid
sequestrants, sucha as cholestyramine (QUESTRAN), colesvelam (WELCHOL),
COLESTID (colestipol)), cholesterol absorption inhibitor (ZETIA (ezetimibe)), lipid
modifying agents, PPAR gamma agonsits, PPAR alpha/gamma agonists, squalene synthase
inhibitors, CETP inhibitors, anti-hypertensives, anti-diabetic agents, including sulphonyl
ureas, insulin, GLP-1 analogs, DDPIV inhibitors, ApoB modulators, MTP inhibitoris and /or
arteriosclerosis obliterans treatments, oncostatin M, estrogen, berbine and therapeutic agents
for an immune-related disorder.
In some aspects, the invention comprises a method of lowering the serum LDL
cholesterol level in a patient. The method comprises administering to a patient in need
thereof a dose of about 10 mg to about 3000 mg of at least one anti-PCSK9 antibody
described herein. In some embodiments, the dose is about 10 mg to about 70 mg of at least
one anti-PCSK9 antibody administered once weekly (QW). In some embodiments, the dose
is about 14 mg to about 45 mg of at least one anti-PCSK9 antibody administered once
weekly. In some embodiments, the dose is about 14 mg to about 35 mg of at least one anti-
PCSK9 antibody administered once weekly. In some embodiments, the dose is about 70 mg
to about 420 mg of at least one anti-PCSK9 antibody administered once every 2 weeks
(Q2W). In some embodiments, the dose is about 70 mg to about 350 mg of at least one anti-
PCSK9 antibody administered once every 2 weeks (Q2W). In some embodiments, the dose
is about 105 mg to about 350 mg of at least one anti-PCSK9 antibody administered once
every 2 weeks (Q2W). In some embodiments, the dose is about 140 mg to about 280 mg of
at least one anti-PCSK9 antibody administered once every 2 weeks (Q2W). In some
embodiments, the dose is about 250 mg to about 480 mg of at least one anti-PCSK9 antibody
administered once every 4 weeks (Q4W). In some embodiments, the dose is about 280 mg to
about 420 mg of at least one anti-PCSK9 antibody administered once every 4 weeks (Q4W).
In some embodiments, the dose is about 350 mg to about 420 mg of at least one anti-PCSK9
antibody administered once every 4 weeks (Q4W). In some embodiments, the dose is about
420 mg to about 3000 mg of at least one anti-PCSK9 antibody administered once every week
(QW). In some embodiments, the dose is about 1000 mg to about 3000 mg of at least one
anti-PCSK9 antibody administered once every week (QW). In some embodiments, the dose
is about 2000 mg to about 3000 mg of at least one anti-PCSK9 antibody administered once
every week (QW). In some embodiments, the dose is about 420 mg to about 3000 mg of at
least one anti-PCSK9 antibody administered once every other week (Q2W). In some
embodiments, the dose is about 1000 mg to about 3000 mg of at least one anti-PCSK9
antibody administered once every other week (Q2W). In some embodiments, the dose is
about 2000 mg to about 3000 mg of at least one anti-PCSK9 antibody administered once
every other week (Q2W). In some embodiments, the dose is about 420 mg to about 3000 mg
of at least one anti-PCSK9 antibody administered once every month (Q4W). In some
embodiments, the dose is about 1000 mg to about 3000 mg of at least one anti-PCSK9
antibody administered once every month (Q4W). In some embodiments, the dose is about
2000 mg to about 3000 mg of at least one anti-PCSK9 antibody administered once every
month (Q4W). In some embodiments, the serum LDL cholesterol level is reduced by at least
about 15% as compared to a predose serum LDL cholesterol level. In some embodiments,
the serum LDL cholesterol level is reduced by at least about 20%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 25%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 30%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 35%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 40%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 45%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 50%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 55%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 60%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 70%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 80%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 85%. %. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 90%.
In some aspects, the invention comprises a method of lowering the serum LDL
cholesterol level in a patient, the method comprising administering to a patient in need
thereof, a dose of at least one anti-PCSK9 antibody, and wherein the dose of anti-PCSK9
antibody is administered on a schedule selected from the group consisting of: (1) at least
about 14 mg every week (QW); (2) at least an amount of about 35 mg every week (QW); (3)
at least an amount of about 45 mg every week (QW); (4) at least an amount of about 70 mg
every other week (Q2W); (5) at least an amount of about 105 mg every two weeks or every
other week (Q2W); (6) at least an amount of about 140 mg every two weeks or every other
week (Q2W); (7) at least an amount of about 150 mg every two weeks or every other week
(Q2W) (8) at least an amount of about 280 mg every two weeks or every other week (Q2W);
and (9) at least an amount of about 150 mg every four weeks (Q4W); (10) at least an amount
of about 160 mg every four weeks (Q4W); (11) at least an amount of about 170 mg every
four weeks (Q4W); (12) at least an amount of about 180 mg every four weeks (Q4W); (13)
at least an amount of about 190 mg every four weeks (Q4W); (14) at least an amount of about
200 mg every four weeks (Q4W); (15) at least an amount of about 280 mg every four weeks
(Q4W); (16) at least an amount of about 350 every four weeks (Q4W); (17) at least an
amount of about 420 mg every four weeks (Q4W); (18) at least an amount of about 1000 mg
every four weeks (Q4W); (19) at least an amount of about 2000 mg every four weeks (Q4W);
and (20) at least an amount of about 3000 mg every four weeks (Q4W). In some
embodiments, the serum LDL cholesterol level is reduced by at least about 15% as compared
to a predose serum LDL cholesterol level. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 20%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 25%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 30%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 35%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 40%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 45%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 50%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 55%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 60%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 65%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 70%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 75%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 80%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 85%. In some embodiments, the serum LDL cholesterol
level is reduced by at least about 90%.
In some aspects, the invention comprises a method of lowering PCSK9 values in a
patient, the method comprising administering to a patient in need thereof, a dose of at least
one anti-PCSK9 antibody, and wherein the dose of anti-PCSK9 antibody is administered on a
schedule selected from the group consisting of: (1) at least about 14 mg every week (QW);
(2) at least an amount of about 35 mg every week (QW); (3) at least an amount of about 45
mg every week (QW); (4) at least an amount of about 70 mg every other week (Q2W); (5) at
least an amount of about 105 mg every two weeks (Q2W); (6) at least an amount of about
140 mg every other week (Q2W); (7) at least an amount of about 150 mg every two weeks or
every other week (Q2W); (8) at least an amount of about 280 mg every two weeks or every
other week (Q2W); (9) at least an amount of about 150 mg every four weeks (Q4W); (10) at
least an amount of about 160 mg every four weeks (Q4W); (11) at least an amount of about
170 mg every four weeks (Q4W); (12) at least an amount of about 180 mg every four weeks
(Q4W); (13) at least an amount of about 190 mg every four weeks (Q4W); (14) at least an
amount of about 200 mg every four weeks (Q4W); (15) at least an amount of about 280 mg
every four weeks (Q4W); (16) at least an amount of about 350 every four weeks (Q4W); (17)
at least an amount of about 420 mg every four weeks (Q4W); (18) at least an amount of about
1000 mg every four weeks (Q4W); (19) at least an amount of about 2000 mg every four
weeks (Q4W); and (20) at least an amount of about 3000 mg every four weeks (Q4W). In
some embodiments, the serum PCSK9 value is reduced by at least about 60% as compared to
a predose serum PCSK9 value. In some embodiments, the serum PCSK9 value is reduced by
at least about 65%. In some embodiments, the serum PCSK9 value is reduced by at least
about 70%. In some embodiments, the serum PCSK9 value is reduced by at least about 75%.
In some embodiments, the serum PCSK9 value is reduced by at least about 80%. In some
embodiments, the serum PCSK9 value is reduced by at least about 85%. In some
embodiments, the serum PCSK9 value is reduced by at least about 90%.
In some aspects, the invention comprises a method of lowering the total cholesterol
level in a patient, the method comprising administering to a patient in need thereof, a dose of
at least one anti-PCSK9 antibody, and wherein the dose of anti-PCSK9 antibody is
administered on a schedule selected from the group consisting of: (1) at least about 14 mg
every week (QW); (2) at least an amount of about 35 mg every week (QW); (3) at least an
amount of about 45 mg every week (QW); (4) at least an amount of about 70 mg every other
week (Q2W); (5) at least an amount of about 105 mg every two weeks (Q2W); (6) at least an
amount of about 140 mg every other week (Q2W); (7) at least an amount of about 150 mg
every two weeks or every other week (Q2W); (8) at least an amount of about 280 mg every
two weeks or every other week (Q2W); (9) at least an amount of about 150 mg every four
weeks (Q4W); (10) at least an amount of about 160 mg every four weeks (Q4W); (11) at
least an amount of about 170 mg every four weeks (Q4W); (12) at least an amount of about
180 mg every four weeks (Q4W); (13) at least an amount of about 190 mg every four weeks
(Q4W); (14) at least an amount of about 200 mg every four weeks (Q4W); (15) at least an
amount of about 280 mg every four weeks (Q4W); (16) at least an amount of about 350 every
four weeks (Q4W); (17) at least an amount of about 420 mg every four weeks (Q4W); (18) at
least an amount of about 1000 mg every four weeks (Q4W); (19) at least an amount of about
2000 mg every four weeks (Q4W); and (20) at least an amount of about 3000 mg every four
weeks (Q4W). In some embodiments, the total cholesterol level is reduced by at least about
% as compared to a predose total cholesterol level. In some embodiments, the total
cholesterol level is reduced by at least about 25%. In some embodiments, the total
cholesterol level is reduced by at least about 30%. In some embodiments, the total
cholesterol level is reduced by at least about 35%. In some embodiments, the total
cholesterol level is reduced by at least about 40%. In some embodiments, the total
cholesterol level is reduced by at least about 45%. In some embodiments, the total
cholesterol level is reduced by at least about 50%. In some embodiments, the total
cholesterol level is reduced by at least about 55%. In some embodiments, the total
cholesterol level is reduced by at least about 60%.
In some aspects, the invention comprises a method of lowering the non-HDL
cholesterol level in a patient, the method comprising administering to a patient in need
thereof, a dose of at least one anti-PCSK9 antibody, and wherein the dose of anti-PCSK9
antibody is administered on a schedule selected from the group consisting of: (1) at least
about 14 mg every week (QW); (2) at least an amount of about 35 mg every week (QW); (3)
at least an amount of about 45 mg every week (QW); (4) at least an amount of about 70 mg
every other week (Q2W); (5) at least an amount of about 105 mg every two weeks (Q2W);
(6) at least an amount of about 140 mg every other week (Q2W); (7) at least an amount of
about 150 mg every two weeks or every other week (Q2W); (8) at least an amount of about
280 mg every two weeks or every other week (Q2W); (9) at least an amount of about 150 mg
every four weeks (Q4W); (10) at least an amount of about 160 mg every four weeks (Q4W);
(11) at least an amount of about 170 mg every four weeks (Q4W); (12) at least an amount of
about 180 mg every four weeks (Q4W); (13) at least an amount of about 190 mg every four
weeks (Q4W); (14) at least an amount of about 200 mg every four weeks (Q4W); (15) at least
an amount of about 280 mg every four weeks (Q4W); (16) at least an amount of about 350
every four weeks (Q4W); (17) at least an amount of about 420 mg every four weeks (Q4W);
(18) at least an amount of about 1000 mg every four weeks (Q4W); (19) at least an amount of
about 2000 mg every four weeks (Q4W); and (20) at least an amount of about 3000 mg every
four weeks (Q4W). In some embodiments, the non-HDL cholesterol level is reduced by at
least about 30% as compared to a predose no-HDL cholesterol level. In some embodiments,
the non-HDL cholesterol level is reduced by at least about 35%. In some embodiments, the
non-HDL cholesterol level is reduced by at least about 40%. In some embodiments, the non-
HDL cholesterol level is reduced by at least about 45%. In some embodiments, the non-
HDL cholesterol level is reduced by at least about 50%. In some embodiments, the non-HDL
cholesterol level is reduced by at least about 55%. In some embodiments, the non-HDL
cholesterol level is reduced by at least about 60%. In some embodiments, the non-HDL
cholesterol level is reduced by at least about 65%. In some embodiments, the non-HDL
cholesterol level is reduced by at least about 70%. In some embodiments, the non-HDL
cholesterol level is reduced by at least about 75%. In some embodiments, the non-HDL
cholesterol level is reduced by at least about 80%. In some embodiments, the non-HDL
cholesterol level is reduced by at least about 85%.
In some aspects, the invention comprises a method of lowering ApoB levels in a
patient, the method comprising administering to a patient in need thereof, a dose of at least
one anti-PCSK9 antibody, and wherein the dose of anti-PCSK9 antibody is administered on a
schedule selected from the group consisting of: (1) at least about 14 mg every week (QW);
(2) at least an amount of about 35 mg every week (QW); (3) at least an amount of about 45
mg every week (QW); (4) at least an amount of about 70 mg every other week (Q2W); (5) at
least an amount of about 105 mg every two weeks (Q2W); (6) at least an amount of about
140 mg every other week (Q2W); (7) at least an amount of about 150 mg every two weeks or
every other week (Q2W); (8) at least an amount of about 280 mg every two weeks or every
other week (Q2W); (9) at least an amount of about 150 mg every four weeks (Q4W); (10) at
least an amount of about 160 mg every four weeks (Q4W); (11) at least an amount of about
170 mg every four weeks (Q4W); (12) at least an amount of about 180 mg every four weeks
(Q4W); (13) at least an amount of about 190 mg every four weeks (Q4W); (14) at least an
amount of about 200 mg every four weeks (Q4W); (15) at least an amount of about 280 mg
every four weeks (Q4W); (16) at least an amount of about 350 every four weeks (Q4W); (17)
at least an amount of about 420 mg every four weeks (Q4W). In some embodiments, the
ApoB level is reduced by at least about 20% as compared to a predose ApoB level. In some
embodiments, the ApoB level is reduced by at least about 25%. In some embodiments, the
ApoB level is reduced by at least about 30%. In some embodiments, the ApoB level is
reduced by at least about 35%. In some embodiments, the ApoB level is reduced by at least
about 40%. In some embodiments, the ApoB level is reduced by at least about 45%. In some
embodiments, the ApoB level is reduced by at least about 50%. In some embodiments, the
ApoB level is reduced by at least about 55%. In some embodiments, the ApoB level is
reduced by at least about 60%. In some embodiments, the ApoB level is reduced by at least
about 65%. In some embodiments, the ApoB level is reduced by at least about 70%. In some
embodiments, the ApoB level is reduced by at least about 75%.
In some aspects, the invention comprises a method of lowering Lipoprotein A
(“Lp(a)”) levels in a patient, the method comprising administering to a patient in need
thereof, a dose of at least one anti-PCSK9 antibody, and wherein the dose of anti-PCSK9
antibody is administered on a schedule selected from the group consisting of: (1) at least
about 14 mg every week (QW); (2) at least an amount of about 35 mg every week (QW); (3)
at least an amount of about 45 mg every week (QW); (4) at least an amount of about 70 mg
every other week (Q2W); (5) at least an amount of about 105 mg every two weeks (Q2W);
(6) at least an amount of about 140 mg every other week (Q2W); (7) at least an amount of
about 150 mg every two weeks or every other week (Q2W); (8) at least an amount of about
280 mg every two weeks or every other week (Q2W); (9) at least an amount of about 150 mg
every four weeks (Q4W); (10) at least an amount of about 160 mg every four weeks (Q4W);
(11) at least an amount of about 170 mg every four weeks (Q4W); (12) at least an amount of
about 180 mg every four weeks (Q4W); (13) at least an amount of about 190 mg every four
weeks (Q4W); (14) at least an amount of about 200 mg every four weeks (Q4W); (15) at least
an amount of about 280 mg every four weeks (Q4W); (16) at least an amount of about 350
every four weeks (Q4W); (17) at least an amount of about 420 mg every four weeks (Q4W);
(18) at least an amount of about 1000 mg every four weeks (Q4W); (19) at least an amount of
about 2000 mg every four weeks (Q4W); and (20) at least an amount of about 3000 mg every
four weeks (Q4W). In some embodiments, the Lp(a) level is reduced by at least about 10%
as compared to a predose Lp(a) level. In some embodiments, the Lp(a) level is reduced by at
least about 15%. In some embodiments, the Lp(a) level is reduced by at least about 20%. In
some embodiments, the Lp(a) level is reduced by at least about 25%. In some embodiments,
the Lp(a) level is reduced by at least about 30%. In some embodiments, the Lp(a) level is
reduced by at least about 35%. In some embodiments, the Lp(a) level is reduced by at least
about 40%. In some embodiments, the Lp(a) level is reduced by at least about 45%. In some
embodiments, the Lp(a) level is reduced by at least about 50%. In some embodiments, the
Lp(a) level is reduced by at least about 55%. In some embodiments, the Lp(a) level is
reduced by at least about 60%. In some embodiments, the Lp(a) level is reduced by at least
about 65%.
In some aspects, the invention comprises a method for treating or preventing a
cholesterol related disorder in a patient, the method comprising administering to a patient in
need thereof a dose of about 10 mg to about 3000 mg of at least one anti-PCSK9 antibody
described herein. In some embodiments, the dose is about 10 mg to about 70 mg of at least
one anti-PCSK9 antibody administered once weekly (QW). In some embodiments, the dose
is about 14 mg to about 45 mg of at least one anti-PCSK9 antibody administered once
weekly. In some embodiments, the dose is about 14 mg to about 35 mg of at least one anti-
PCSK9 antibody administered once weekly. In some embodiments, the dose is about 70 mg
to about 420 mg of at least one anti-PCSK9 antibody administered once every two weeks
(Q2W). In some embodiments, the dose is about 70 mg to about 350 mg of at least one anti-
PCSK9 antibody administered once every two weeks (Q2W). In some embodiments, the
dose is about 105 mg to about 350 mg of at least one anti-PCSK9 antibody administered once
every two weeks (Q2W). In some embodiments, the dose is about 140 mg to about 280 mg
of at least one anti-PCSK9 antibody administered once every two weeks (Q2W). In some
embodiments, the dose is about 150 mg to about 280 mg of at least one anti-PCSK9 antibody
administered once every two weeks (Q2W). In some embodiments, the dose is about 150 mg
to about 200 mg of at least one anti-PCSK9 antibody administered once every two weeks
(Q2W). In some embodiments, the dose is about 150 mg to about 480 mg of at least one anti-
PCSK9 antibody administered once every four weeks (Q4W). In some embodiments, the
dose is about 150 mg to about 200 mg of at least one anti-PCSK9 antibody administered once
every four weeks (Q4W). In some embodiments, the dose is about 200 mg to about 480 mg
of at least one anti-PCSK9 antibody administered once every four weeks (Q4W). In some
embodiments, the dose is about 250 mg to about 480 mg of at least one anti-PCSK9 antibody
administered once every four weeks (Q4W). In some embodiments, the dose is about 280 mg
to about 420 mg of at least one anti-PCSK9 antibody administered once every four weeks
(Q4W). In some embodiments, the dose is about 350 mg to about 420 mg of at least one anti-
PCSK9 antibody administered once every four weeks. In some embodiments, the dose is
about 1000 mg every four weeks (Q4W). In some embodiments, the dose is about about
2000 mg every four weeks (Q4W). In some embodiments, the dose is about 3000 mg every
four weeks (Q4W). In some embodiments, the serum LDL cholesterol level is reduced by at
least about 15% as compared to a predose serum LDL cholesterol level. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 20%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 25%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 30%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 35%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 40%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 45%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 50%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 55%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 60%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 65%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 70%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 75%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 80%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 85%. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 90%. In some
embodiments, the cholesterol related disorder is heterozygous familial hypercholesterolemia,
homozygous familial hypercholesterolemia, non-familial hypercholesterolemia, hyperlipidemia
or dyslipidemia.
In some aspects, the invention comprises a method of treating or preventing a
cholesterol related disorder in a patient, the method comprising administering to a patient in
need thereof, a dose of at least one anti-PCSK9 antibody, and wherein the dose of anti-
PCSK9 antibody is administered on a schedule selected from the group consisting of: (1) at
least about 14 mg every week (QW); (2) at least an amount of about 35 mg every week (QW);
(3) at least an amount of about 45 mg every week (QW); (4) at least an amount of about 70
mg every other week (Q2W); (5) at least an amount of about 105 mg every two weeks
(Q2W); (6) at least an amount of about 140 mg every other week (Q2W); (7) at least an
amount of about 150 mg every two weeks or every other week (Q2W); (8) at least an amount
of about 280 mg every two weeks or every other week (Q2W); (9) at least an amount of about
150 mg every four weeks (Q4W); (10) at least an amount of about 160 mg every four weeks
(Q4W); (11) at least an amount of about 170 mg every four weeks (Q4W); (12) at least an
amount of about 180 mg every four weeks (Q4W); (13) at least an amount of about 190 mg
every four weeks (Q4W); (14) at least an amount of about 200 mg every four weeks (Q4W);
(15) at least an amount of about 280 mg every four weeks (Q4W); (16) at least an amount of
about 350 every four weeks (Q4W); (17) at least an amount of about 420 mg every four
weeks (Q4W); (18) at least an amount of about 1000 mg every four weeks (Q4W); (19) at
least an amount of about 2000 mg every four weeks (Q4W); and (20) at least an amount of
about 3000 mg every four weeks (Q4W). In some embodiments, the serum LDL cholesterol
level is reduced by at least about 15% as compared to a predose serum LDL cholesterol level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about 20%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 25%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 30%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 35%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 40%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 45%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 50%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 55%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 60%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 65%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 70%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 75%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 80%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 85%. In
some embodiments, the serum LDL cholesterol level is reduced by at least about 90%.
In some embodiments, the anti-PCSK9 antibody is 21B12, 26H5, 31H4, 8A3, 11F1
and/or 8A1.
In some embodiments, the cholesterol related disorder is heterozygous familial
hypercholesterolemia, homozygous familial hypercholesterolemia, non-familial
hypercholesterolemia, hyperlipidemia or dyslipidemia.
In some aspects, the invention comprises pharmaceutical formulations comprising at
least one anti-PCSK9 antibody selected from the group consisting of 21B12, 26H5, 31H4,
8A3, 11F1 and 8A1.
Other embodiments of this invention will be readily apparent from the disclosure
provided herewith.
BRIEF DESCRIPTION OF THE FIGURES
depicts an amino acid sequence of the mature form of the PCSK9 with the
pro-domain underlined.
FIGs. 1B -1B depict amino acid and nucleic acid sequences of PCSK9 with the pro-
domain underlined and the signal sequence in bold.
FIGs. 2A-2D are sequence comparison tables of various light chains of various
antigen binding proteins. continues the sequence started in .
continues the sequence started on .
FIGs. 3A-3D are sequence comparison tables of various heavy chains of various
antigen binding proteins. continues the sequence started in .
continues the sequence started on .
FIGs. 3E-3JJ depict the amino acid and nucleic acid sequences for the variable
domains of some embodiments of the antigen binding proteins.
K depicts the amino acid sequences for various constant domains.
FIGs. 3LL-3BBB depict the amino acid and nucleic acid sequences for the variable
domains of some embodiments of the antigen binding proteins.
FIGs. 3CCC-3JJJ are sequence comparison tables of various heavy and light chains of
some embodiments of the antigen binding proteins.
is a binding curve of an antigen binding protein to human PCSK9.
is a binding curve of an antigen binding protein to human PCSK9.
is a binding curve of an antigen binding protein to cynomolgus PCSK9.
is a binding curve of an antigen binding protein to cynomolgus PCSK9.
is a binding curve of an antigen binding protein to mouse PCSK9.
is a binding curve of an antigen binding protein to mouse PCSK9.
depicts the results of an SDS PAGE experiment involving PCSK9 and
various antigen binding proteins demonstrating the relative purity and concentration of the
proteins.
and 5C depict graphs from Biacore solution equilibrium assays for 21B12.
depicts the graph of the kinetics from a Biacore capture assay.
depicts a bar graph depicting binning results for three ABPs.
is an inhibition curve of antigen binding protein 31H4 IgG2 to PCSK9 in an
in vitro PCSK9:LDLR binding assay
is an inhibition curve of antigen binding protein 31H4 IgG4 to PCSK9 in an
in vitro PCSK9:LDLR binding assay.
is an inhibition curve of antigen binding protein 21B12 IgG2 to PCSK9 in an
in vitro PCSK9:LDLR binding assay.
is an inhibition curve of antigen binding protein 21B12 IgG4 to PCSK9 in an
in vitro PCSK9:LDLR binding assay.
is an inhibition curve of antigen binding protein 31H4 IgG2 in the cell LDL
uptake assay showing the effect of the ABP to reduce the LDL uptake blocking effects of
PCSK9
is an inhibition curve of antigen binding protein 31H4 IgG4 in the cell LDL
uptake assay showing the effect of the ABP to reduce the LDL uptake blocking effects of
PCSK9
is an inhibition curve of antigen binding protein 21B12 IgG2 in the cell LDL
uptake assay showing the effect of the ABP to reduce the LDL uptake blocking effects of
PCSK9
is an inhibition curve of antigen binding protein 21B12 IgG4 in the cell LDL
uptake assay showing the effect of the ABP to reduce the LDL uptake blocking effects of
PCSK9
is a graph depicting the serum cholesterol lowering ability in mice of ABP
31H4, changes relative to the IgG control treated mice (* p< 0.01).
is a graph depicting the serum cholesterol lowering ability in mice of ABP
31H4, changes relative to time = zero hours (# p, 0.05).
is a graph depicting the effect of ABP 31H4 on HDL cholesterol levels in
C57B1/6 mice (* p< 0.01).
is a graph depicting the effect of ABP 31H4 on HDL cholesterol levels in
C57Bl/6 mice (# p< 0.05).
depicts a western blot analysis of the ability of ABP 31H4 to enhance the
amount of liver LDLR protein present after various time points.
A is a graph depicting the ability of an antigen binding protein 31H4 to lower
total serum cholesterol in wild type mice, relative.
B is a graph depicting the ability of an antigen binding protein 31H4 to lower
HDL in wild type mice.
C is a graph depicting the serum cholesterol lowering ability of various
antigen binding proteins 31H4 and 16F12.
A depicts an injection protocol for testing the duration and ability of antigen
binding proteins to lower serum cholesterol.
B is a graph depicting the results of the protocol in A.
A depicts LDLR levels in response to the combination of a statin and ABP
21B12 in HepG2 cells.
B depicts LDLR levels in response to the combination of a statin and ABP
31H4 in HepG2 cells.
C depicts LDLR levels in response to the combination of a statin and ABP
25A7.1, a non-neutralizing antibody, (in contrast the “25A7” a neutralizing antibody) in
HepG2 cells.
D depicts LDLR levels in response to the combination of a statin and ABP
21B12 in HepG2 cells over expressing PCSK9.
E depicts LDLR levels in response to the combination of a statin and ABP
31H4 in HepG2 cells over expressing PCSK9.
F depicts LDLR levels in response to the combination of a statin and ABP
25A7.1, a non-neutralizing antibody, (in contrast the “25A7” a neutralizing antibody) in
HepG2 cells over expressing PCSK9.
A depicts the various light chain amino acid sequences of various ABPs to
PCSK9. The dots (.) indicate no amino acid.
B depicts a light chain cladogram for various ABPs to PCSK9.
C depicts the various heavy chain amino acid sequences of various ABPs to
PCSK9. The dots (.) indicate no amino acid.
D depicts a heavy chain dendrogram for various ABPs to PCSK9.
E depicts a comparison of light and heavy CDRs and designation of groups
from which to derive consensus.
F depicts the consensus sequences for Groups 1 and 2.
G depicts the consensus sequences for Groups 3 and 4.
H depicts the consensus sequences for Groups 1 and 2. The dots (.) indicated
identical residues.
I depicts the consensus sequences for Group 2. The dots (.) indicated identical
residues.
J depicts the consensus sequences for Groups 3 and 4. The dots (.) indicated
identical residues.
is a graph showing the reduction of LDL-c levels in patients receiving
multiple-doses of an anti-PCSK9 antibody (21B12).
is a graph showing the reduction of LDL-c levels in patients on low to
moderate and high-dose statins receiving multiple-doses of an anti-PCSK9 antibody (21B12).
is a graph showing the reduction of ApoB levels in patients receiving
multiple-doses of an anti-PCSK9 antibody (21B12).
is a bar graph showing the reduction of lipoprotein a (“Lp(a)”) levels in
patients on low to moderate and high-dose statins receiving multiple-doses of an anti-PCSK9
antibody (21B12).
is a graph showing the reduction of LDL-c levels in patients having
heterozygous familial hypercholesterolemia (“HeFH”) receiving multiple-doses of an anti-
PCSK9 antibody (21B12).
is a graph showing the reduction of PCSK9 levels in patients having
heterozygous familial hypercholesterolemia (“HeFH”) receiving multiple-doses of an anti-
PCSK9 antibody (21B12).
is a graph showing the reduction of total cholesterol levels in patients having
heterozygous familial hypercholesterolemia (“HeFH”) receiving multiple-doses of an anti-
PCSK9 antibody (21B12).
is a graph showing the reduction of non-HDL cholesterol levels in patients
having heterozygous familial hypercholesterolemia (“HeFH”) receiving multiple-doses of an
anti-PCSK9 antibody (21B12).
is a graph showing the reduction of ApoB levels in patients having
heterozygous familial hypercholesterolemia (“HeFH”) receiving multiple-doses of an anti-
PCSK9 antibody (21B12).
is a bar graph showing the reduction of lipoprotein a (“Lp(a)”) in patients
having heterozygous familial hypercholesterolemia (“HeFH”) receiving multiple-doses of an
anti-PCSK9 antibody (21B12).
A is a graph showing the aggregate data relating to LDL-C reduction in
patients from four studies described in Examples 22-25 who received various doses of an
anti-PCSK9 antibody (21B12) every other week (Q2W) over a 12 week period.
B is a graph showing the aggregate data relating to LDL-C reduction in
patients from four studies described in Examples 22-25 who received various doses of an
anti-PCSK9 antibody (21B12) every four weeks (Q4W) over a 12 week period.
A is a bar graph showing the aggregate data relating to Lp(a) reduction in
patients from four studies described in Examples 22-25 who received various doses of an
anti-PCSK9 antibody (21B12) either every other week (Q2W) or every 4 weeks (Q4W) over
a 12 week period.
B is a bar graph showing the aggregate data relating to HDL-C reduction in
patients from four studies described in Examples 22-25 who received various doses of an
anti-PCSK9 antibody (21B12) either every other week (Q2W) or every 4 weeks (Q4W) over
a 12 week period.
C is a bar graph showing the aggregate data relating to triglyceride reduction
in patients from four studies described in Examples 22-25 who received various doses of an
anti-PCSK9 antibody (21B12) either every other week (Q2W) or every 4 weeks (Q4W) over
a 12 week period.
D is a bar graph showing the aggregate data relating to VLDL-C reduction in
patients from four studies described in Examples 22-25 who received various doses of an
anti-PCSK9 antibody (21B12) either every other week (Q2W) or every 4 weeks (Q4W) over
a 12 week period.
is a bar graph showing the viscosity of anti-PCSK9 antibody (21B12)
formulations containing various stabilizers/excipients.
is a graph showing the the stabilizer/excipient, proline, has the ability to
lower viscosity of anti-PCSK9 antibody (21B12) formulations having high protein
concentrations.
A is a graph showing the viscosity of various concentrations of anti-PCSK9
antibody, 21B12, in a formulation comprising 10 mM sodium acetate, and 9% Sucrose pH
5.2 at 25ºC and 40ºC.
Figure 28B is a graph showing the viscosity of various concentrations of anti-PCSK9
antibody, 21B12, in a formulation comprising 10 mM sodium acetate, and 9% Sucrose pH
.2 at 25ºC and 40ºC, as compared to a formulation comprising 10 mM sodium acetate, 125
mM arginine, and 3% Sucrose pH 5.0 at 25ºC and 40ºC.
Figure 28C is a graph showing the viscosity of various concentrations of anti-PCSK9
antibody, 21B12, in a formulation comprising 10 mM sodium acetate, and 9% Sucrose pH
.2 at 25ºC and 40ºC, as compared to a formulation comprising 10 mM sodium acetate, 100
mM methionine, and 4% Sucrose pH 5.0 at 25ºC and 40ºC.
Figure 28D is a graph showing the viscosity of various concentrations of anti-PCSK9
antibody, 21B12, in a formulation comprising 10 mM sodium acetate, and 9% Sucrose pH
.2 at 25ºC and 40ºC, as compared to a formulation comprising 10 mM sodium acetate and
250 mM proline, pH 5.0 at 25ºC and 40ºC.
A is a bar graph showing the number of 10 µm particles in various
formulations of anti-PCSK9 antibody (i.e., 21B12) formulations over a period of 6 months.
B is a bar graph showing the number of 25 µm particles in various
formulations of anti-PCSK9 antibody (i.e., 21B12) formulations over a period of 6 months.
A is a bar graph showing the number of 10 µm particles in various
formulations of anti-PCSK9 antibody (i.e., 11F1) formulations over a period of 4 months.
B is a bar graph showing the number of 25 µm particles in various
formulations of anti-PCSK9 antibody (i.e.,11F1) formulations over a period of 4 months.
Figure 31 is a graph illustrating the binding specificity of 11F1 in a competition assay
with PCSKP, PCSK2, PCSK1 PCSK7 and Furin with OD plotted on the vertical axis and
concentration of PCSK9 (ug/ml) plotted on the horizontal axis.
Figure 32 is a graph showing the dose response curve for inhibition of LDLR:D374Y
PCSK9 binding by 11F1 in a competition assay with OD plotted on the vertical axis and
Log [11F1] (pM) plotted on the horizontal axis.
Figure 33is a graph depicting the dose response curve for the inhibition of LDLR: WT
PCSK9 binding by 11F1in a competition assay with OD plotted on the vertical axis and
Log [11f1] (pM) plotted on the horizontal axis.
Figure 34 is a graph depicting the dose response curve for the ability of 11F1 to block
human D374Y PCSK9-mediated reduction of LDL uptake in HepG2 cells with relative
fluorescence units (x10 ) plotted on the vertical axis and Log [11F1] (nM) plotted on the
horizontal axis.
Figure 35 is a graph depicting the dose response curve for the ability of 11F1 to block
human WT PCSK9-mediated reduction of LDL uptake in HepG2 cells with relative
fluorescence units plotted (x10 ) on the vertical axis and Log [11F1] (nM) plotted on the
horizontal axis.
Figure 36 is a bar graph depicting the effect of 11F1 and 8A3 on serum non-HDL
cholesterol in mice expressing human PCSK9 by AAV with non-HDL-C serum concentration
(mg/ml) on the vertical axis and time following injection (days) plotted on the horizontal
axis.
Figure 37 is a bar graph depicting the effect of 11F1 and 8A3 on Serum Total
Cholesterol in mice expressing human PCSK9 by AAV with Serum Total Cholesterol
(mg/ml) on the vertical axis and time following injection (days) plotted on the horizontal
axis.
Figure 38 is a bar graph depicting the effect of 11F1 and 8A3 on Serum HDL
Cholesterol (HDL-C) in mice expressing human PCSK9 by AAV with HDL-C (mg/ml) on
the vertical axis and time following injection (days) plotted on the horizontal axis.
Figure 39 is a graph depicting IgG2, 8A3 and 11F1 antibody concentration profiles in
mice expressing human PCSK9 by AAV with serum antibody concentration (ng/mL) plotted
on the vertical axis and time following injection in days plotted on the horizontal axis.
Figure 40 is a table summarizing PK parameters for IgG2, 11F1 and 8A3 in mice
expressing human PCSK9 by AAV.
Figure 41 is a graph depicting the effect of a single subcutaneous administration of an
anti-KLH antibody (control), 21B12, 8A3 and 11F1 on serum LDL concentration (LDL-C) in
cynomolgus monkeys with LDL-C (mg/dl) plotted on the vertical axis and time following
administration in days on the horizontal axis.
Figure 42 is a graph depicting the effect of a single subcutaneous administration of an
anti-KLH antibody (control), 21B12, 8A3 and 11F1 on Serum Total Cholesterol in
cynomolgus monkeys with Total Cholesterol concentration (mg/dl) plotted on the vertical
axis and time following administration in days on the horizontal axis.
Figure 43 is a graph depicting the effect of a single subcutaneous administration of an
anti-KLH antibody (control), 21B12, 8A3 and 11F1 on Serum HDL Cholesterol in
cynomolgus monkeys with HDL-C (mg/dl) plotted on the vertical axis and time following
administration in days on the horizontal axis.
Figure 44 is a graph depicting the effect of a single subcutaneous administration of an
anti-KLH antibody (control), 21B12, 8A3 and 11F1 on Serum Triglycerides in cynomolgus
monkeys with Serum Triglyceride concentration (mg/dl) plotted on the vertical axis and time
following administration in days on the horizontal axis.
Figure 45 is a graph depicting the effect of a single subcutaneous administration of an
anti-KLH antibody (control), 21B12, 8A3 and 11F1 on Apolipoprotein B (ApoB) in
cynomolgus monkeys with APOB concentration (mg/dl) plotted on the vertical axis and time
following administration in days on the horizontal axis.
Figure 46 is a graph depicting the mean pharmacokinetic profiles for the anti--KLH
antibody (control), 21B12, 8A3 and 11F1 in cynomolgus monkeys with antibody
concentrations (ng/ml) plotted on the vertical axis and time following administration in days
on the horizontal axis.
Figure 47 is a table summarizing PK parameters for the anti--KLH antibody (control),
21B12, 8A3 and 11F1 in cynomolgus monkeys.
Figure 48A depicts a comparison of light chain amino acid sequences of 8A1, 8A3
and 11F1, as well as a consensus sequence derived from the the comparison. CDR sequences
are underlined.
Figure 48B depicts a comparison of heavy chain amino acid sequences of 8A1, 8A3
and 11F1, as well as a consensus sequence derived from the the comparison. CDR sequences
are underlined.
DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
Antigen binding proteins (such as antibodies and functional binding fragments
thereof) that bind to PCSK9 are disclosed herein. In some embodiments, the antigen binding
proteins bind to PCSK9 and prevent PCSK9 from functioning in various ways. In some
embodiments, the antigen binding proteins block or reduce the ability of PCSK9 to interact
with other substances. For example, in some embodiments, the antigen binding protein binds
to PCSK9 in a manner that prevents or reduces the likelihood that PCSK9 will bind to LDLR.
In other embodiments, antigen binding proteins bind to PCSK9 but do not block PCSK9’s
ability to interact with LDLR. In some embodiments, the antigen binding proteins are human
monoclonal antibodies.
As will be appreciated by one of skill in the art, in light of the present disclosure,
altering the interactions between PCSK9 and LDLR can increase the amount of LDLR
available for binding to LDL, which in turn decreases the amount of serum LDL in a subject,
resulting in a reduction in the subject’s serum cholesterol level. As such, antigen binding
proteins to PCSK9 can be used in various methods and formulations for treating subjects with
elevated serum cholesterol levels, at risk of elevated serum cholesterol levels, or which could
benefit from a reduction in their serum cholesterol levels. Thus, various methods and
techniques for lowering, maintaining, or preventing an increase in serum cholesterol are also
described herein. In some embodiments, the antigen binding protein allows for binding
between PCSK9 and LDLR, but the antigen binding protein prevents or reduces the adverse
activity of PCSK9 on LDLR. In some embodiments, the antigen binding protein prevents or
reduces the binding of PCSK9 to LDLR.
For convenience, the following sections generally outline the various meanings of the
terms used herein. Following this discussion, general aspects regarding antigen binding
proteins are discussed, followed by specific examples demonstrating the properties of various
embodiments of the antigen binding proteins and how they can be employed.
Definitions and Embodiments
It is to be understood that both the foregoing general description and the following
detailed description are exemplary and explanatory only and are not restrictive of the
invention as claimed. In this application, the use of the singular includes the plural unless
specifically stated otherwise. In this application, the use of “or” means “and/or” unless
stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such
as “includes” and “included”, is not limiting. Also, terms such as “element” or “component”
encompass both elements and components comprising one unit and elements and components
that comprise more than one subunit unless specifically stated otherwise. Also, the use of the
term “portion” can include part of a moiety or the entire moiety.
The section headings used herein are for organizational purposes only and are not to
be construed as limiting the subject matter described. All documents, or portions of
documents, cited in this application, including but not limited to patents, patent applications,
articles, books, and treatises, are hereby expressly incorporated by reference in their entirety
for any purpose. As utilized in accordance with the present disclosure, the following terms,
unless otherwise indicated, shall be understood to have the following meanings:
The term “proprotein convertase subtilisin kexin type 9” or “PCSK9” refers to a
polypeptide as set forth in SEQ ID NO: 1 and/or 3 or fragments thereof, as well as related
polypeptides, which include, but are not limited to, allelic variants, splice variants, derivative
variants, substitution variants, deletion variants, and/or insertion variants including the
addition of an N-terminal methionine, fusion polypeptides, and interspecies homologs. In
certain embodiments, a PCSK9 polypeptide includes terminal residues, such as, but not
limited to, leader sequence residues, targeting residues, amino terminal methionine residues,
lysine residues, tag residues and/or fusion protein residues. “PCSK9” has also been referred
to as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin/kexin type 9, and neural
apoptosis regulated convertase 1. The PCSK9 gene encodes a proprotein convertase protein
that belongs to the proteinase K subfamily of the secretory subtilase family. The term
“PCSK9” denotes both the proprotein and the product generated following autocatalysis of
the proprotein. When only the autocatalyzed product is being referred to (such as for an
antigen binding protein that selectively binds to the cleaved PCSK9), the protein can be
referred to as the “mature,” “cleaved”, “processed” or “active” PCSK9. When only the
inactive form is being referred to, the protein can be referred to as the “inactive”, “pro-form”,
or “unprocessed” form of PCSK9. The term PCSK9 as used herein also includes naturally
occurring alleles, such as the mutations D374Y, S127R and F216L. The term PCSK9 also
encompasses PCSK9 molecules incorporating post-translational modifications of the PCSK9
amino acid sequence, such as PCSK9 sequences that have been glycosylated, PEGylated,
PCSK9 sequences from which its signal sequence has been cleaved, PCSK9 sequence from
which its pro domain has been cleaved from the catalytic domain but not separated from the
catalytic domain (e.g., FIGs. 1A and 1B).
The term “PCSK9 activity” includes any biological effect of PCSK9. In certain
embodiments, PCSK9 activity includes the ability of PCSK9 to interact or bind to a substrate
or receptor. In some embodiments, PCSK9 activity is represented by the ability of PCSK9 to
bind to a LDL receptor (LDLR). In some embodiments, PCSK9 binds to and catalyzes a
reaction involving LDLR. In some embodiments, PCSK9 activity includes the ability of
PCSK9 to alter (e.g., reduce) the availability of LDLR. In some embodiments, PCSK9
activity includes the ability of PCSK9 to increase the amount of LDL in a subject. In some
embodiments, PCSK9 activity includes the ability of PCSK9 to decrease the amount of
LDLR that is available to bind to LDL. In some embodiments, “PCSK9 activity” includes
any biological activity resulting from PCSK9 signaling. Exemplary activities include, but are
not limited to, PCSK9 binding to LDLR, PCSK9 enzyme activity that cleaves LDLR or other
proteins, PCSK9 binding to proteins other than LDLR that facilitate PCSK9 action, PCSK9
altering APOB secretion (Sun X-M et al, “Evidence for effect of mutant PCSK9 on
apoliprotein B secretion as the cause of unusually severe dominant hypercholesterolemia,
Human Molecular Genetics 14: 1161-1169, 2005 and Ouguerram K et al, “Apolipoprotein
B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in
PCSK9, Arterioscler thromb Vasc Biol. 24: 1448-1453, 2004), PCSK9’s role in liver
regeneration and neuronal cell differentiation (Seidah NG et al, “The secretory proprotein
convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and
neuronal differentiation” PNAS 100: 928-933, 2003), and PCSK9s role in hepatic glucose
metabolism (Costet et al., “Hepatic PCSK9 expression is regulated by nutritional status via
insulin and sterol regulatory element-binding protein 1c” J. Biol. Chem. 281(10):6211-18,
2006).
The term “hypercholesterolemia,” as used herein, refers to a condition in which
cholesterol levels are elevated above a desired level. In some embodiments, this denotes that
serum cholesterol levels are elevated. In some embodiments, the desired level takes into
account various “risk factors” that are known to one of skill in the art (and are described or
referenced herein).
The term “polynucleotide” or “nucleic acid” includes both single-stranded and
double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be
ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said
modifications include base modifications such as bromouridine and inosine derivatives,
ribose modifications such as 2’,3’-dideoxyribose, and internucleotide linkage modifications
such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
The term “oligonucleotide” means a polynucleotide comprising 200 or fewer
nucleotides. In some embodiments, oligonucleotides are 10 to 60 bases in length. In other
embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in
length. Oligonucleotides can be single stranded or double stranded, e.g., for use in the
construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides.
An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or
an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR
primers, cloning primers or hybridization probes.
An “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA,
cDNA, or synthetic origin or some combination thereof which is not associated with all or a
portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is
linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure,
it should be understood that “a nucleic acid molecule comprising” a particular nucleotide
sequence does not encompass intact chromosomes. Isolated nucleic acid molecules
“comprising” specified nucleic acid sequences can include, in addition to the specified
sequences, coding sequences for up to ten or even up to twenty other proteins or portions
thereof, or can include operably linked regulatory sequences that control expression of the
coding region of the recited nucleic acid sequences, and/or can include vector sequences.
Unless specified otherwise, the left-hand end of any single-stranded polynucleotide
sequence discussed herein is the 5’ end; the left-hand direction of double-stranded
polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition
of nascent RNA transcripts is referred to as the transcription direction; sequence regions on
the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of
the RNA transcript are referred to as “upstream sequences;” sequence regions on the DNA
strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA
transcript are referred to as “downstream sequences.”
The term “control sequence” refers to a polynucleotide sequence that can affect the
expression and processing of coding sequences to which it is ligated. The nature of such
control sequences can depend upon the host organism. In particular embodiments, control
sequences for prokaryotes can include a promoter, a ribosomal binding site, and a
transcription termination sequence. For example, control sequences for eukaryotes can
include promoters comprising one or a plurality of recognition sites for transcription factors,
transcription enhancer sequences, and transcription termination sequence. “Control
sequences” can include leader sequences and/or fusion partner sequences.
The term “vector” means any molecule or entity (e.g., nucleic acid, plasmid,
bacteriophage or virus) used to transfer protein coding information into a host cell.
The term “expression vector” or “expression construct” refers to a vector that is
suitable for transformation of a host cell and contains nucleic acid sequences that direct
and/or control (in conjunction with the host cell) expression of one or more heterologous
coding regions operatively linked thereto. An expression construct can include, but is not
limited to, sequences that affect or control transcription, translation, and, if introns are
present, affect RNA splicing of a coding region operably linked thereto.
As used herein, “operably linked” means that the components to which the term is
applied are in a relationship that allows them to carry out their inherent functions under
suitable conditions. For example, a control sequence in a vector that is “operably linked” to a
protein coding sequence is ligated thereto so that expression of the protein coding sequence is
achieved under conditions compatible with the transcriptional activity of the
control sequences.
The term “host cell” means a cell that has been transformed, or is capable of being
transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term
includes the progeny of the parent cell, whether or not the progeny is identical in morphology
or in genetic make-up to the original parent cell, so long as the gene of interest is present.
The term “transfection” means the uptake of foreign or exogenous DNA by a cell, and
a cell has been “transfected” when the exogenous DNA has been introduced inside the cell
membrane. A number of transfection techniques are well known in the art and are disclosed
herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular
Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular
Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce
one or more exogenous DNA moieties into suitable host cells.
The term “transformation” refers to a change in a cell's genetic characteristics, and a
cell has been transformed when it has been modified to contain new DNA or RNA. For
example, a cell is transformed where it is genetically modified from its native state by
introducing new genetic material via transfection, transduction, or other techniques.
Following transfection or transduction, the transforming DNA can recombine with that of the
cell by physically integrating into a chromosome of the cell, or can be maintained transiently
as an episomal element without being replicated, or can replicate independently as a plasmid.
A cell is considered to have been “stably transformed” when the transforming DNA is
replicated with the division of the cell.
The terms “polypeptide” or “protein” means a macromolecule having the amino acid
sequence of a native protein, that is, a protein produced by a naturally-occurring and non-
recombinant cell; or it is produced by a genetically-engineered or recombinant cell, and
comprise molecules having the amino acid sequence of the native protein, or molecules
having deletions from, additions to, and/or substitutions of one or more amino acids of the
native sequence. The term also includes amino acid polymers in which one or more amino
acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers.
The terms “polypeptide” and “protein” specifically encompass PCSK9 antigen binding
proteins, antibodies, or sequences that have deletions from, additions to, and/or substitutions
of one or more amino acid of antigen-binding protein. The term “polypeptide fragment”
refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion,
and/or an internal deletion as compared with the full-length native protein. Such fragments
can also contain modified amino acids as compared with the native protein. In certain
embodiments, fragments are about five to 500 amino acids long. For example, fragments can
be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino
acids long. Useful polypeptide fragments include immunologically functional fragments of
antibodies, including binding domains. In the case of a PCSK9-binding antibody, useful
fragments include but are not limited to a CDR region, a variable domain of a heavy and/or
light chain, a portion of an antibody chain or just its variable region including two CDRs, and
the like.
The term “isolated protein” referred means that a subject protein (1) is free of at least
some other proteins with which it would normally be found, (2) is essentially free of other
proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a
different species, (4) has been separated from at least about 50 percent of polynucleotides,
lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably
associated (by covalent or noncovalent interaction) with a polypeptide with which it is not
associated in nature, or (6) does not occur in nature. Typically, an “isolated protein”
constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of
a given sample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any
combination thereof can encode such an isolated protein. Preferably, the isolated protein is
substantially free from proteins or polypeptides or other contaminants that are found in its
natural environment that would interfere with its therapeutic, diagnostic, prophylactic,
research or other use.
The term “amino acid” includes its normal meaning in the art.
A “variant” of a polypeptide (e.g., an antigen binding protein, or an antibody)
comprises an amino acid sequence wherein one or more amino acid residues are inserted into,
deleted from and/or substituted into the amino acid sequence relative to another polypeptide
sequence. Variants include fusion proteins.
The term “identity” refers to a relationship between the sequences of two or more
polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and
comparing the sequences. “Percent identity” means the percent of identical residues between
the amino acids or nucleotides in the compared molecules and is calculated based on the size
of the smallest of the molecules being compared. For these calculations, gaps in alignments
(if any) are preferably addressed by a particular mathematical model or computer program
(i.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned
nucleic acids or polypeptides include those described in Computational Molecular Biology,
(Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and
Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis
of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey:
Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York:
Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991,
New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.
In calculating percent identity, the sequences being compared are typically aligned in
a way that gives the largest match between the sequences. One example of a computer
program that can be used to determine percent identity is the GCG program package, which
includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group,
University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align the
two polypeptides or polynucleotides for which the percent sequence identity is to be
determined. The sequences are aligned for optimal matching of their respective amino acid
or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty
(which is calculated as 3x the average diagonal, wherein the “average diagonal” is the
average of the diagonal of the comparison matrix being used; the “diagonal” is the score or
number assigned to each perfect amino acid match by the particular comparison matrix) and a
gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a
comparison matrix such as PAM 250 or BLOSum 62 are used in conjunction with the
algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978,
Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix;
Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSum 62
comparison matrix) is also used by the algorithm.
Examples of parameters that can be employed in determining percent identity for
polypeptides or nucleotide sequences using the GAP program are the following:
Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453
Comparison matrix: BLOSum 62 from Henikoff et al., 1992, supra
Gap Penalty: 12 (but with no penalty for end gaps)
Gap Length Penalty: 4
Threshold of Similarity: 0
Certain alignment schemes for aligning two amino acid sequences may result in
matching of only a short region of the two sequences, and this small aligned region may have
very high sequence identity even though there is no significant relationship between the two
full-length sequences. Accordingly, the selected alignment method (GAP program) can be
adjusted if so desired to result in an alignment that spans at least 50 or other number of
contiguous amino acids of the target polypeptide.
As used herein, the twenty conventional (e.g., naturally occurring) amino acids and
their abbreviations follow conventional usage. See Immunology--A Synthesis (2nd Edition, E.
S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is
incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the
twenty conventional amino acids, unnatural amino acids such as -, -disubstituted amino
acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be
suitable components for polypeptides of the present invention. Examples of unconventional
amino acids include: 4-hydroxyproline, -carboxyglutamate, -N,N,N-trimethyllysine, -N-
acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-
hydroxylysine, -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-
hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino
terminal direction and the right-hand direction is the carboxy-terminal direction, in
accordance with standard usage and convention.
Similarly, unless specified otherwise, the left-hand end of single-stranded
polynucleotide sequences is the 5’ end; the left-hand direction of double-stranded
polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition
of nascent RNA transcripts is referred to as the transcription direction; sequence regions on
the DNA strand having the same sequence as the RNA and which are 5’ to the 5’ end of the
RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand
having the same sequence as the RNA and which are 3’ to the 3’ end of the RNA transcript
are referred to as “downstream sequences.”
Conservative amino acid substitutions can encompass non-naturally occurring amino
acid residues, which are typically incorporated by chemical peptide synthesis rather than by
synthesis in biological systems. These include peptidomimetics and other reversed or
inverted forms of amino acid moieties.
Naturally occurring residues can be divided into classes based on common side chain
properties:
1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
3) acidic: Asp, Glu;
4) basic: His, Lys, Arg;
) residues that influence chain orientation: Gly, Pro; and
6) aromatic: Trp, Tyr, Phe.
For example, non-conservative substitutions can involve the exchange of a member of one of
these classes for a member from another class. Such substituted residues can be introduced,
for example, into regions of a human antibody that are homologous with non-human
antibodies, or into the non-homologous regions of the molecule.
In making changes to the antigen binding protein or the PCSK9 protein, according to
certain embodiments, the hydropathic index of amino acids can be considered. Each amino
acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge
characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-
0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and
arginine (-4.5).
The importance of the hydropathic amino acid index in conferring interactive
biological function on a protein is understood in the art. Kyte et al., J. Mol. Biol., 157:105-
131 (1982). It is known that certain amino acids can be substituted for other amino acids
having a similar hydropathic index or score and still retain a similar biological activity. In
making changes based upon the hydropathic index, in certain embodiments, the substitution
of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments,
those which are within ±1 are included, and in certain embodiments, those within ±0.5 are
included.
It is also understood in the art that the substitution of like amino acids can be made
effectively on the basis of hydrophilicity, particularly where the biologically functional
protein or peptide thereby created is intended for use in immunological embodiments, as in
the present case. In certain embodiments, the greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e., with a biological property of the protein.
The following hydrophilicity values have been assigned to these amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine
(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making
changes based upon similar hydrophilicity values, in certain embodiments, the substitution of
amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments,
those which are within ±1 are included, and in certain embodiments, those within ±0.5 are
included. One can also identify epitopes from primary amino acid sequences on the basis of
hydrophilicity. These regions are also referred to as "epitopic core regions."
Exemplary amino acid substitutions are set forth in Table 1.
TABLE 1
Amino Acid Substitutions
Original Residues Exemplary Substitutions Preferred Substitutions
Ala Val, Leu, Ile Val
Arg Lys, Gln, Asn Lys
Asn Gln Gln
Asp Glu Glu
Cys Ser, Ala Ser
Gln Asn Asn
Glu Asp Asp
Gly Pro, Ala Ala
His Asn, Gln, Lys, Arg Arg
Leu, Val, Met, Ala,
Ile Leu
Phe, Norleucine
Norleucine, Ile,
Leu Ile
Val, Met, Ala, Phe
Arg, 1,4 Diamino-butyric
Lys Arg
Acid, Gln, Asn
Met Leu, Phe, Ile Leu
Leu, Val, Ile, Ala,
Phe Leu
Pro Ala Gly
Ser Thr, Ala, Cys Thr
Thr Ser Ser
Trp Tyr, Phe Tyr
Tyr Trp, Phe, Thr, Ser Phe
Ile, Met, Leu, Phe,
Val Leu
Ala, Norleucine
The term “derivative” refers to a molecule that includes a chemical modification other
than an insertion, deletion, or substitution of amino acids (or nucleic acids). In certain
embodiments, derivatives comprise covalent modifications, including, but not limited to,
chemical bonding with polymers, lipids, or other organic or inorganic moieties. In certain
embodiments, a chemically modified antigen binding protein can have a greater circulating
half-life than an antigen binding protein that is not chemically modified. In certain
embodiments, a chemically modified antigen binding protein can have improved targeting
capacity for desired cells, tissues, and/or organs. In some embodiments, a derivative antigen
binding protein is covalently modified to include one or more water soluble polymer
attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or
polypropylene glycol. See, e.g., U.S. Patent Nos: 4,640,835, 4,496,689, 4,301,144,
4,670,417, 4,791,192 and 4,179,337. In certain embodiments, a derivative antigen binding
protein comprises one or more polymer, including, but not limited to, monomethoxy-
polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl
pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene
oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl
alcohol, as well as mixtures of such polymers.
In certain embodiments, a derivative is covalently modified with polyethylene glycol
(PEG) subunits. In certain embodiments, one or more water-soluble polymer is bonded at
one or more specific position, for example at the amino terminus, of a derivative. In certain
embodiments, one or more water-soluble polymer is randomly attached to one or more side
chains of a derivative. In certain embodiments, PEG is used to improve the therapeutic
capacity for an antigen binding protein. In certain embodiments, PEG is used to improve the
therapeutic capacity for a humanized antibody. Certain such methods are discussed, for
example, in U.S. Patent No. 6,133,426, which is hereby incorporated by reference for any
purpose.
Peptide analogs are commonly used in the pharmaceutical industry as non-peptide
drugs with properties analogous to those of the template peptide. These types of non-peptide
compound are termed “peptide mimetics” or “peptidomimetics.” Fauchere, J., Adv. Drug
Res., 15:29 (1986); Veber & Freidinger, TINS, p.392 (1985); and Evans et al., J. Med.
Chem., 30:1229 (1987), which are incorporated herein by reference for any purpose. Such
compounds are often developed with the aid of computerized molecular modeling. Peptide
mimetics that are structurally similar to therapeutically useful peptides can be used to
produce a similar therapeutic or prophylactic effect. Generally, peptidomimetics are
structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical
property or pharmacological activity), such as human antibody, but have one or more peptide
linkages optionally replaced by a linkage selected from: --CH NH--, --CH S--, --CH -CH
2 2 2 2
--, --CH=CH-(cis and trans), --COCH --, --CH(OH)CH --, and --CH SO--, by methods well
2 2 2
known in the art. Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be
used in certain embodiments to generate more stable peptides. In addition, constrained
peptides comprising a consensus sequence or a substantially identical consensus sequence
variation can be generated by methods known in the art (Rizo and Gierasch, Ann. Rev.
Biochem., 61:387 (1992), incorporated herein by reference for any purpose); for example, by
adding internal cysteine residues capable of forming intramolecular disulfide bridges which
cyclize the peptide.
The term “naturally occurring” as used throughout the specification in connection
with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to
materials which are found in nature or a form of the materials that is found in nature.
An “antigen binding protein” (“ABP”) as used herein means any protein that binds a
specified target antigen. In the instant application, the specified target antigen is the PCSK9
protein or fragment thereof. “Antigen binding protein” includes but is not limited to
antibodies and binding parts thereof, such as immunologically functional fragments.
Peptibodies are another example of antigen binding proteins. The term “immunologically
functional fragment” (or simply “fragment”) of an antibody or immunoglobulin chain (heavy
or light chain) antigen binding protein, as used herein, is a species of antigen binding protein
comprising a portion (regardless of how that portion is obtained or synthesized) of an
antibody that lacks at least some of the amino acids present in a full-length chain but which is
still capable of specifically binding to an antigen. Such fragments are biologically active in
that they bind to the target antigen and can compete with other antigen binding proteins,
including intact antibodies, for binding to a given epitope. In some embodiments, the
fragments are neutralizing fragments. In some embodiments, the fragments can block or
reduce the likelihood of the interaction between LDLR and PCSK9. In one aspect, such a
fragment will retain at least one CDR present in the full-length light or heavy chain, and in
some embodiments will comprise a single heavy chain and/or light chain or portion thereof.
These biologically active fragments can be produced by recombinant DNA techniques, or can
be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact
antibodies. Immunologically functional immunoglobulin fragments include, but are not
limited to, Fab, a diabody (heavy chain variable domain on the same polypeptide as a light
chain variable domain, connected via a short peptide linker that is too short to permit pairing
between the two domains on the same chain), Fab’, F(ab’) , Fv, domain antibodies and single-
chain antibodies, and can be derived from any mammalian source, including but not limited
to human, mouse, rat, camelid or rabbit. It is further contemplated that a functional portion of
the antigen binding proteins disclosed herein, for example, one or more CDRs, could be
covalently bound to a second protein or to a small molecule to create a therapeutic agent
directed to a particular target in the body, possessing bifunctional therapeutic properties, or
having a prolonged serum half-life. As will be appreciated by one of skill in the art, an
antigen binding protein can include nonprotein components. In some sections of the present
disclosure, examples of ABPs are described herein in terms of “number/letter/number” (e.g.,
25A7). In these cases, the exact name denotes a specific antibody. That is, an ABP named
25A7 is not necessarily the same as an antibody named 25A7.1, (unless they are explicitly
taught as the same in the specification, e.g., 25A7 and 25A7.3). As will be appreciated by
one of skill in the art, in some embodiments LDLR is not an antigen binding protein. In some
embodiments, binding subsections of LDLR are not antigen binding proteins, e.g., EGFa. In
some embodiments, other molecules through which PCSK9 signals in vivo are not antigen
binding proteins. Such embodiments will be explicitly identified as such.
Certain antigen binding proteins described herein are antibodies or are derived from
antibodies. In certain embodiments, the polypeptide structure of the antigen binding proteins
is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific
antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein
as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies,
antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments
thereof, respectively. In some embodiments, the ABP comprises or consists of avimers
(tightly binding peptide). These various antigen binding proteins are further described herein.
An “Fc” region comprises two heavy chain fragments comprising the C 1 and C 2
domains of an antibody. The two heavy chain fragments are held together by two or more
disulfide bonds and by hydrophobic interactions of the C 3 domains.
A “Fab fragment” comprises one light chain and the C 1 and variable regions of one
heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another
heavy chain molecule.
A “Fab’ fragment” comprises one light chain and a portion of one heavy chain that
contains the VH domain and the C 1 domain and also the region between the C 1 and C 2
H H H
domains, such that an interchain disulfide bond can be formed between the two heavy chains
of two Fab’ fragments to form an F(ab’) molecule.
A “F(ab’) fragment” contains two light chains and two heavy chains containing a
portion of the constant region between the C 1 and C 2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A F(ab’) fragment thus is composed
of two Fab’ fragments that are held together by a disulfide bond between the two
heavy chains.
The “Fv region” comprises the variable regions from both the heavy and light chains,
but lacks the constant regions.
“Single-chain antibodies” are Fv molecules in which the heavy and light chain
variable regions have been connected by a flexible linker to form a single polypeptide chain,
which forms an antigen binding region. Single chain antibodies are discussed in detail in
International Patent Application Publication No. WO 88/01649 and United States Patent Nos.
4,946,778 and No. 5,260,203, the disclosures of which are incorporated by reference.
A “domain antibody” is an immunologically functional immunoglobulin fragment
containing only the variable region of a heavy chain or the variable region of a light chain. In
some instances, two or more V regions are covalently joined with a peptide linker to create a
bivalent domain antibody. The two V regions of a bivalent domain antibody can target the
same or different antigens.
A “bivalent antigen binding protein” or “bivalent antibody” comprises two antigen
binding sites. In some instances, the two binding sites have the same antigen specificities.
Bivalent antigen binding proteins and bivalent antibodies can be bispecific, see, infra. A
bivalent antibody other than a “multispecific” or “multifunctional” antibody, in certain
embodiments, typically is understood to have each of its binding sites identical.
A “multispecific antigen binding protein” or “multispecific antibody” is one that
targets more than one antigen or epitope.
A “bispecific,” “dual-specific” or “bifunctional” antigen binding protein or antibody
is a hybrid antigen binding protein or antibody, respectively, having two different antigen
binding sites. Bispecific antigen binding proteins and antibodies are a species of
multispecific antigen binding protein antibody and can be produced by a variety of methods
including, but not limited to, fusion of hybridomas or linking of Fab’ fragments. See, e.g.,
Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J.
Immunol. 148:1547-1553. The two binding sites of a bispecific antigen binding protein or
antibody will bind to two different epitopes, which can reside on the same or different protein
targets.
An antigen binding protein is said to “specifically bind” its target antigen when the
dissociation constant (K ) is ≤10 M. The ABP specifically binds antigen with “high
-9 -10
affinity” when the K is ≤5 x 10 M, and with “very high affinity” when the K is ≤5x 10
M. In one embodiment, the ABP has a K of ≤10 M. In one embodiment, the off-rate is <1
x 10 . In other embodiments, the ABPs will bind to human PCSK9 with a K of between
-9 -13
about 10 M and 10 M, and in yet another embodiment the ABPs will bind with a K ≤5 x
. As will be appreciated by one of skill in the art, in some embodiments, any or all of the
antigen binding fragments can specifically bind to PCSK9.
An antigen binding protein is “selective” when it binds to one target more tightly than
it binds to a second target.
“Antigen binding region” means a protein, or a portion of a protein, that specifically
binds a specified antigen (e.g., a paratope). For example, that portion of an antigen binding
protein that contains the amino acid residues that interact with an antigen and confer on the
antigen binding protein its specificity and affinity for the antigen is referred to as “antigen
binding region.” An antigen binding region typically includes one or more “complementary
binding regions” (“CDRs”). Certain antigen binding regions also include one or more
“framework” regions. A “CDR” is an amino acid sequence that contributes to antigen
binding specificity and affinity. “Framework” regions can aid in maintaining the proper
conformation of the CDRs to promote binding between the antigen binding region and an
antigen. Structurally, framework regions can be located in antibodies between CDRs.
Examples of framework and CDR regions are shown in FIGs. 2A-3D, 3CCC-3JJJ. In some
embodiments, the sequences for CDRs for the light chain of antibody 3B6 are as follows:
CDR1 TLSSGYSSYEVD (SEQ ID NO: 279); CDR2 VDTGGIVGSKGE (SEQ ID
NO: 280); CDR3 GADHGSGTNFVVV (SEQ ID NO: 281), and the FRs are as follows:
FR1 QPVLTQPLFASASLGASVTLTC (SEQ ID NO: 282); FR2
WYQQRPGKGPRFVMR (SEQ ID NO: 283); FR3
GIPDRFSVLGSGLNRYLTIKNIQEEDESDYHC (SEQ ID NO: 284); and FR4
FGGGTKLTVL (SEQ ID NO: 285).
In certain aspects, recombinant antigen binding proteins that bind PCSK9, for
example human PCSK9, are provided. In this context, a “recombinant antigen binding
protein” is a protein made using recombinant techniques, i.e., through the expression of a
recombinant nucleic acid as described herein. Methods and techniques for the production of
recombinant proteins are well known in the art.
The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment
thereof that can compete with the intact antibody for specific binding to the target antigen,
and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An
“antibody” is a species of an antigen binding protein. An intact antibody will generally
comprise at least two full-length heavy chains and two full-length light chains, but in some
instances can include fewer chains such as antibodies naturally occurring in camelids which
can comprise only heavy chains. Antibodies can be derived solely from a single source, or
can be “chimeric,” that is, different portions of the antibody can be derived from two
different antibodies as described further below. The antigen binding proteins, antibodies, or
binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by
enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term
“antibody” includes, in addition to antibodies comprising two full-length heavy chains and
two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples
of which are described below. Furthermore, unless explicitly excluded, antibodies include
monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic
antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies,
humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as
“antibody conjugates”), and fragments thereof, respectively. In some embodiments, the term
also encompasses peptibodies.
Naturally occurring antibody structural units typically comprise a tetramer. Each such
tetramer typically is composed of two identical pairs of polypeptide chains, each pair having
one full-length “light” (in certain embodiments, about 25 kDa) and one full-length “heavy”
chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain
typically includes a variable region of about 100 to 110 or more amino acids that typically is
responsible for antigen recognition. The carboxy-terminal portion of each chain typically
defines a constant region that can be responsible for effector function. Human light chains
are typically classified as kappa and lambda light chains. Heavy chains are typically
classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM,
IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited
to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and
IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and
IgA2. Within full-length light and heavy chains, typically, the variable and constant regions
are joined by a “J” region of about 12 or more amino acids, with the heavy chain also
including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology,
Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its
entirety for all purposes). The variable regions of each light/heavy chain pair typically form
the antigen binding site.
The variable regions typically exhibit the same general structure of relatively
conserved framework regions (FR) joined by three hyper variable regions, also called
complementarity determining regions or CDRs. The CDRs from the two chains of each pair
typically are aligned by the framework regions, which can enable binding to a specific
epitope. From N-terminal to C-terminal, both light and heavy chain variable regions
typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The
assignment of amino acids to each domain is typically in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987);
Chothia et al., Nature, 342:878-883 (1989).
In certain embodiments, an antibody heavy chain binds to an antigen in the absence of
an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen
in the absence of an antibody heavy chain. In certain embodiments, an antibody binding
region binds to an antigen in the absence of an antibody light chain. In certain embodiments,
an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In
certain embodiments, an individual variable region specifically binds to an antigen in the
absence of other variable regions.
In certain embodiments, definitive delineation of a CDR and identification of residues
comprising the binding site of an antibody is accomplished by solving the structure of the
antibody and/or solving the structure of the antibody-ligand complex. In certain
embodiments, that can be accomplished by any of a variety of techniques known to those
skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of
analysis can be employed to identify or approximate the CDR regions. Examples of such
methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM
definition and the contact definition.
The Kabat definition is a standard for numbering the residues in an antibody and is
typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28:
214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia
definition takes into account positions of certain structural loop regions. See, e.g., Chothia et
al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM
definition uses an integrated suite of computer programs produced by Oxford Molecular
Group that model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA),
86:9268-9272 (1989); “AbM , A Computer Program for Modeling Variable Regions of
Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary
structure of an antibody from primary sequence using a combination of knowledge databases
and ab initio methods, such as those described by Samudrala et al., “Ab Initio Protein
Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure,
Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an
analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol.
Biol., 5:732-45 (1996).
By convention, the CDR regions in the heavy chain are typically referred to as H1,
H2, and H3 and are numbered sequentially in the direction from the amino terminus to the
carboxy terminus. The CDR regions in the light chain are typically referred to as L1, L2, and
L3 and are numbered sequentially in the direction from the amino terminus to the carboxy
terminus.
The term “light chain” includes a full-length light chain and fragments thereof having
sufficient variable region sequence to confer binding specificity. A full-length light chain
includes a variable region domain, V , and a constant region domain, C . The variable region
domain of the light chain is at the amino-terminus of the polypeptide. Light chains include
kappa chains and lambda chains.
The term “heavy chain” includes a full-length heavy chain and fragments thereof
having sufficient variable region sequence to confer binding specificity. A full-length heavy
chain includes a variable region domain, V , and three constant region domains, C 1, C 2,
H H H
and C 3. The V domain is at the amino-terminus of the polypeptide, and the C domains
H H H
are at the carboxyl-terminus, with the C 3 being closest to the carboxy-terminus of the
polypeptide. Heavy chains can be of any isotype, including IgG (including IgG1, IgG2, IgG3
and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
A bispecific or bifunctional antibody typically is an artificial hybrid antibody having
two different heavy/light chain pairs and two different binding sites. Bispecific antibodies
can be produced by a variety of methods including, but not limited to, fusion of hybridomas
or linking of Fab' fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79: 315-321
(1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).
Some species of mammals also produce antibodies having only a single heavy chain.
Each individual immunoglobulin chain is typically composed of several
“immunoglobulin domains,” each consisting of roughly 90 to 110 amino acids and having a
characteristic folding pattern. These domains are the basic units of which antibody
polypeptides are composed. In humans, the IgA and IgD isotypes contain four heavy chains
and four light chains; the IgG and IgE isotypes contain two heavy chains and two light
chains; and the IgM isotype contains five heavy chains and five light chains. The heavy
chain C region typically comprises one or more domains that can be responsible for effector
function. The number of heavy chain constant region domains will depend on the isotype.
IgG heavy chains, for example, contain three C region domains known as C 1, C 2 and C 3.
H H H
The antibodies that are provided can have any of these isotypes and subtypes. In certain
embodiments of the present invention, an anti-PCSK9 antibody is of the IgG2 or IgG4
subtype.
The term “variable region” or “variable domain” refers to a portion of the light and/or
heavy chains of an antibody, typically including approximately the amino-terminal 120 to
130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the
light chain. In certain embodiments, variable regions of different antibodies differ
extensively in amino acid sequence even among antibodies of the same species. The
variable region of an antibody typically determines specificity of a particular antibody for its
target
The term “neutralizing antigen binding protein” or “neutralizing antibody” refers to
an antigen binding protein or antibody, respectively, that binds to a ligand and prevents or
reduces the biological effect of that ligand. This can be done, for example, by directly
blocking a binding site on the ligand or by binding to the ligand and altering the ligand’s
ability to bind through indirect means (such as structural or energetic alterations in the
ligand). In some embodiments, the term can also denote an antigen binding protein that
prevents the protein to which it is bound from performing a biological function. In assessing
the binding and/or specificity of an antigen binding protein, e.g., an antibody or
immunologically functional fragment thereof, an antibody or fragment can substantially
inhibit binding of a ligand to its binding partner when an excess of antibody reduces the
quantity of binding partner bound to the ligand by at least about 1-20, 20-30%, 30-40%, 40-
50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or
more (as measured in an in vitro competitive binding assay). In some embodiments, in the
case of PCSK9 antigen binding proteins, such a neutralizing molecule can diminish the
ability of PCSK9 to bind the LDLR. In some embodiments, the neutralizing ability is
characterized and/or described via a competition assay. In some embodiments, the
neutralizing ability is described in terms of an IC or EC value. In some embodiments,
50 50
ABPs 27B2, 13H1, 13B5 and 3C4 are non-neutralizing ABPs, 3B6, 9C9 and 31A4 are weak
neutralizers, and the remaining ABPs in Table 2 are strong neutralizers. In some
embodiments, the antibodies or antigen binding proteins neutralize by binding to PCSK9 and
preventing PCSK9 from binding to LDLR (or reducing the ability of PCSK9 to bind to
LDLR). In some embodiments, the antibodies or ABPs neutralize by binding to PCSK9, and
while still allowing PCSK9 to bind to LDLR, preventing or reducing the PCSK9 mediated
degradation of LDLR. Thus, in some embodiments, a neutralizing ABP or antibody can still
permit PCSK9/LDLR binding, but will prevent (or reduce) subsequent PCSK9 involved
degradation of LDLR.
The term “target” refers to a molecule or a portion of a molecule capable of being
bound by an antigen binding protein. In certain embodiments, a target can have one or more
epitopes. In certain embodiments, a target is an antigen. The use of “antigen” in the phrase
“antigen binding protein” simply denotes that the protein sequence that comprises the antigen
can be bound by an antibody. In this context, it does not require that the protein be foreign or
that it be capable of inducing an immune response.
The term “compete” when used in the context of antigen binding proteins (e.g.,
neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same
epitope means competition between antigen binding proteins as determined by an assay in
which the antigen binding protein (e.g., antibody or immunologically functional fragment
thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference
antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g.,
PCSK9 or a fragment thereof). Numerous types of competitive binding assays can be used to
determine if one antigen binding protein competes with another, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme
immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in
Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986,
J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled
sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold
Spring Harbor Press); solid phase direct label RIA using I-125 label (see, e.g., Morel et al.,
1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et
al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand.
J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to
a solid surface or cells bearing either of these, an unlabelled test antigen binding protein and a
labeled reference antigen binding protein. Competitive inhibition is measured by determining
the amount of label bound to the solid surface or cells in the presence of the test antigen
binding protein. Usually the test antigen binding protein is present in excess. Antigen
binding proteins identified by competition assay (competing antigen binding proteins) include
antigen binding proteins binding to the same epitope as the reference antigen binding proteins
and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the
epitope bound by the reference antigen binding protein for steric hindrance to occur.
Additional details regarding methods for determining competitive binding are provided in the
examples herein. Usually, when a competing antigen binding protein is present in excess, it
will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common
antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or
more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%,
or 97% or more.
The term “antigen” refers to a molecule or a portion of a molecule capable of being
bound by a selective binding agent, such as an antigen binding protein (including, e.g., an
antibody or immunological functional fragment thereof). In some embodiments, the antigen
is capable of being used in an animal to produce antibodies capable of binding to that antigen.
An antigen can possess one or more epitopes that are capable of interacting with different
antigen binding proteins, e.g., antibodies.
The term “epitope” includes any determinant capable being bound by an antigen
binding protein, such as an antibody or to a T-cell receptor. An epitope is a region of an
antigen that is bound by an antigen binding protein that targets that antigen, and when the
antigen is a protein, includes specific amino acids that directly contact the antigen binding
protein. Most often, epitopes reside on proteins, but in some instances can reside on other
kinds of molecules, such as nucleic acids. Epitope determinants can include chemically
active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or
sulfonyl groups, and can have specific three dimensional structural characteristics, and/or
specific charge characteristics. Generally, antibodies specific for a particular target antigen
will preferentially recognize an epitope on the target antigen in a complex mixture of proteins
and/or macromolecules.
As used herein, “substantially pure” means that the described species of molecule is
the predominant species present, that is, on a molar basis it is more abundant than any other
individual species in the same mixture. In certain embodiments, a substantially pure
molecule is a composition wherein the object species comprises at least 50% (on a molar
basis) of all macromolecular species present. In other embodiments, a substantially pure
composition will comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecular
species present in the composition. In other embodiments, the object species is purified to
essential homogeneity wherein contaminating species cannot be detected in the composition
by conventional detection methods and thus the composition consists of a single detectable
macromolecular species.
The term “agent” is used herein to denote a chemical compound, a mixture of
chemical compounds, a biological macromolecule, or an extract made from biological
materials.
As used herein, the terms “label” or “labeled” refers to incorporation of a detectable
marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of
biotin moieties that can be detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by optical or colorimetric
methods). In certain embodiments, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the art and can be used.
Examples of labels for polypeptides include, but are not limited to, the following:
3 14 15 35 90 99 111 125 131
radioisotopes or radionuclides (e.g., H, C, N, S, Y, Tc, In, I, I), fluorescent
labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish
peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl
groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine
zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope
tags). In certain embodiments, labels are attached by spacer arms of various lengths to
reduce potential steric hindrance.
The term “biological sample”, as used herein, includes, but is not limited to, any
quantity of a substance from a living thing or formerly living thing. Such living things
include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such
substances include, but are not limited to, blood, serum, urine, cells, organs, tissues, bone,
bone marrow, lymph nodes, and skin.
The term “pharmaceutical agent composition” (or agent or drug) as used herein refers
to a chemical compound, composition, agent or drug capable of inducing a desired
therapeutic effect when properly administered to a patient. It does not necessarily require
more than one type of ingredient.
The term “therapeutically effective amount” refers to the amount of a PCSK9 antigen
binding protein determined to produce a therapeutic response in a mammal. Such
therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
The term “modulator,” as used herein, is a compound that changes or alters the
activity or function of a molecule. For example, a modulator can cause an increase or
decrease in the magnitude of a certain activity or function of a molecule compared to the
magnitude of the activity or function observed in the absence of the modulator. In certain
embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one
activity or function of a molecule. Certain exemplary activities and functions of a molecule
include, but are not limited to, binding affinity, enzymatic activity, and signal transduction.
Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies,
peptibodies, carbohydrates or small organic molecules. Peptibodies are described in, e.g.,
U.S. Patent No. 6,660,843 (corresponding to PCT Application No. WO 01/83525).
The terms “patient” and “subject” are used interchangeably and include human and
non-human animal subjects as well as those with formally diagnosed disorders, those without
formally recognized disorders, those receiving medical attention, those at risk of developing
the disorders, etc.
The term “treat” and “treatment” includes therapeutic treatments, prophylactic
treatments, and applications in which one reduces the risk that a subject will develop a
disorder or other risk factor. Treatment does not require the complete curing of a disorder
and encompasses embodiments in which one reduces symptoms or underlying risk factors.
The term “prevent” does not require the 100% elimination of the possibility of an
event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced
in the presence of the compound or method.
Standard techniques can be used for recombinant DNA, oligonucleotide synthesis,
and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions
and purification techniques can be performed according to manufacturer's specifications or as
commonly accomplished in the art or as described herein. The foregoing techniques and
procedures can be generally performed according to conventional methods well known in the
art and as described in various general and more specific references that are cited and
discussed throughout the present specification. See, e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless
specific definitions are provided, the nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are those well known and
commonly used in the art. Standard techniques can be used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
Antigen Binding Proteins to PCSK9
Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serine protease involved in
regulating the levels of the low density lipoprotein receptor (LDLR) protein (Horton et al.,
2007; Seidah and Prat, 2007). PCSK9 is a prohormone-proprotein convertase in the subtilisin
(S8) family of serine proteases (Seidah et al., 2003). An exemplary human PCSK9 amino
acid sequence is presented as SEQ ID NOs: 1 and 3. in (depicting the “pro” domain
of the protein as underlined) and (depicting the signal sequence in bold and the pro
domain underlined). An exemplary human PCSK9 coding sequence is presented as SEQ ID
NO: 2 (). As described herein, PCSK9 proteins can also include fragments of the full
length PCSK9 protein. The structure of the PCSK9 protein was solved by two groups
(Cunningham et al., Nature Structural & Molecular Biology, 2007, and Piper et al., Structure,
15:1-8, 2007), the entireties of both of which are herein incorporated by reference. PCSK9
includes a signal sequence, a N-terminal prodomain, a subtilisin-like catalytic domain and a
C-terminal domain.
Antigen binding proteins (ABPs) that bind PCSK9, including human PCSK9, are
provided herein. In some embodiments, the antigen binding proteins provided are
polypeptides which comprise one or more complementary determining regions (CDRs), as
described herein. In some antigen binding proteins, the CDRs are embedded into a
“framework” region, which orients the CDR(s) such that the proper antigen binding
properties of the CDR(s) is achieved. In some embodiments, antigen binding proteins
provided herein can interfere with, block, reduce or modulate the interaction between PCSK9
and LDLR. Such antigen binding proteins are denoted as “neutralizing.” In some
embodiments, binding between PCSK9 and LDLR can still occur, even though the antigen
binding protein is neutralizing and bound to PCSK9. For example, in some embodiments, the
ABP prevents or reduces the adverse influence of PCSK9 on LDLR without blocking the
LDLR binding site on PCSK9. Thus, in some embodiments, the ABP modulates or alters
PCSK9’s ability to result in the degradation of LDLR, without having to prevent the binding
interaction between PCSK9 and LDLR. Such ABPs can be specifically described as “non-
competitively neutralizing” ABPs. In some embodiments, the neutralizing ABP binds to
PCSK9 in a location and/or manner that prevents PCSK9 from binding to LDLR. Such ABPs
can be specifically described as “competitively neutralizing” ABPs. Both of the above
neutralizers can result in a greater amount of free LDLR being present in a subject, which
results in more LDLR binding to LDL (thereby reducing the amount of LDL in the subject).
In turn, this results in a reduction in the amount of serum cholesterol present in a subject.
In some embodiments, the antigen binding proteins provided herein are capable of
inhibiting PCSK9-mediated activity (including binding). In some embodiments, antigen
binding proteins binding to these epitopes inhibit, inter alia, interactions between PCSK9 and
LDLR and other physiological effects mediated by PCSK9. In some embodiments, the
antigen binding proteins are human, such as fully human antibodies to PCSK9.
In some embodiments, the ABP binds to the catalytic domain of PCSK9. In some
embodiments, the ABP binds to the mature form of PCSK9. In some embodiments the ABP
binds in the prodomain of PCSK9. In some embodiments, the ABP selectively binds to the
mature form of PCSK9. In some embodiments, the ABP binds to the catalytic domain in a
manner such that PCSK9 cannot bind or bind as efficiently to LDLR. In some embodiments,
the antigen binding protein does not bind to the c-terminus of the catalytic domain. In some
embodiments, the antigen binding protein does not bind to the n-terminus of the catalytic
domain. In some embodiments, the ABP does not bind to the n- or c-terminus of the PCSK9
protein. In some embodiments, the ABP binds to any one of the epitopes bound by the
antibodies discussed herein. In some embodiments, this can be determined by competition
assays between the antibodies disclosed herein and other antibodies. In some embodiments,
the ABP binds to an epitope bound by one of the antibodies described in Table 2. In some
embodiments, the antigen binding proteins bind to a specific conformational state of PCSK9
so as to prevent PCSK9 from interacting with LDLR. In some embodiments, the ABP binds
to the V domain of PCSK9. In some embodiments, the ABP binds to the V domain of
PCSK9 and prevents (or reduces) PCSK9 from binding to LDLR. In some embodiments, the
ABP binds to the V domain of PCSK9, and while it does not prevent (or reduce) the binding
of PCSK9 to LDLR, the ABP prevents or reduces the adverse activities mediated through
PCSK9 on LDLR.
The antigen binding proteins that are disclosed herein have a variety of utilities.
Some of the antigen binding proteins, for instance, are useful in specific binding assays,
affinity purification of PCSK9, in particular human PCSK9 or its ligands and in screening
assays to identify other antagonists of PCSK9 activity. Some of the antigen binding proteins
are useful for inhibiting binding of PCSK9 to LDLR, or inhibiting PCSK9-mediated
activities.
The antigen binding proteins can be used in a variety of therapeutic applications, as
explained herein. For example, in some embodiments the PCSK9 antigen binding proteins
are useful for treating conditions associated with PCSK9, such as cholesterol related
disorders (or “serum cholesterol related disorders”) such as hypercholesterolemia, as further
described herein. Other uses for the antigen binding proteins include, for example, diagnosis
of PCSK9-associated diseases or conditions and screening assays to determine the presence
or absence of PCSK9. Some of the antigen binding proteins described herein are useful in
treating consequences, symptoms, and/or the pathology associated with PCSK9 activity.
In some embodiments, the antigen binding proteins that are provided comprise one or
more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs). In some embodiments, the antigen binding protein
comprises (a) a polypeptide structure and (b) one or more CDRs that are inserted into and/or
joined to the polypeptide structure. The polypeptide structure can take a variety of different
forms. For example, it can be, or comprise, the framework of a naturally occurring antibody,
or fragment or variant thereof, or can be completely synthetic in nature. Examples of various
polypeptide structures are further described below.
In certain embodiments, the polypeptide structure of the antigen binding proteins is an
antibody or is derived from an antibody, including, but not limited to, monoclonal antibodies,
bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes
referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies,
antibody fusions (sometimes referred to as “antibody conjugates”), and portions or fragments
of each, respectively. In some instances, the antigen binding protein is an immunological
fragment of an antibody (e.g., a Fab, a Fab’, a F(ab’) , or a scFv). The various structures are
further described and defined herein.
Certain of the antigen binding proteins as provided herein specifically and/or
selectively bind to human PCSK9. In some embodiments, the antigen binding protein
specifically and/or selectively binds to human PCSK9 protein having and/or consisting of
residues 153-692 of SEQ ID NO: 3. In some embodiments the ABP specifically and/or
selectively binds to human PCSK9 having and/or consisting of residues 31-152 of SEQ ID
NO: 3. In some embodiments, the ABP selectively binds to a human PCSK9 protein as
depicted in (SEQ ID NO: 1). In some embodiments, the antigen binding protein
specifically binds to at least a fragment of the PCSK9 protein and/or a full length PCSK9
protein, with or without a signal sequence.
In embodiments where the antigen binding protein is used for therapeutic
applications, an antigen binding protein can inhibit, interfere with or modulate one or more
biological activities of PCSK9. In one embodiment, an antigen binding protein binds
specifically to human PCSK9 and/or substantially inhibits binding of human PCSK9 to
LDLR by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by
measuring binding in an in vitro competitive binding assay). Some of the antigen binding
proteins that are provided herein are antibodies. In some embodiments, the ABP has a K of
-7 -8 -9 -10 -11 -12 -13
less (binding more tightly) than 10 , 10 , 10 , 10 , 10 , 10 , 10 M. In some
embodiments, the ABP has an IC for blocking the binding of LDLR to PCSK9 (D374Y,
high affinity variant) of less than 1 microM, 1000 nM to 100 nM, 100nM to 10 nM, 10nM to
1 nM, 1000pM to 500pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM
to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM.
One example of an IgG2 heavy chain constant domain of an anti-PCSK9 antibody of
the present invention has the amino acid sequence as shown in SEQ ID NO: 154, K.
One example of an IgG4 heavy chain constant domain of an anti-PCSK9 antibody of
the present invention has the amino acid sequence as shown in SEQ ID NO: 155, K.
One example of a kappa light chain constant domain of an anti-PCSK9 antibody has
the amino acid sequence as shown in SEQ ID NO: 157, K.
One example of a lambda light chain constant domain of an anti-PCSK9 antibody has
the amino acid sequence as shown in SEQ ID NO: 156, K.
Variable regions of immunoglobulin chains generally exhibit the same overall
structure, comprising relatively conserved framework regions (FR) joined by three
hypervariable regions, more often called “complementarity determining regions” or CDRs.
The CDRs from the two chains of each heavy chain/light chain pair mentioned above
typically are aligned by the framework regions to form a structure that binds specifically with
a specific epitope on the target protein (e.g., PCSK9). From N-terminal to C-terminal,
naturally-occurring light and heavy chain variable regions both typically conform with the
following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A
numbering system has been devised for assigning numbers to amino acids that occupy
positions in each of these domains. This numbering system is defined in Kabat Sequences of
Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia &
Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883.
Various heavy chain and light chain variable regions are provided herein and are
depicted in FIGs. 2A-3JJ and 3LL-3BBB. In some embodiments, each of these variable
regions can be attached to the above heavy and light chain constant regions to form a
complete antibody heavy and light chain, respectively. Further, each of the so generated
heavy and light chain sequences can be combined to form a complete antibody structure.
Specific examples of some of the variable regions of the light and heavy chains of the
antibodies that are provided and their corresponding amino acid sequences are summarized in
TABLE 2.
TABLE 2: Exemplary Heavy and Light Chain Variable Regions
Antibody Light/Heavy
SEQ ID NO
30A4 5/74
3C4 7/85
23B5 9/71
25G4 10/72
31H4 12/67
27B2 13/87
25A7 15/58
27H5 16/52
26H5 17/51
31D1 18/53
20D10 19/48
27E7 20/54
30B9 21/55
19H9 22/56
26E10 23/49
21B12 23/49
17C2 24/57
23G1 26/50
13H1 28/91
9C9 30/64
9H6 31/62
31A4 32/89
1A12 33/65
16F12 35/79
22E2 36/80
27A6 37/76
28B12 38/77
28D6 39/78
31G11 40/83
13B5 42/69
31B12 44/81
3B6 46/60
5H5 421/419
24F7 425/423
22B11 429/427
30F1 433/431
24B9.1 437/435
24B9.2 441/439
20A5.1 445/443
20A5.2 449/447
20E5.1 453/451
20E5.2 457/455
8A3 461/459
11F1 465/463
12H11 469/467
11H4 473/471
11H8 477/475
11G1 481/479
8A1 485/483
Again, each of the exemplary variable heavy chains listed in Table 2 can be combined
with any of the exemplary variable light chains shown in Table 2 to form an antibody. Table
2 shows exemplary light and heavy chain pairings found in several of the antibodies disclosed
herein. In some instances, the antibodies include at least one variable heavy chain and one
variable light chain from those listed in Table 2. In other instances, the antibodies contain
two identical light chains and two identical heavy chains. As an example, an antibody or
antigen binding protein can include a heavy chain and a light chain, two heavy chains, or two
light chains. In some embodiments the antigen binding protein comprises (and/or consists) of
1, 2, and/or 3 heavy and/or light CDRs from at least one of the sequences listed in Table 2
(CDRs for the sequences are outlined in FIGs. 2A-3D, and other embodiments in FIGs.
3CCC-3JJJ and 15A-15D). In some embodiments, all 6 CDRs (CDR1-3 from the light
(CDRL1, CDRL2, CDRL3) and CDR1-3 from the heavy (CDRH1, CDRH2, and CDRH3))
are part of the ABP. In some embodiments, 1, 2, 3, 4, 5, or more CDRs are included in the
ABP. In some embodiments, one heavy and one light CDR from the CDRs in the sequences
in Table 2 is included in the ABP (CDRs for the sequences in table 2 are outlined in FIGs.
2A-3D). In some embodiments, additional sections (e.g., as depicted in -2D, 3A-3D,
and other embodiments in 3CCC-3JJJ and 15A-15D) are also included in the ABP.
Examples of CDRs and FRs for the heavy and light chains noted in Table 2 are outlined in
FIGs. 2A-3D (and other embodiments in FIGs. 3CCC-3JJJ and 15A-15D). Optional light
chain variable sequences (including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4) can be
selected from the following: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,
, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 421, 425, 429, 433, 437, 441, 445, 449, 453,
457, 461,465, 469, 473, 477, 481, and 485.. Optional heavy chain variable sequences
(including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4) can be selected from the
following: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65,
79, 80, 76, 77, 78, 83, 69, 81,60, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, 463,
467, 471, 475, 479, and 483.. In some of the entries in -3D, variations of the
sequences or alternative boundaries of the CDRs and FRs are identified. These alternatives
are identified with a “v1” following the ABP name. As most of these alternatives are minor
in nature, only sections with differences are displayed in the table. It is understood that the
remaining section of the light or heavy chain is the same as shown for the base ABP in the
other panels. Thus, for example, 19H9v1 in has the same FR1, CDR1, and FR2 as
19H9 in as the only difference is noted in . For three of the nucleic acid
sequences (ABPs 26E10, 30B9, and 31B12), additional alternative nucleic acid sequences are
provided in the figures. As will be appreciated by one of skill in the art, no more than one
such sequence need actually be used in the creation of an antibody or ABP. Indeed, in some
embodiments, only one or neither of the specific heavy or light chain nucleic acids need be
present.
In some embodiments, the ABP is encoded by a nucleic acid sequence that can
encode any of the protein sequences in Table 2.
In some embodiments, the ABP binds selectively to the form of PCSK9 that binds to
LDLR (e.g., the autocatalyzed form of the molecule). In some embodiments, the antigen
binding protein does not bind to the c-terminus of the catalytic domain (e.g., the 5. 5-10, 10-
, 15-20, 20-25, 25-30, 30-40 most amino acids in the c-terminus). In some embodiments,
the antigen binding protein does not bind to the n-terminus of the catalytic domain (e.g., the
5. 5-10, 10-15, 15-20, 20-25, 25-30, 30-40 most amino acids in the n-terminus). In some
embodiments, the ABP binds to amino acids within amino acids 1-100 of the mature form of
PCSK9. In some embodiments, the ABP binds to amino acids within (and/or amino acid
sequences consisting of) amino acids 31-100, 100-200, 31-152, 153-692, 200-300, 300-400,
452-683, 400-500, 500-600, 31-692, 31-449, and/or 600-692. In some embodiments, the
ABP binds to the catalytic domain. In some embodiments, the neutralizing and/or non-
neutralizing ABP binds to the prodomain. In some embodiments, the ABP binds to both the
catalytic and pro domains. In some embodiments, the ABP binds to the catalytic domain so
as to obstruct an area on the catalytic domain that interacts with the pro domain. In some
embodiments, the ABP binds to the catalytic domain at a location or surface that the pro-
domain interacts with as outlined in Piper et al. (Structure 15:1-8 (2007), the entirety of
which is hereby incorporated by reference, including the structural representations therein).
In some embodiments, the ABP binds to the catalytic domain and restricts the mobility of the
prodomain. In some embodiments, the ABP binds to the catalytic domain without binding to
the pro-domain. In some embodiments, the ABP binds to the catalytic domain, without
binding to the pro-domain, while preventing the pro-domain from reorienting to allow
PCSK9 to bind to LDLR. In some embodiments, the ABP binds in the same epitope as those
surrounding residues 149-152 of the pro-domain in Piper et al. In some embodiments, the
ABPs bind to the groove (as outlined in Piper et al.) on the V domain. In some embodiments,
the ABPs bind to the histidine-rich patch proximal to the groove on the V domain. In some
embodiments, such antibodies (that bind to the V domain) are not neutralizing. In some
embodiments, antibodies that bind to the V domain are neutralizing. In some embodiments,
the neutralizing ABPs prevent the binding of PCSK9 to LDLR. In some embodiments, the
neutralizing ABPs, while preventing the PCSK9 degradation of LDLR, do not prevent the
binding of PCSK9 to LDLR (for example ABP 31A4). In some embodiments, the ABP binds
to or blocks at least one of the histidines depicted in Figure 4 of the Piper et al. paper. In
some embodiments, the ABP blocks the catalytic triad in PCSK9.
In some embodiments, the antibody binds selectively to variant PCSK9 proteins, e.g.,
D374Y over wild type PCSK9. In some embodiments, these antibodies bind to the variant at
least twice as strongly as the wild type, and preferably 2-5, 5-10, 10-100, 100-1000, 1000-
,000 fold or more to the mutant than the wild type (as measured via a K ). In some
embodiments, the antibody selectively inhibits variant D374Y PCSK9 from interacting with
LDLR over wild type PCSK9’s ability to interact with LDLR. In some embodiments, these
antibodies block the variant’s ability to bind to LDLR more strongly than the wild type’s
ability, e.g., at least twice as strongly as the wild type, and preferably 2-5, 5-10, 10-100, 100-
1000 fold or more to the mutant than the wild type (as measured via an IC ). In some
embodiments, the antibody binds to and neutralizes both wild type PCSK9 and variant forms
of PCSK9, such as D374Y at similar levels. In some embodiments, the antibody binds to
PCSK9 to prevent variants of LDLR from binding to PCSK9. In some embodiments, the
variants of LDLR are at least 50% identical to human LDLR. It is noted that variants of
LDLR are known to those of skill in the art (e.g., Brown MS et al, “Calcium cages, acid baths
and recycling receptors” Nature 388: 629-630, 1997). In some embodiments, the ABP can
raise the level of effective LDLR in heterozygote familial hypercholesterolemia (where a
loss-of function variant of LDLR is present).
In some embodiments, the ABP binds to (but does not block) variants of PCSK9 that
are at least 50%, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99, or greater percent identity to the
form of PCSK9 depicted in and/or . In some embodiments, the ABP binds
to (but does not block) variants of PCSK9 that are at least 50%, 50-60, 60-70, 70-80, 80-90,
90-95, 95-99, or greater percent identity to the mature form of PCSK9 depicted in
and/or . In some embodiments, the ABP binds to and prevents variants of PCSK9
that are at least 50%, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99, or greater percent identity to
the form of PCSK9 depicted in and/or from interacting with LDLR. In
some embodiments, the ABP binds to and prevents variants of PCSK9 that are at least 50, 50-
60, 60-70, 70-80, 80-90, 90-95, 95-99, or greater percent identity to the mature form of
PCSK9 depicted in from interacting with LDLR. In some embodiments, the variant
of PCSK9 is a human variant, such as variants at position 474, E620G, and/or E670G. In
some embodiments, the amino acid at position 474 is valine (as in other humans) or threonine
(as in cyno and mouse). Given the cross-reactivity data presented herein, it is believed that
the present antibodies will readily bind to the above variants.
In some embodiments, the ABP binds to an epitope bound by one of the antibodies
described in Table 2. In some embodiments, the antigen binding proteins bind to a specific
conformational state of PCSK9 so as to prevent PCSK9 from interacting with LDLR.
Humanized Antigen Binding Proteins (e.g., Antibodies)
As described herein, an antigen binding protein to PCSK9 can comprise a humanized
antibody and/or part thereof. An important practical application of such a strategy is the
“humanization” of the mouse humoral immune system.
In certain embodiments, a humanized antibody is substantially non-immunogenic in
humans. In certain embodiments, a humanized antibody has substantially the same affinity
for a target as an antibody from another species from which the humanized antibody is
derived. See, e.g., U.S. Patent 5,530,101, U.S. Patent 5,693,761; U.S. Patent 5,693,762; U.S.
Patent 5,585,089.
In certain embodiments, amino acids of an antibody variable domain that can be
modified without diminishing the native affinity of the antigen binding domain while
reducing its immunogenicity are identified. See, e.g., U.S. Patent Nos. 5,766,886 and
,869,619.
In certain embodiments, modification of an antibody by methods known in the art is
typically designed to achieve increased binding affinity for a target and/or to reduce
immunogenicity of the antibody in the recipient. In certain embodiments, humanized
antibodies are modified to eliminate glycosylation sites in order to increase affinity of the
antibody for its cognate antigen. See, e.g., Co et al., Mol. Immunol., 30:1361-1367 (1993).
In certain embodiments, techniques such as “reshaping,” “hyperchimerization,” or
“veneering/resurfacing” are used to produce humanized antibodies. See, e.g., Vaswami et al.,
Annals of Allergy, Asthma, & Immunol. 81:105 (1998); Roguska et al., Prot. Engineer.,
9:895-904 (1996); and U.S. Patent No. 6,072,035. In certain such embodiments, such
techniques typically reduce antibody immunogenicity by reducing the number of foreign
residues, but do not prevent anti-idiotypic and anti-allotypic responses following repeated
administration of the antibodies. Certain other methods for reducing immunogenicity are
described, e.g., in Gilliland et al., J. Immunol ., 62(6): 3663-71 (1999).
In certain instances, humanizing antibodies results in a loss of antigen binding
capacity. In certain embodiments, humanized antibodies are “back mutated.” In certain such
embodiments, the humanized antibody is mutated to include one or more of the amino acid
residues found in the donor antibody. See, e.g., Saldanha et al., Mol Immunol 36:709-19
(1999).
In certain embodiments the complementarity determining regions (CDRs) of the light
and heavy chain variable regions of an antibody to PCSK9 can be grafted to framework
regions (FRs) from the same, or another, species. In certain embodiments, the CDRs of the
light and heavy chain variable regions of an antibody to PCSK9 can be grafted to consensus
human FRs. To create consensus human FRs, in certain embodiments, FRs from several
human heavy chain or light chain amino acid sequences are aligned to identify a consensus
amino acid sequence. In certain embodiments, the FRs of an antibody to PCSK9 heavy chain
or light chain are replaced with the FRs from a different heavy chain or light chain. In certain
embodiments, rare amino acids in the FRs of the heavy and light chains of an antibody to
PCSK9 are not replaced, while the rest of the FR amino acids are replaced. Rare amino acids
are specific amino acids that are in positions in which they are not usually found in FRs. In
certain embodiments, the grafted variable regions from an antibody to PCSK9 can be used
with a constant region that is different from the constant region of an antibody to PCSK9. In
certain embodiments, the grafted variable regions are part of a single chain Fv antibody.
CDR grafting is described, e.g., in U.S. Patent Nos. 6,180,370, 6,054,297, 5,693,762,
,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101, and in Jones et al., Nature, 321:
522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988), Winter, FEBS Letts., 430:92-94 (1998), which are hereby
incorporated by reference for any purpose.
Human Antigen Binding Proteins (e.g., Antibodies)
As described herein, an antigen binding protein that binds to PCSK9 can comprise a
human (i.e., fully human) antibody and/or part thereof. In certain embodiments, nucleotide
sequences encoding, and amino acid sequences comprising, heavy and light chain
immunoglobulin molecules, particularly sequences corresponding to the variable regions are
provided. In certain embodiments, sequences corresponding to complementarity determining
regions (CDR's), specifically from CDR1 through CDR3, are provided. According to certain
embodiments, a hybridoma cell line expressing such an immunoglobulin molecule is
provided. According to certain embodiments, a hybridoma cell line expressing such a
monoclonal antibody is provided. In certain embodiments a hybridoma cell line is selected
from at least one of the cell lines described in Table 2, e.g., 21B12, 16F12 and 31H4. In
certain embodiments, a purified human monoclonal antibody to human PCSK9 is provided.
One can engineer mouse strains deficient in mouse antibody production with large
fragments of the human Ig loci in anticipation that such mice would produce human
antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the
large variable gene diversity as well as the proper regulation of antibody production and
expression. By exploiting the mouse machinery for antibody diversification and selection
and the lack of immunological tolerance to human proteins, the reproduced human antibody
repertoire in these mouse strains can yield high affinity fully human antibodies against any
antigen of interest, including human antigens. Using the hybridoma technology, antigen-
specific human MAbs with the desired specificity can be produced and selected. Certain
exemplary methods are described in WO 98/24893, U.S. Patent No. 5,545,807, EP 546073,
and EP 546073.
In certain embodiments, one can use constant regions from species other than human
along with the human variable region(s).
The ability to clone and reconstruct megabase sized human loci in yeast artificial
chromosomes (YACs) and to introduce them into the mouse germline provides an approach
to elucidating the functional components of very large or crudely mapped loci as well as
generating useful models of human disease. Furthermore, the utilization of such technology
for substitution of mouse loci with their human equivalents could provide insights into the
expression and regulation of human gene products during development, their communication
with other systems, and their involvement in disease induction and progression.
Human antibodies avoid some of the problems associated with antibodies that possess
murine or rat variable and/or constant regions. The presence of such murine or rat derived
proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an
immune response against the antibody by a patient. In order to avoid the utilization of
murine or rat derived antibodies, fully human antibodies can be generated through the
introduction of functional human antibody loci into a rodent, other mammal or animal so that
the rodent, other mammal or animal produces fully human antibodies.
Humanized antibodies are those antibodies that, while initially starting off containing
antibody amino acid sequences that are not human, have had at least some of these nonhuman
antibody amino acid sequences replaced with human antibody sequences. This is in contrast
with human antibodies, in which the antibody is encoded (or capable of being encoded) by
genes possessed a human.
Antigen Binding Protein Variants
Other antibodies that are provided are variants of the ABPs listed above formed by
combination or subparts of the variable heavy and variable light chains shown in Table 2 and
comprise variable light and/or variable heavy chains that each have at least 50%, 50-60, 60-
70, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or above 99% identity to the amino
acid sequences of the sequences in Table 2 (either the entire sequence or a subpart of the
sequence, e.g., one or more CDR). In some instances, such antibodies include at least one
heavy chain and one light chain, whereas in other instances the variant forms contain two
identical light chains and two identical heavy chains (or subparts thereof). In some
embodiments, the sequence comparison in -3D (and 13A-13J, other embodiments in
15A-15D and FIGs. 48A and 48B) can be used in order to identify sections of the antibodies
that can be modified by observing those variations that impact binding and those variations
that do not appear to impact binding. For example, by comparing similar sequences, one can
identify those sections (e.g., particular amino acids) that can be modified and how they can be
modified while still retaining (or improving) the functionality of the ABP. In some
embodiments, variants of ABPs include those consensus groups and sequences depicted in
FIGs. 13A, 13C, 13F, 13G, 13H, 13I, 13J, and/or 48A and 48B and variations are allowed in
the positions identified as variable in the figures. The CDRs shown in FIGs. 13A, 13C, 13F,
13G, 48A and 48B were defined based upon a hybrid combination of the Chothia method
(based on the location of the structural loop regions, see, e.g., “Standard conformations for
the canonical structures of immunoglobulins,” Bissan Al-Lazikani, Arthur M. Lesk and Cyrus
Chothia, Journal of Molecular Biology, 273(4): 927-948, 7 November (1997)) and the Kabat
method (based on sequence variability, see, e.g., Sequences of Proteins of Immunological
Interest, Fifth Edition. NIH Publication No. 91-3242, Kabat et al., (1991)). Each residue
determined by either method, was included in the final list of CDR residues (and is presented
in FIGs. 13A, 13C, 13F, 13G, and 48A and 48B). The CDRs in FIGs. 13H, 13I, and 13J
were obtained by the Kabat method alone. Unless specified otherwise, the defined consensus
sequences, CDRs, and FRs in FIGs. 13H-13J will define and control the noted CDRs and FRs
for the referenced ABPs in .
In certain embodiments, an antigen binding protein comprises a heavy chain
comprising a variable region comprising an amino acid sequence at least 90% identical to an
amino acid sequence selected from at least one of the sequences of SEQ ID NO: 74, 85, 71,
72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83,
69, 81, and 60. In certain embodiments, an antigen binding protein comprises a heavy chain
comprising a variable region comprising an amino acid sequence at least 95% identical to an
amino acid sequence selected from at least one of the sequences of SEQ ID NO: 74, 85, 71,
72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83,
69, 81, and 60. In certain embodiments, an antigen binding protein comprises a heavy chain
comprising a variable region comprising an amino acid sequence at least 99% identical to an
amino acid sequence selected from at least one of the sequences of SEQ ID NO: 74, 85, 71,
72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83,
69, 81, and 60.
In some embodiments, the antigen binding protein comprises a sequence that is at
least 90%, 90-95%, and/or 95-99% identical to one or more CDRs from the CDRs in at least
one of sequences of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57,
50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In some embodiments, 1, 2, 3, 4,
, or 6 CDR (each being at least 90%, 90-95%, and/or 95-99% identical to the above
sequences) is present.
In some embodiments, the antigen binding protein comprises a sequence that is at
least 90%, 90-95%, and/or 95-99% identical to one or more FRs from the FRs in at least one
of sequences of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50,
91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In some embodiments, 1, 2, 3, or 4
FR (each being at least 90%, 90-95%, and/or 95-99% identical to the above sequences) is
present.
In certain embodiments, an antigen binding protein comprises a light chain
comprising a variable region comprising an amino acid sequence at least 90% identical to an
amino acid sequence selected from at least one of the sequences of SEQ ID NO: 5, 7, 9, 10,
12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,
44, and 46. In certain embodiments, an antigen binding protein comprises a light chain
comprising a variable region comprising an amino acid sequence at least 95% identical to an
amino acid sequence selected from at least one of the sequences of SEQ ID NO: 5, 7, 9, 10,
12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,
44, and 46. In certain embodiments, an antigen binding protein comprises a light chain
comprising a variable region comprising an amino acid sequence at least 99% identical to an
amino acid sequence selected from at least one of the sequences of SEQ ID NO: 5, 7, 9, 10,
12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,
44, and 46.
In some embodiments, the antigen binding protein comprises a sequence that is at
least 90%, 90-95%, and/or 95-99% identical to one or more CDRs from the CDRs in at least
one of sequences of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26,
28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, 1, 2, 3, 4, 5,
or 6 CDR (each being at least 90%, 90-95%, and/or 95-99% identical to the above sequences)
is present.
In some embodiments, the antigen binding protein comprises a sequence that is at
least 90%, 90-95%, and/or 95-99% identical to one or more FRs from the FRs in at least one
of sequences of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,
, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, 1, 2, 3, or 4 FR
(each being at least 90%, 90-95%, and/or 95-99% identical to the above sequences) is
present.
In light of the present disclosure, a skilled artisan will be able to determine suitable
variants of the ABPs as set forth herein using well-known techniques. In certain
embodiments, one skilled in the art can identify suitable areas of the molecule that may be
changed without destroying activity by targeting regions not believed to be important for
activity. In certain embodiments, one can identify residues and portions of the molecules that
are conserved among similar polypeptides. In certain embodiments, even areas that can be
important for biological activity or for structure can be subject to conservative amino acid
substitutions without destroying the biological activity or without adversely affecting the
polypeptide structure.
Additionally, one skilled in the art can review structure-function studies identifying
residues in similar polypeptides that are important for activity or structure. In view of such a
comparison, one can predict the importance of amino acid residues in a protein that
correspond to amino acid residues which are important for activity or structure in similar
proteins. One skilled in the art can opt for chemically similar amino acid substitutions for
such predicted important amino acid residues.
One skilled in the art can also analyze the three-dimensional structure and amino acid
sequence in relation to that structure in similar ABPs. In view of such information, one
skilled in the art can predict the alignment of amino acid residues of an antibody with respect
to its three dimensional structure. In certain embodiments, one skilled in the art can choose
not to make radical changes to amino acid residues predicted to be on the surface of the
protein, since such residues can be involved in important interactions with other molecules.
Moreover, one skilled in the art can generate test variants containing a single amino acid
substitution at each desired amino acid residue. The variants can then be screened using
activity assays known to those skilled in the art. Such variants can be used to gather
information about suitable variants. For example, if one discovered that a change to a
particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable
activity, variants with such a change can be avoided. In other words, based on information
gathered from such routine experiments, one skilled in the art can readily determine the
amino acids where further substitutions should be avoided either alone or in combination
with other mutations.
A number of scientific publications have been devoted to the prediction of secondary
structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al.,
Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974);
Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.
Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979). Moreover,
computer programs are currently available to assist with predicting secondary structure. One
method of predicting secondary structure is based upon homology modeling. For example,
two polypeptides or proteins which have a sequence identity of greater than 30%, or
similarity greater than 40% often have similar structural topologies. The recent growth of the
protein structural database (PDB) has provided enhanced predictability of secondary
structure, including the potential number of folds within a polypeptide’s or protein’s
structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested
(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited number
of folds in a given polypeptide or protein and that once a critical number of structures have
been resolved, structural prediction will become dramatically more accurate.
Additional methods of predicting secondary structure include “threading” (Jones, D.,
Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)),
“profile analysis” (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym.,
183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci. USA, 84(13):4355-4358 (1987)),
and “evolutionary linkage” (See Holm, supra (1999), and Brenner, supra (1997)).
In certain embodiments, antigen binding protein variants include glycosylation
variants wherein the number and/or type of glycosylation site has been altered compared to
the amino acid sequences of a parent polypeptide. In certain embodiments, protein variants
comprise a greater or a lesser number of N-linked glycosylation sites than the native protein.
An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr,
wherein the amino acid residue designated as X can be any amino acid residue except proline.
The substitution of amino acid residues to create this sequence provides a potential new site
for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which
eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided
is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked
glycosylation sites (typically those that are naturally occurring) are eliminated and one or
more new N-linked sites are created. Additional preferred antibody variants include cysteine
variants wherein one or more cysteine residues are deleted from or substituted for another
amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants
can be useful when antibodies must be refolded into a biologically active conformation such
as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer
cysteine residues than the native protein, and typically have an even number to minimize
interactions resulting from unpaired cysteines.
According to certain embodiments, amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding
affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or
modify other physicochemical or functional properties on such polypeptides. According to
certain embodiments, single or multiple amino acid substitutions (in certain embodiments,
conservative amino acid substitutions) can be made in the naturally-occurring sequence (in
certain embodiments, in the portion of the polypeptide outside the domain(s) forming
intermolecular contacts). In certain embodiments, a conservative amino acid substitution
typically may not substantially change the structural characteristics of the parent sequence
(e.g., a replacement amino acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that characterizes the parent
sequence). Examples of art-recognized polypeptide secondary and tertiary structures are
described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman
and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze,
eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354:105
(1991), which are each incorporated herein by reference.
In some embodiments, the variants are variants of the nucleic acid sequences of the
ABPs disclosed herein. One of skill in the art will appreciate that the above discussion can be
used for identifying, evaluating, and/creating ABP protein variants and also for nucleic acid
sequences that can encode for those protein variants. Thus, nucleic acid sequences encoding
for those protein variants (as well as nucleic acid sequences that encode for the ABPs in
Table 2, but are different from those explicitly disclosed herein) are contemplated. For
example, an ABP variant can have at least 80, 80-85, 85-90, 90-95, 95-97, 97-99 or greater
identity to at least one nucleic acid sequence described in SEQ ID NOs: 152, 153, 92, 93, 94,
95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,
115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151
or at least one to six (and various combinations thereof) of the CDR(s) encoded by the
nucleic acid sequences in SEQ ID NOs: 152, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,
120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, and 151.
In some embodiments, the antibody (or nucleic acid sequence encoding it) is a variant
if the nucleic acid sequence that encodes the particular ABP (or the nucleic acid sequence
itself) can selectively hybridize to any of the nucleic acid sequences that encode the proteins
in Table 2 (such as, but not limited to SEQ ID NO: 152, 153, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, and 151) under
stringent conditions. In one embodiment, suitable moderately stringent conditions include
prewashing in a solution of 5XSSC; 0.5% SDS, 1.0 mM EDTA (pH 8:0); hybridizing at 50
C, -65 C, 5xSSC, overnight or, in the event of cross-species homology, at 45 C with
0.5xSSC; followed by washing twice at 65 C for 20 minutes with each of 2x, 0.5x and
0.2xSSC containing 0.1% SDS. Such hybridizing DNA sequences are also within the scope
of this invention, as are nucleotide sequences that, due to code degeneracy, encode an
antibody polypeptide that is encoded by a hybridizing DNA sequence and the amino acid
sequences that are encoded by these nucleic acid sequences. In some embodiments, variants
of CDRs include nucleic acid sequences and the amino acid sequences encoded by those
sequences, that hybridize to one or more of the CDRs within the sequences noted above
(individual CDRs can readily be determined in light of FIGs. 2A-3D, and other embodiments
in FIGs. 3CCC-3JJJ and 15A-15D). The phrase "selectively hybridize" referred to in this
context means to detectably and selectively bind. Polynucleotides, oligonucleotides and
fragments thereof in accordance with the invention selectively hybridize to nucleic acid
strands under hybridization and wash conditions that minimize appreciable amounts of
detectable binding to nonspecific nucleic acids. High stringency conditions can be used to
achieve selective hybridization conditions as known in the art and discussed herein.
Generally, the nucleic acid sequence homology between the polynucleotides,
oligonucleotides, and fragments of the invention and a nucleic acid sequence of interest will
be at least 80%, and more typically with preferably increasing homologies of at least 85%,
90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is a partial or
complete identity between their sequences. For example, 85% homology means that 85% of
the amino acids are identical when the two sequences are aligned for maximum matching.
Gaps (in either of the two sequences being matched) are allowed in maximizing matching;
gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and
preferably, two protein sequences (or polypeptide sequences derived from them of at least 30
amino acids in length) are homologous, as this term is used herein, if they have an alignment
score of at more than 5 (in standard deviation units) using the program ALIGN with the
mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of
Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research
Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. The two sequences or parts
thereof are more preferably homologous if their amino acids are greater than or equal to 50%
identical when optimally aligned using the ALIGN program. The term "corresponds to" is
used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not
strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or
that a polypeptide sequence is identical to a reference polypeptide sequence. In
contradistinction, the term "complementary to" is used herein to mean that the
complementary sequence is homologous to all or a portion of a reference polynucleotide
sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference
sequence "TATAC" and is complementary to a reference sequence "GTATA".
Preparation of Antigen Binding Proteins (e.g., Antibodies)
In certain embodiments, antigen binding proteins (such as antibodies) are produced by
immunization with an antigen (e.g., PCSK9). In certain embodiments, antibodies can be
produced by immunization with full-length PCSK9, a soluble form of PCSK9, the catalytic
domain alone, the mature form of PCSK9 shown in , a splice variant form of PCSK9,
or a fragment thereof. In certain embodiments, the antibodies of the invention can be
polyclonal or monoclonal, and/or can be recombinant antibodies. In certain embodiments,
antibodies of the invention are human antibodies prepared, for example, by immunization of
transgenic animals capable of producing human antibodies (see, for example, PCT Published
Application No. W0 93/12227).
In certain embodiments, certain strategies can be employed to manipulate inherent
properties of an antibody, such as the affinity of an antibody for its target. Such strategies
include, but are not limited to, the use of site-specific or random mutagenesis of the
polynucleotide molecule encoding an antibody to generate an antibody variant. In certain
embodiments, such generation is followed by screening for antibody variants that exhibit the
desired change, e.g. increased or decreased affinity.
In certain embodiments, the amino acid residues targeted in mutagenic strategies are
those in the CDRs. In certain embodiments, amino acids in the framework regions of the
variable domains are targeted. In certain embodiments, such framework regions have been
shown to contribute to the target binding properties of certain antibodies. See, e.g., Hudson,
Curr. Opin. Biotech., 9:395-402 (1999) and references therein.
In certain embodiments, smaller and more effectively screened libraries of antibody
variants are produced by restricting random or site-directed mutagenesis to hyper-mutation
sites in the CDRs, which are sites that correspond to areas prone to mutation during the
somatic affinity maturation process. See, e.g., Chowdhury & Pastan, Nature Biotech., 17:
568-572 (1999) and references therein. In certain embodiments, certain types of DNA
elements can be used to identify hyper-mutation sites including, but not limited to, certain
direct and inverted repeats, certain consensus sequences, certain secondary structures, and
certain palindromes. For example, such DNA elements that can be used to identify hyper-
mutation sites include, but are not limited to, a tetrabase sequence comprising a purine (A or
G), followed by guainine (G), followed by a pyrimidine (C or T), followed by either
adenosine or thymidine (A or T) (i.e., A/G-G-C/T-A/T). Another example of a DNA element
that can be used to identify hyper-mutation sites is the serine codon, A-G-C/T.
Preparation of Fully Human ABPs (e.g., Antibodies)
In certain embodiments, a phage display technique is used to generate monoclonal
antibodies. In certain embodiments, such techniques produce fully human monoclonal
antibodies. In certain embodiments, a polynucleotide encoding a single Fab or Fv antibody
fragment is expressed on the surface of a phage particle. See, e.g., Hoogenboom et al., J.
Mol. Biol., 227: 381 (1991); Marks et al., J Mol Biol 222: 581 (1991); U.S. Patent No.
,885,793. In certain embodiments, phage are “screened” to identify those antibody
fragments having affinity for target. Thus, certain such processes mimic immune selection
through the display of antibody fragment repertoires on the surface of filamentous
bacteriophage, and subsequent selection of phage by their binding to target. In certain such
procedures, high affinity functional neutralizing antibody fragments are isolated. In certain
such embodiments (discussed in more detail below), a complete repertoire of human antibody
genes is created by cloning naturally rearranged human V genes from peripheral blood
lymphocytes. See, e.g., Mullinax et al., Proc Natl Acad Sci (USA), 87: 8095-8099 (1990).
According to certain embodiments, antibodies of the invention are prepared through
the utilization of a transgenic mouse that has a substantial portion of the human antibody
producing genome inserted but that is rendered deficient in the production of endogenous,
murine antibodies. Such mice, then, are capable of producing human immunoglobulin
molecules and antibodies and are deficient in the production of murine immunoglobulin
molecules and antibodies. Technologies utilized for achieving this result are disclosed in the
patents, applications and references disclosed in the specification, herein. In certain
embodiments, one can employ methods such as those disclosed in PCT Published
Application No. WO 98/24893 or in Mendez et al., Nature Genetics, 15:146-156 (1997),
which are hereby incorporated by reference for any purpose.
Generally, fully human monoclonal ABPs (e.g., antibodies) specific for PCSK9 can
be produced as follows. Transgenic mice containing human immunoglobulin genes are
immunized with the antigen of interest, e.g. PCSK9, lymphatic cells (such as B-cells) from
the mice that express antibodies are obtained. Such recovered cells are fused with a myeloid-
type cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are
screened and selected to identify hybridoma cell lines that produce antibodies specific to the
antigen of interest. In certain embodiments, the production of a hybridoma cell line that
produces antibodies specific to PCSK9 is provided.
In certain embodiments, fully human antibodies are produced by exposing human
splenocytes (B or T cells) to an antigen in vitro, and then reconstituting the exposed cells in
an immunocompromised mouse, e.g. SCID or nod/SCID. See, e.g., Brams et al., J.Immunol.
160: 2051-2058 (1998); Carballido et al., Nat. Med., 6: 103-106 (2000). In certain such
approaches, engraftment of human fetal tissue into SCID mice (SCID-hu) results in long-term
hematopoiesis and human T-cell development. See, e.g., McCune et al., Science, 241:1532-
1639 (1988); Ifversen et al., Sem. Immunol., 8:243-248 (1996). In certain instances, humoral
immune response in such chimeric mice is dependent on co-development of human T-cells in
the animals. See, e.g., Martensson et al., Immunol., 83:1271-179 (1994). In certain
approaches, human peripheral blood lymphocytes are transplanted into SCID mice. See, e.g.,
Mosier et al., Nature, 335:256-259 (1988). In certain such embodiments, when such
transplanted cells are treated either with a priming agent, such as Staphylococcal Enterotoxin
A (SEA), or with anti-human CD40 monoclonal antibodies, higher levels of B cell production
is detected. See, e.g., Martensson et al., Immunol., 84: 224-230 (1995); Murphy et al., Blood,
86:1946-1953 (1995).
Thus, in certain embodiments, fully human antibodies can be produced by the
expression of recombinant DNA in host cells or by expression in hybridoma cells. In other
embodiments, antibodies can be produced using the phage display techniques described
herein.
The antibodies described herein were prepared through the utilization of the
XenoMouse technology, as described herein. Such mice, then, are capable of producing
human immunoglobulin molecules and antibodies and are deficient in the production of
murine immunoglobulin molecules and antibodies. Technologies utilized for achieving the
same are disclosed in the patents, applications, and references disclosed in the background
section herein. In particular, however, a preferred embodiment of transgenic production of
mice and antibodies therefrom is disclosed in U.S. Patent Application Serial No. 08/759,620,
filed December 3, 1996 and International Patent Application Nos. WO 98/24893, published
June 11, 1998 and WO 00/76310, published December 21, 2000, the disclosures of which are
hereby incorporated by reference. See also Mendez et al., Nature Genetics, 15:146-156
(1997), the disclosure of which is hereby incorporated by reference.
Through the use of such technology, fully human monoclonal antibodies to a variety
of antigens have been produced. Essentially, XenoMouse lines of mice are immunized with
an antigen of interest (e.g. PCSK9), lymphatic cells (such as B-cells) are recovered from the
hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell
line to prepare immortal hybridoma cell lines. These hybridoma cell lines are screened and
selected to identify hybridoma cell lines that produced antibodies specific to the antigen of
interest. Provided herein are methods for the production of multiple hybridoma cell lines that
produce antibodies specific to PCSK9 Further, provided herein are characterization of the
antibodies produced by such cell lines, including nucleotide and amino acid sequence
analyses of the heavy and light chains of such antibodies.
The production of the XenoMouse strains of mice is further discussed and delineated
in U.S. Patent Application Serial Nos. 07/466,008, filed January 12, 1990, 07/610,515, filed
November 8, 1990, 07/919,297, filed July 24, 1992, 07/922,649, filed July 30, 1992,
08/031,801, filed March 15, 1993, 08/112,848, filed August 27, 1993, 08/234,145, filed April
28, 1994, 08/376,279, filed January 20, 1995, 08/430, 938, filed April 27, 1995, 08/464,584,
filed June 5, 1995, 08/464,582, filed June 5, 1995, 08/463,191, filed June 5, 1995,
08/462,837, filed June 5, 1995, 08/486,853, filed June 5, 1995, 08/486,857, filed June 5,
1995, 08/486,859, filed June 5, 1995, 08/462,513, filed June 5, 1995, 08/724,752, filed
October 2, 1996, 08/759,620, filed December 3, 1996, U.S. Publication 2003/0093820, filed
November 30, 2001 and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and
,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also
European Patent No., EP 0 463 151 B1, grant published June 12, 1996, International Patent
Application No., WO 94/02602, published February 3, 1994, International Patent Application
No., WO 96/34096, published October 31, 1996, WO 98/24893, published June 11, 1998,
WO 00/76310, published December 21, 2000. The disclosures of each of the above-cited
patents, applications, and references are hereby incorporated by reference in their entirety.
In an alternative approach, others, including GenPharm International, Inc., have
utilized a “minilocus” approach. In the minilocus approach, an exogenous Ig locus is
mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or
more V genes, one or more D genes, one or more J genes, a mu constant region, and
H H H
usually a second constant region (preferably a gamma constant region) are formed into a
construct for insertion into an animal. This approach is described in U.S. Patent No.
,545,807 to Surani et al. and U.S. Patent Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425,
,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each to
Lonberg & Kay, U.S. Patent No. 5,591,669 and 6,023.010 to Krimpenfort & Berns, U.S.
Patent Nos. 5,612,205, 5,721,367, and 5,789,215 to Berns et al., and U.S. Patent No.
,643,763 to Choi & Dunn, and GenPharm International U.S. Patent Application Serial Nos.
07/574,748, filed August 29, 1990, 07/575,962, filed August 31, 1990, 07/810,279, filed
December 17, 1991, 07/853,408, filed March 18, 1992, 07/904,068, filed June 23, 1992,
07/990,860, filed December 16, 1992, 08/053,131, filed April 26, 1993, 08/096,762, filed
July 22, 1993, 08/155,301, filed November 18, 1993, 08/161,739, filed December 3, 1993,
08/165,699, filed December 10, 1993, 08/209,741, filed March 9, 1994, the disclosures of
which are hereby incorporated by reference. See also European Patent No. 0 546 073 B1,
International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO
92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and
WO 98/24884 and U.S. Patent No. 5,981,175, the disclosures of which are hereby
incorporated by reference in their entirety. See further Taylor et al., 1992, Chen et al., 1993,
Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al., (1994), and
Tuaillon et al., (1995), Fishwild et al., (1996), the disclosures of which are hereby
incorporated by reference in their entirety.
Kirin has also demonstrated the generation of human antibodies from mice in which,
through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been
introduced. See European Patent Application Nos. 773 288 and 843 961, the disclosures of
which are hereby incorporated by reference. Additionally, KM mice, which are the result
of cross-breeding of Kirin’s Tc mice with Medarex’s minilocus (Humab) mice have been
generated. These mice possess the human IgH transchromosome of the Kirin mice and the
kappa chain transgene of the Genpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-
102).
Human antibodies can also be derived by in vitro methods. Suitable examples include
but are not limited to phage display (CAT, Morphosys, Dyax, Biosite/Medarex, Xoma,
Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display (CAT), yeast display,
and the like.
In some embodiments, the antibodies described herein possess human IgG4 heavy
chains as well as IgG2 heavy chains. Antibodies can also be of other human isotypes,
including IgG1. The antibodies possessed high affinities, typically possessing a Kd of from
-6 -13
about 10 through about 10 M or below, when measured by various techniques.
As will be appreciated, antibodies can be expressed in cell lines other than hybridoma
cell lines. Sequences encoding particular antibodies can be used to transform a suitable
mammalian host cell. Transformation can be by any known method for introducing
polynucleotides into a host cell, including, for example packaging the polynucleotide in a
virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by
transfection procedures known in the art, as exemplified by U.S. Patent Nos. 4,399,216,
4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by
reference). The transformation procedure used depends upon the host to be transformed.
Methods for introducing heterologous polynucleotides into mammalian cells are well known
in the art and include dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
Mammalian cell lines available as hosts for expression are well known in the art and
include many immortalized cell lines available from the American Type Culture Collection
(ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma
cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Cell
lines of particular preference are selected through determining which cell lines have high
expression levels and produce antibodies with constitutive PCSK9 binding properties.
In certain embodiments, antibodies and/or ABP are produced by at least one of the
following hybridomas: 21B12, 31H4, 16F12, any the other hybridomas listed in Table 2 or
disclosed in the examples. In certain embodiments, antigen binding proteins bind to PCSK9
with a dissociation constant (K ) of less than approximately 1 nM, e.g., 1000pM to 100 pM,
100 pM to 10 pM, 10 pM to 1 pM, and/or 1 pM to 0.1 pM or less.
In certain embodiments, antigen binding proteins comprise an immunoglobulin
molecule of at least one of the IgG1, IgG2, IgG3, IgG4, Ig E, IgA, IgD, and IgM isotype. In
certain embodiments, antigen binding proteins comprise a human kappa light chain and/or a
human heavy chain. In certain embodiments, the heavy chain is of the IgG1, IgG2, IgG3,
IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, antigen binding proteins have
been cloned for expression in mammalian cells. In certain embodiments, antigen binding
proteins comprise a constant region other than any of the constant regions of the IgG1, IgG2,
IgG3, IgG4, IgE, IgA, IgD, and IgM isotype.
In certain embodiments, antigen binding proteins comprise a human lambda light
chain and a human IgG2 heavy chain. In certain embodiments, antigen binding proteins
comprise a human lambda light chain and a human IgG4 heavy chain. In certain
embodiments, antigen binding proteins comprise a human lambda light chain and a human
IgG1, IgG3, IgE, IgA, IgD or IgM heavy chain. In other embodiments, antigen binding
proteins comprise a human kappa light chain and a human IgG2 heavy chain. In certain
embodiments, antigen binding proteins comprise a human kappa light chain and a human
IgG4 heavy chain. In certain embodiments, antigen binding proteins comprise a human
kappa light chain and a human IgG1, IgG3, IgE, IgA, IgD or IgM heavy chain. In certain
embodiments, antigen binding proteins comprise variable regions of antibodies ligated to a
constant region that is neither the constant region for the IgG2 isotype, nor the constant
region for the IgG4 isotype. In certain embodiments, antigen binding proteins have been
cloned for expression in mammalian cells.
In certain embodiments, conservative modifications to the heavy and light chains of
antibodies from at least one of the hybridoma lines: 21B12, 31H4 and 16F12 (and
corresponding modifications to the encoding nucleotides) will produce antibodies to PCSK9
having functional and chemical characteristics similar to those of the antibodies from the
hybridoma lines: 21B12, 31H4 and 16F12. In contrast, in certain embodiments, substantial
modifications in the functional and/or chemical characteristics of antibodies to PCSK9 can be
accomplished by selecting substitutions in the amino acid sequence of the heavy and light
chains that differ significantly in their effect on maintaining (a) the structure of the molecular
backbone in the area of the substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side
chain.
For example, a “conservative amino acid substitution” can involve a substitution of a
native amino acid residue with a nonnative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position. Furthermore, any native residue
in the polypeptide can also be substituted with alanine, as has been previously described for
“alanine scanning mutagenesis.”
Desired amino acid substitutions (whether conservative or non-conservative) can be
determined by those skilled in the art at the time such substitutions are desired. In certain
embodiments, amino acid substitutions can be used to identify important residues of
antibodies to PCSK9, or to increase or decrease the affinity of the antibodies to PCSK9 as
described herein.
In certain embodiments, antibodies of the present invention can be expressed in cell
lines other than hybridoma cell lines. In certain embodiments, sequences encoding particular
antibodies can be used for transformation of a suitable mammalian host cell. According to
certain embodiments, transformation can be by any known method for introducing
polynucleotides into a host cell, including, for example packaging the polynucleotide in a
virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by
transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216,
4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by
reference for any purpose). In certain embodiments, the transformation procedure used can
depend upon the host to be transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are well known in the art and include, but are not
limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene
mediated transfection, protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
Mammalian cell lines available as hosts for expression are well known in the art and
include, but are not limited to, many immortalized cell lines available from the American
Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO)
cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. In certain
embodiments, cell lines can be selected through determining which cell lines have high
expression levels and produce antibodies with constitutive HGF binding properties.
Appropriate expression vectors for mammalian host cells are well known.
In certain embodiments, antigen binding proteins comprise one or more polypeptides.
In certain embodiments, any of a variety of expression vector/host systems can be utilized to
express polynucleotide molecules encoding polypeptides comprising one or more ABP
components or the ABP itself. Such systems include, but are not limited to, microorganisms,
such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors; yeast transformed with yeast expression vectors; insect cell systems
infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with
virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV)
or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell
systems.
In certain embodiments, a polypeptide comprising one or more ABP components or
the ABP itself is recombinantly expressed in yeast. Certain such embodiments use
commercially available expression systems, e.g., the Pichia Expression System (Invitrogen,
San Diego, CA), following the manufacturer's instructions. In certain embodiments, such a
system relies on the pre-pro-alpha sequence to direct secretion. In certain embodiments,
transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction
by methanol.
In certain embodiments, a secreted polypeptide comprising one or more ABP
components or the ABP itself is purified from yeast growth medium. In certain
embodiments, the methods used to purify a polypeptide from yeast growth medium is the
same as those used to purify the polypeptide from bacterial and mammalian cell supernatants.
In certain embodiments, a nucleic acid encoding a polypeptide comprising one or
more ABP components or the ABP itself is cloned into a baculovirus expression vector, such
as pVL1393 (PharMingen, San Diego, CA). In certain embodiments, such a vector can be
used according to the manufacturer's directions (PharMingen) to infect Spodoptera
frugiperda cells in sF9 protein-free media and to produce recombinant polypeptide. In
certain embodiments, a polypeptide is purified and concentrated from such media using a
heparin-Sepharose column (Pharmacia).
In certain embodiments, a polypeptide comprising one or more ABP components or
the ABP itself is expressed in an insect system. Certain insect systems for polypeptide
expression are well known to those of skill in the art. In one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes
in Spodoptera frugiperda cells or in Trichoplusia larvae. In certain embodiments, a nucleic
acid molecule encoding a polypeptide can be inserted into a nonessential gene of the virus,
for example, within the polyhedrin gene, and placed under control of the promoter for that
gene. In certain embodiments, successful insertion of a nucleic acid molecule will render the
nonessential gene inactive. In certain embodiments, that inactivation results in a detectable
characteristic. For example, inactivation of the polyhedrin gene results in the production of
virus lacking coat protein.
In certain embodiments, recombinant viruses can be used to infect S. frugiperda cells
or Trichoplusia larvae. See, e.g., Smith et al., J. Virol., 46: 584 (1983); Engelhard et al.,
Proc. Nat. Acad. Sci. (USA), 91: 3224-7 (1994).
In certain embodiments, polypeptides comprising one or more ABP components or
the ABP itself made in bacterial cells are produced as insoluble inclusion bodies in the
bacteria. In certain embodiments, host cells comprising such inclusion bodies are collected
by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with
0.1 mg/ml lysozyme (Sigma, St. Louis, MO) for 15 minutes at room temperature. In certain
embodiments, the lysate is cleared by sonication, and cell debris is pelleted by centrifugation
for 10 minutes at 12,000 X g. In certain embodiments, the polypeptide-containing pellet is
resuspended in 50 mM Tris, pH 8, and 10 mM EDTA; layered over 50% glycerol; and
centrifuged for 30 minutes at 6000 X g. In certain embodiments, that pellet can be
++ ++
resuspended in standard phosphate buffered saline solution (PBS) free of Mg and Ca . In
certain embodiments, the polypeptide is further purified by fractionating the resuspended
pellet in a denaturing SDS polyacrylamide gel (See, e.g., Sambrook et al., supra). In certain
embodiments, such a gel can be soaked in 0.4 M KCl to visualize the protein, which can be
excised and electroeluted in gel-running buffer lacking SDS. According to certain
embodiments, a Glutathione-S-Transferase (GST) fusion protein is produced in bacteria as a
soluble protein. In certain embodiments, such GST fusion protein is purified using a GST
Purification Module (Pharmacia).
In certain embodiments, it is desirable to “refold” certain polypeptides, e.g.,
polypeptides comprising one or more ABP components or the ABP itself. In certain
embodiments, such polypeptides are produced using certain recombinant systems discussed
herein. In certain embodiments, polypeptides are “refolded” and/or oxidized to form desired
tertiary structure and/or to generate disulfide linkages. In certain embodiments, such
structure and/or linkages are related to certain biological activity of a polypeptide. In certain
embodiments, refolding is accomplished using any of a number of procedures known in the
art. Exemplary methods include, but are not limited to, exposing the solubilized polypeptide
agent to a pH typically above 7 in the presence of a chaotropic agent. An exemplary
chaotropic agent is guanidine. In certain embodiments, the refolding/oxidation solution also
contains a reducing agent and the oxidized form of that reducing agent. In certain
embodiments, the reducing agent and its oxidized form are present in a ratio that will
generate a particular redox potential that allows disulfide shuffling to occur. In certain
embodiments, such shuffling allows the formation of cysteine bridges. Exemplary redox
couples include, but are not limited to, cysteine/cystamine, glutathione/dithiobisGSH, cupric
chloride, dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME. In
certain embodiments, a co-solvent is used to increase the efficiency of refolding. Exemplary
cosolvents include, but are not limited to, glycerol, polyethylene glycol of various molecular
weights, and arginine.
In certain embodiments, one substantially purifies a polypeptide comprising one or
more ABP components or the ABP itself. Certain protein purification techniques are known
to those of skill in the art. In certain embodiments, protein purification involves crude
fractionation of polypeptide fractionations from non-polypeptide fractions. In certain
embodiments, polypeptides are purified using chromatographic and/or electrophoretic
techniques. Exemplary purification methods include, but are not limited to, precipitation
with ammonium sulphate; precipitation with PEG; immunoprecipitation; heat denaturation
followed by centrifugation; chromatography, including, but not limited to, affinity
chromatography (e.g., Protein-A-Sepharose), ion exchange chromatography, exclusion
chromatography, and reverse phase chromatography; gel filtration; hydroxyapatite
chromatography; isoelectric focusing; polyacrylamide gel electrophoresis; and combinations
of such and other techniques. In certain embodiments, a polypeptide is purified by fast
protein liquid chromatography or by high pressure liquid chromotography (HPLC). In
certain embodiments, purification steps can be changed or certain steps can be omitted, and
still result in a suitable method for the preparation of a substantially purified polypeptide.
In certain embodiments, one quantitates the degree of purification of a polypeptide
preparation. Certain methods for quantifying the degree of purification are known to those of
skill in the art. Certain exemplary methods include, but are not limited to, determining the
specific binding activity of the preparation and assessing the amount of a polypeptide within
a preparation by SDS/PAGE analysis. Certain exemplary methods for assessing the amount
of purification of a polypeptide preparation comprise calculating the binding activity of a
preparation and comparing it to the binding activity of an initial extract. In certain
embodiments, the results of such a calculation are expressed as “fold purification.” The units
used to represent the amount of binding activity depend upon the particular assay performed.
In certain embodiments, a polypeptide comprising one or more ABP components or
the ABP itself is partially purified. In certain embodiments, partial purification can be
accomplished by using fewer purification steps or by utilizing different forms of the same
general purification scheme. For example, in certain embodiments, cation-exchange column
chromatography performed utilizing an HPLC apparatus will generally result in a greater
“fold purification” than the same technique utilizing a low-pressure chromatography system.
In certain embodiments, methods resulting in a lower degree of purification can have
advantages in total recovery of polypeptide, or in maintaining binding activity of a
polypeptide.
In certain instances, the electrophoretic migration of a polypeptide can vary,
sometimes significantly, with different conditions of SDS/PAGE. See, e.g., Capaldi et al.,
Biochem. Biophys. Res. Comm., 76: 425 (1977). It will be appreciated that under different
electrophoresis conditions, the apparent molecular weights of purified or partially purified
polypeptide can be different.
Exemplary Epitopes
Epitopes to which anti-PCSK9 antibodies bind are provided. In some embodiments,
epitopes that are bound by the presently disclosed antibodies are particularly useful. In some
embodiments, antigen binding proteins that bind to any of the epitopes that are bound by the
antibodies described herein are useful. In some embodiments, the epitopes bound by any of
the antibodies listed in Table 2 and FIGs. 2 and 3 are especially useful. In some
embodiments, the epitope is on the catalytic domain PCSK9.
In certain embodiments, a PCSK9 epitope can be utilized to prevent (e.g., reduce)
binding of an anti-PCSK9 antibody or antigen binding protein to PCSK9. In certain
embodiments, a PCSK9 epitope can be utilized to decrease binding of an anti-PCSK9
antibody or antigen binding protein to PCSK9. In certain embodiments, a PCSK9 epitope
can be utilized to substantially inhibit binding of an anti-PCSK9 antibody or antigen binding
protein to PCSK9.
In certain embodiments, a PCSK9 epitope can be utilized to isolate antibodies or
antigen binding proteins that bind to PCSK9. In certain embodiments, a PCSK9 epitope can
be utilized to generate antibodies or antigen binding proteins which bind to PCSK9. In
certain embodiments, a PCSK9 epitope or a sequence comprising a PCSK9 epitope can be
utilized as an immunogen to generate antibodies or antigen binding proteins that bind to
PCSK9. In certain embodiments, a PCSK9 epitope can be administered to an animal, and
antibodies that bind to PCSK9 can subsequently be obtained from the animal. In certain
embodiments, a PCSK9 epitope or a sequence comprising a PCSK9 epitope can be utilized to
interfere with normal PCSK9-mediated activity, such as association of PCSK9 with the
LDLR.
In some embodiments, antigen binding proteins disclosed herein bind specifically to
N-terminal prodomain, a subtilisin-like catalytic domain and/or a C-terminal domain. In
some embodiments, the antigen binding protein binds to the substrate-binding groove of
PCSK-9 (described in Cunningham et al., incorporated herein in its entirety by reference).
In some embodiments, the domain(s)/region(s) containing residues that are in contact
with or are buried by an antibody can be identified by mutating specific residues in PCSK9
(e.g., a wild-type antigen) and determining whether the antigen binding protein can bind the
mutated or variant PCSK9 protein. By making a number of individual mutations, residues
that play a direct role in binding or that are in sufficiently close proximity to the antibody
such that a mutation can affect binding between the antigen binding protein and antigen can
be identified. From knowledge of these amino acids, the domain(s) or region(s) of the
antigen that contain residues in contact with the antigen binding protein or covered by the
antibody can be elucidated. Such a domain can include the binding epitope of an antigen
binding protein. One specific example of this general approach utilizes an arginine/glutamic
acid scanning protocol (see, e.g., Nanevicz, T., et al., 1995, J. Biol. Chem., 270:37, 21619-
21625 and Zupnick, A., et al., 2006, J. Biol. Chem., 281:29, 20464-20473). In general,
arginine and glutamic acids are substituted (typically individually) for an amino acid in the
wild-type polypeptide because these amino acids are charged and bulky and thus have the
potential to disrupt binding between an antigen binding protein and an antigen in the region
of the antigen where the mutation is introduced. Arginines that exist in the wild-type antigen
are replaced with glutamic acid. A variety of such individual mutants are obtained and the
collected binding results analyzed to determine what residues affect binding.
An alteration (for example a reduction or increase) in binding between an antigen
binding protein and a variant PCSK9 as used herein means that there is a change in binding
affinity (e.g., as measured by known methods such as Biacore testing or the bead based assay
described below in the examples), EC , and/or a change (for example a reduction) in the
total binding capacity of the antigen binding protein (for example, as evidenced by a decrease
in Bmax in a plot of antigen binding protein concentration versus antigen concentration). A
significant alteration in binding indicates that the mutated residue is directly involved in
binding to the antigen binding protein or is in close proximity to the binding protein when the
binding protein is bound to antigen.
In some embodiments, a significant reduction in binding means that the binding
affinity, EC50, and/or capacity between an antigen binding protein and a mutant PCSK9
antigen is reduced by greater than 10%, greater than 20%, greater than 40 %, greater than 50
%, greater than 55 %, greater than 60 %, greater than 65 %, greater than 70 %, greater than75
%, greater than 80 %, greater than 85 %, greater than 90% or greater than 95% relative to
binding between the antigen binding protein and a wild type PCSK9 (e.g., shown in SEQ ID
NO: 1 and/or SEQ ID NO: (303). In certain embodiments, binding is reduced below
detectable limits. In some embodiments, a significant reduction in binding is evidenced when
binding of an antigen binding protein to a variant PCSK9 protein is less than 50% (for
example, less than 40%, 35%, 30%, 25%, 20%, 15% or 10%) of the binding observed
between the antigen binding protein and a wild-type PCSK9 protein (for example, the protein
of SEQ ID NO: 1 and/or SEQ ID NO: (303). Such binding measurements can be made using
a variety of binding assays known in the art.
In some embodiments, antigen binding proteins are provided that exhibit significantly
lower binding for a variant PCSK9 protein in which a residue in a wild-type PCSK9 protein
(e.g., SEQ ID NO: 1 or SEQ ID NO: 303 is substituted with arginine or glutamic acid. In
some embodiments, binding of an antigen binding protein is significantly reduced or
increased for a variant PCSK9 protein having any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
, or 244) of the following mutations: R207E, D208R, R185E, R439E, E513R, V538R,
E539R, T132R, S351R, A390R, A413R, E582R, D162R, R164E, E167R, S123R, E129R,
A311R, D313R, D337R, R519E, H521R, and Q554R as compared to a wild-type PCSK9
protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303. In the shorthand notation used here, the
format is: Wild type residue: Position in polypeptide: Mutant residue, with the numbering of
the residues as indicated in SEQ ID NO: 1or SEQ ID NO: 303.
In some embodiments, binding of an antigen binding protein is significantly reduced
or increased for a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, or more)
mutations at the following positions: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390,
413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554, as shown in SEQ ID
NO: 1 as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303.
In some embodiments, binding of an antigen binding protein is reduced or increased for a
mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, or more) mutations at the
following positions: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162,
164, 167, 123, 129, 311, 313, 337, 519, 521, and 554, as shown in SEQ ID NO: 1 as
compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303. In some
embodiments, binding of an antigen binding protein is substantially reduced or increased for
a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, or more) mutations at the
following positions: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162,
164, 167, 123, 129, 311, 313, 337, 519, 521, and 554, within SEQ ID NO: 1 as compared to a
wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303.
In some embodiments, binding of an ABP is significantly reduced or increased for a
mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, etc.) of the following mutations:
R207E, D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R,
E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R,
and Q554R within SEQ ID NO: 1 or SEQ ID NO: 303, as compared to a wild-type PCSK9
protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303).
In some embodiments, binding of an ABP is significantly reduced or increased for a
mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, etc.) of the following mutations:
R207E, D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R,
and E582R within SEQ ID NO: 1 or SEQ ID NO: 303, as compared to a wild-type PCSK9
protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303). In some embodiments, the binding is
reduced. In some embodiments, the reduction in binding is observed as a change in EC50. In
some embodiments, the change in EC50 is an increase in the numerical value of the EC50
(and thus is a decrease in binding).
In some embodiments, binding of an ABP is significantly reduced or increased for a
mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, etc.) of the following mutations:
D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and
Q554R within SEQ ID NO: 1, as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO:
1 or SEQ ID NO: 303). In some embodiments, the binding is reduced. In some
embodiments, the reduction in binding is observed as a change in Bmax. In some
embodiments, the shift in Bmax is a reduction of the maximum signal generated by the ABP.
In some embodiments, for an amino acid to be part of an epitope, the Bmax is reduced by at
least 10%, for example, reductions of at least any of the following amounts: 20, 30, 40, 50,
60, 70, 80, 90, 95, 98, 99, or 100 percent can, in some embodiments, indicate that the residue
is part of the epitope.
Although the variant forms just listed are referenced with respect to the wild-type
sequence shown in SEQ ID NO: 1 or SEQ ID NO: 303, it will be appreciated that in an allelic
variant of PCSK9 the amino acid at the indicated position could differ. Antigen binding
proteins showing significantly lower binding for such allelic forms of PCSK9 are also
contemplated. Accordingly, in some embodiments, any of the above embodiments can be
compared to an allelic sequence, rather than purely the wild-type sequence shown in
In some embodiments, binding of an antigen binding protein is significantly reduced
for a variant PCSK9 protein in which the residue at a selected position in the wild-type
PCSK9 protein is mutated to any other residue. In some embodiments, the herein described
arginine/glutamic acid replacements are used for the identified positions. In some
embodiments, alanine is used for the identified positions.
As noted above, residues directly involved in binding or covered by an antigen
binding protein can be identified from scanning results. These residues can thus provide an
indication of the domains or regions of SEQ ID NO: 1 (or SEQ ID NO: 303 or SEQ ID NO:
3) that contain the binding region(s) to which antigen binding proteins bind. As can be seen
from the results summarized in Example 39, in some embodiments an antigen binding protein
binds to a domain containing at least one of amino acids: 207, 208, 185, 181, 439, 513, 538,
539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 of
SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, the antigen binding protein binds
to a region containing at least one of amino acids 207, 208, 185, 181, 439, 513, 538, 539,
132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 of SEQ
ID NO: 1 or SEQ ID NO: 303.
In some embodiments, the antigen binding protein binds to a region containing at least
one of amino acids 162, 164, 167, 207 and/or 208 of SEQ ID NO: 1 or SEQ ID NO: 303. In
some embodiments, more than one (e.g., 2, 3, 4, or 5) of the identified residues are part of the
region that is bound by the ABP. In some embodiments, the ABP competes with ABP
21B12.
In some embodiments, the antigen binding protein binds to a region containing at least
one of amino acid 185 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, the
ABP competes with ABP 31H4.
In some embodiments, the antigen binding protein binds to a region containing at least
one of amino acids 439, 513, 538, and/or 539 of SEQ ID NO: 1 or SEQ ID NO: 303. In some
embodiments, more than one (e.g., 2, 3, or 4) of the identified residues are part of the region
that is bound by the ABP. In some embodiments, the ABP competes with ABP 31A4.
In some embodiments, the antigen binding protein binds to a region containing at least
one of amino acids 123, 129, 311, 313, 337, 132, 351, 390, and/or 413 of SEQ ID NO: 1 or
SEQ ID NO: 303. In some embodiments, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of the
identified residues are part of the region that is bound by the ABP. In some embodiments,
the ABP competes with ABP 12H11.
In some embodiments, the antigen binding protein binds to a region containing at least
one of amino acid 582, 519, 521, and/or 554 of SEQ ID NO: 1 or SEQ ID NO: 303. In some
embodiments, more than one (e.g., 2, 3, or 4) of the identified residues are part of the region
that is bound by the ABP. In some embodiments, the ABP competes with ABP 3C4.
In some embodiments, the antigen binding proteins binds to the foregoing regions
within a fragment or the full length sequence of SEQ ID NO: 1 or SEQ ID NO: 303. In other
embodiments, antigen binding proteins bind to polypeptides consisting of these regions. The
reference to “SEQ ID NO: 1 or SEQ ID NO: 303” denotes that one or both of these sequences
can be employed or relevant. The phrase does not denote that only one should be employed.
As noted above, the above description references specific amino acid positions with
reference to SEQ ID NO: 1. However, throughout the specification generally, reference is
made to a Pro/Cat domain that commences at position 31, which is provided in SEQ ID NO:
3. As noted below, SEQ ID NO: 1 and SEQ ID NO: 303 lack the signal sequence of PCSK9.
As such, any comparison between these various disclosures should take this difference in
numbering into account. In particular, any amino acid position in SEQ ID NO: 1, will
correspond to an amino acid position 30 amino acids further into the protein in SEQ ID NO:
3. For example, position 207 of SEQ ID NO: 1, corresponds to position 237 of SEQ ID NO:
3 (the full length sequence, and the numbering system used in the present specification
generally). Table 39.6 outlines how the above noted positions, which reference SEQ ID NO:
1 (and/or SEQ ID NO: 303) correspond to SEQ ID NO: 3 (which includes the signal
sequence). Thus, any of the above noted embodiments that are described in regard to SEQ ID
NO: 1 (and/or SEQ ID NO: 303), are described in reference to SEQ ID NO: 3, by the noted
corresponding positions.
In some embodiments, ABP 21B12 binds to an epitope including residues 162-167
(e.g., residues D162-E167 of SEQ ID NO: 1). In some embodiments, ABP 12H11 binds to
an epitope that includes residues 123-132 (e.g., S123-T132 of SEQ ID NO: 1). In some
embodiments, ABP 12H11 binds to an epitope that includes residues 311-313 (e.g., A311-
D313 of SEQ ID NO: 1). In some embodiments, ABPs can bind to an epitope that includes
any one of these strands of sequences.
Competing Antigen Binding Proteins
In another aspect, antigen binding proteins are provided that compete with one of the
exemplified antibodies or functional fragments binding to the epitope described herein for
specific binding to PCSK9. Such antigen binding proteins can also bind to the same epitope
as one of the herein exemplified antigen binding proteins, or an overlapping epitope. Antigen
binding proteins and fragments that compete with or bind to the same epitope as the
exemplified antigen binding proteins are expected to show similar functional properties. The
exemplified antigen binding proteins and fragments include those described above, including
those with the heavy and light chains, variable region domains and CDRs included in TABLE
2 and/or FIGs. 2-3. Thus, as a specific example, the antigen binding proteins that are
provided include those that compete with an antibody or antigen binding protein having:
(a) all 6 of the CDRs listed for an antibody listed in FIGs. 2-3;
(b) a VH and a VL listed for an antibody listed in Table 2; or
(c) two light chains and two heavy chains as specified for an antibody listed in Table 2.
Therapeutic Pharmaceutical Formulations and Administration
The present invention provides pharmaceutical formulations containing antigen
binding proteins to PCSK9. As used herein, “pharmaceutical formulation” is a sterile
composition of a pharmaceutically active drug, namely, at least one antigen binding protein
to PCSK9, that is suitable for parenteral administration (including but not limited to
intravenous, intramuscular, subcutaneous, aerosolized, intrapulmonary, intranasal, or
intrathecal) to a patient in need thereof and includes only pharmaceutically acceptable
excipients, diluents, and other additives deemed safe by the Federal Drug Administration or
other foreign national authorities. Pharmaceutical formulations include liquid, e.g., aqueous,
solutions that may be directly administered, and lyophilized powders which may be
reconstituted into solutions by adding a diluent before administration. Specifically excluded
from the scope of the term “pharmaceutical formulation” are compositions for topical
administration to patients, compositions for oral ingestion, and compositions for parenteral
feeding.
In certain embodiments, the pharmaceutical formulation is a stable pharmaceutical
formulation. As used herein, the phrases, “stable pharmaceutical formulation, “stable
formulation” or “a pharmaceutical formulation is stable” refers to a pharmaceutical
formulation of biologically active proteins that exhibit increased aggregation and/or reduced
loss of biological activity of not more than 5% when stored at 2-8ºC for at least 1 month, or 2
months, or 3 months, or 6 months, or 1 year or 2 years compared with a control formula
sample. Formulation stability can be easily determed by a person of skill in the art using any
number of standard assays, including but not limited to size exclusion HPLC (“SEC-HPLC”),
cation-exchange HPLC (CEX-HPLC), Subvisible Particle Detection by Light Obscuration
(“HIAC”) and/or visual inspection.
In certain embodiments, the pharmaceutical formulation comprises any of the antigen
binding proteins to PCSK9 depicted in Table 2 and FIGs. 2 and/or 3 and FIGs 48A and 48B.
In certain oher embodiments, the pharmaceutical formulation may comprise other antigen
binding proteins to PCSK9; namely an antibody comprised of a light chain variable domain,
SEQ ID NO:588 and a heavy chain variable domain, SEQ ID NO:589. In some embodiments
the pharmaceutical formulation comprises any one of 21B12, 26H5, 31H4, 8A3, 11F1 or
8A1.
In some embodiments, the pharmaceutical formulation comprises more than one
different antigen binding protein to PCSK9. In certain embodiments, pharmaceutical
formulations comprise more than one antigen binding protein to PCSK9 wherein the antigen
binding proteins to PCSK9 bind more than one epitope. In some embodiments, the various
antigen binding proteins will not compete with one another for binding to PCSK9. In some
embodiments, any of the antigen binding proteins depicted in Table 2 and FIGs. 2 and/or 3
can be combined together in a pharmaceutical formulation.
In certain embodiments, an antigen binding protein to PCSK9 and/or a therapeutic
molecule is linked to a half-life extending vehicle known in the art. Such vehicles include,
but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of the ABP), and
dextran. Such vehicles are described, e.g., in U.S. Application Serial No. 09/428,082, now
US Patent No. 6,660,843 and published PCT Application No. WO 99/25044, which are
hereby incorporated by reference for any purpose.
In certain embodiments, acceptable formulation materials preferably are nontoxic to
recipients at the dosages and concentrations employed. In some embodiments, the
formulation material(s) are for s.c. and/or I.V. administration. In certain embodiments, the
pharmaceutical formulation comprises formulation materials for modifying, maintaining or
preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
In certain embodiments, suitable formulation materials include, but are not limited to,
amino acids (such as proline, arginine, lysine, methionine, taurine, glycine, glutamine, or
asparagine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium
hydrogen-sulfite); buffers (such as borate, bicarbonate, sodium phosphate (“NaOAC”), Tris-
HCl, Tris buffer, citrates, phosphate buffer, phosphate-buffered saline (i.e., PBS buffer) or
other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as
ethylenediamine tetra acetic acid (EDTA)); complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;
monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose,, fructose,
lactose, mannose, trehelose, or dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic
polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming
counter ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid,
salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or
polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents;
surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as
polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal);
stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as
alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery
vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical
Sciences, 18 Edition, A.R. Gennaro, ed., Mack Publishing Company (1995).
In certain embodiments, the optimal pharmaceutical formulation will be determined
by one skilled in the art depending upon, for example, the intended route of administration,
delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences,
supra. In certain embodiments, such formulations may influence the physical state, stability,
rate of in vivo release and rate of in vivo clearance of the antibodies of the invention.
In one aspect, the pharmaceutical formulation comprises high concentrations of
antigen binding protein to PCSK9. In certain embodiments, ABP concentration ranges from
about 70 mg/ml to about 250 mg/ml, e.g., about 70 mg/ml, about 80 mg/ml, about 90 mg/ml,
about 100 mg/ml, about 100 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml,
about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml,
about 200 mg/ml, about 210 mg/ml, about 220 mg/ml, about 230 mg/ml, about 240 mg/ml, or
about 250 mg/ml, and including all values in between. In some embodiments, the
concentration of 21B12, 26H5, or 31H4 ranges from about 100 mg/ml to about 150 mg/ml,
e.g., 100 mg/ml, about 100 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, or
about 150 mg/ml. In some embodiments, the concentration of 8A3, 11F1 or 8A1 ranges from
about 140 mg/ml to about 220 mg/ml, e.g., 140 mg/ml, about 150 mg/ml, about 160 mg/ml,
about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 210 mg/ml,
about 220 mg/ml, or about 250 mg/ml.
In another aspect, the pharmaceutical formulation comprises at least one buffering
agent such as, for example, sodium acetate, sodium chloride, phosphates, phosphate buffered
saline (“PBS”), and/or Tris buffer of about pH 7.0-8.5. The buffer serves to maintain a
physiologically suitable pH. In addition, the buffer can serve to enhance isotonicity and
chemical stability of the pharmaceutical formulation. In certain embodiments, the buffering
agent ranges from about 0.05 mM to about 40 mM, e.g., about 0.05 mM, about 0.1 mM,
about 0.5 mM, about 1.0 mM, about 5.0 mM, about 10 mM, about 15 mM, about 20 mM,
about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about
90 mM, or about 100nM buffering agent, inclusiveof all values in between. In certain
embodiments, the bufferning agent is NaOAC. Exemplary pHs of the pharmaceutical
formulation include from about 4 to about 6, or from about 4.8 to about 5.8, or from about
5.0 to about 5.2, or about 5, or about 5.2.
In certain embodiments, the pharmaceutical formulation is isotonic with an osmolality
ranging from between about 250 to about 350 miliosmol/kg, e.g., about 250 mOsm/kg, about
260 mOsm/kg, about 270 mOsm/kg, about 280 mOsm/kg, about 290 mOsm/kg, about 300
mOsm/kg, about 310 mOsm/kg, about 320 mOsm/kg, about 330 mOsm/kg, about 340
mOsm/kg, or about 350 mOsm/kg, and including all values in between. As used herein,
“osmolality” is the measure of the ratio of solutes to volume fluid. In other words, it is the
number of molecules and ions (or molecules) per kilogram of a solution. Osmolality may be
measured on an analytical instrument called an osmometer, such as Advanced Instruments
2020 Multi-sample Osmometer, Norwood, MA. The Advanced Instrumetns 2020 Multi-
sample Osmometer measures osmolality by using the Freezing Point Depression method. The
higher the osmolytes in a solution, the temperature in which it will freeze drops. Osmolality
may also be measured using any other methods and in any other units known in the art such
as linear extrapolation.
In still another aspect, the pharmaceutical formulation comprises at least one
surfactant including but not limited to Polysorbate-80, Polysorbate-60, Polysorbate-40, and
Polysorbate-20. In certain embodiments, the pharmaceutical formulation comprises a
surfactant at a concentration that ranges from about 0.004% to about 10% weight per volume
(“w/v”) of the formulation, e.g., about 0.004%, about 0.005%, about 0.006%, about 0.007%,
about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%,
about 5%, or about 10% surfactant w/v of the formulation. In certain embodiments, the
pharmaceutical formulation comprises polysorbate 80 at a concentration that ranges from
about 0.004% to about 0.1% w/v of the formulation. In certain embodiments, the
pharmaceutical formulation comprises polysorbate 20 at a concentration that ranges from
about 0.004% to about 0.1% w/v of the formulation.
In certain embodiments, the pharmaceutical formulation comprises at least one
stabilizing agent, such as a polyhydroxy hydrocarbon (including but not limited to sorbitol,
mannitol, glycerol and dulcitol) and/or a disaccharide (including but not limited to sucrose,
lactose, maltose and threhalose) and/or an amino acid (including but not limited to proline,
arginine, lysine, methionine, and taurine) and or benzyl alcohol; the total of said
polyhydroxy hydrocarbon and/or disaccharide and/or amino acid and/or benzyl alchol being
about 0.5% to about 10% w/v of the formulation. In certain embodiments, the
pharmaceutical formulation comprises a stabilizing agent at a concentration of about 1%,
about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about
10% sucrose. In certain embodiments, the pharmaceutical formulation comprises a
stabilizing agent at a concentration of about 5% sucrose. In certain embodiments, the
pharmaceutical formulation comprises a a stabilizing agent at a concentration of about 1%,
about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about
% sorbital. In certain embodiments, the pharmaceutical formulation comprises a
stabilizing agent at a concentration of about 9% sorbital. In certain embodiments, the
pharmaceutical formulation comprises a a stabilizing agent at a concentration of about 1%,
about 2%, about 3%, about 4%, about 5% proline, arginine, lysine, methionine, and/or
taurine. In certain embodiments, the pharmaceutical formulation comprises a stabilizing
agent at a concentration of between about 2-3% proline. In certain embodiments, the
pharmaceutical formulation comprises a a stabilizing agent at a concentration of about 1%,
about 2%, about 3%, about 4%, about 5% benzyl alcohol. In certain embodiments, the
pharmaceutical formulation comprises a stabilizing agent at a concentration of between about
1-2% benzyl alcohol.
In one aspect, the pharmaceutical formulation has a viscosity level of less than about
centipoise (cP) as measured at room temperature (i.e., 25C). As used herein, “viscosity” is
a fluid’s resistance to flow, and may be measured in units of centipoise (cP) or milliPascal-
second (mPa-s), where 1 cP=1 mPa-s, at a given shear rate. Viscosity may be measured by
using a viscometer, e.g., Brookfield Engineering Dial Reading Viscometer, model LVT.
Viscosity may also be measured using any other methods and in any other units known in the
art (e.g., absolute, kinematic or dynamic viscosity or absolute viscosity). In certain
embodiments, the pharmaceutical formulation has a viscosity level of less than about 25 cP,
about 20 cP, about 18 cP, about 15 cP, about 12 cP, about 10 cP; about 8 cP, about 6 cP,
about 4 cP; about 2 cP; or about 1 cP.
In one aspecet, the pharmaceutical formulation is stable as measured by at least one
stability assay known to one of skill in the art, such as assays that examne the biophysical or
biochemical characteristics of biologically active proteins over time. As mentioned above, a
stable pharmaceutical formulation of the present invention is a pharmaceutical formulation of
biologically active proteins that exhibits increased aggregation and/or reduced loss of
biological activity of not more than 5% when stored at 2-8ºC for at least 1 month, or 2
months, or 3 months, or 6 months, or 1 year or 2 years compared with a control formula
sample. In certain embodiments, the pharmaceutical formulation stability is measured using
size exclusion HPLC (“SEC-HPLC”). SEC-HPLC separates proteins based on differences in
their hydrodynamic volumes. Molecules with larger hydrodynamic proteins volumes elute
earlier than molecules with smaller volumes. In the case of SEC-HPLC, a stable
pharmaceutical formulation should exhibit no more than about a 5% increase in high
molecular weight species as compared to a control sample. In certain other embodiments, the
pharmaceutical formulation should exhibit no more than about a 4%, no more than about a
3%, no more than about a 2%, no more than about a 1%, no more than about a 0.5% increase
in high molecular weight speciies as compared to a control sample.
In certain embodiments, the pharmaceutical formulation stability is measured using
cation-exchange HPLC (CEX-HPLC). CEX-HPLC separates proteins based on differences
in their surface charge. At a set pH, charged isoforms of an anti-PCSK9 ABP are separated
on a cation-exchange column and eluted using a salt gradient. The eluent is monitored by UV
absorbance. The charged isoform distribution is evaluated by determining the peak area of
each isoform as a percent of the total peak area. In the case of CEX-HPLC, a stable
pharmaceutical formulation should exhibit no more than about a 5% decrease in the main
isoform peak as compared to a control sample. In certain other embodiments, a stable
pharmaceutical formulation should exhibit no more than about a 3% to about a 5% decrease
in the main isoform peak as compared to a control sample. In certain embodiments, the
pharmaceutical formulation should exhibit no more than about a 4% decrease, no more than
about a 3% decrease, no more than about a 2% decrease, no more than about a 1% decrease,
no more than about a 0.5% decrease in the main isoform peak as compared to a control
sample.
In certain embodiments, the pharmaceutical formulation stability is measured using
Subvisible Particle Detection by Light Obscuration (“HIAC”). An electronic, liquid-borne
particle-counting system (HIAC/Royco 9703 or equivalent) containing a light-obscuration
sensor (HIAC/Royco HRLD-150 or equivalent) with a liquid sampler quantifies the number
of particles and their size range in a given test sample. When particles in a liquid pass
between the light source and the detector they diminish or “obscure” the beam of light that
falls on the detector. When the concentration of particles lies within the normal range of the
sensor, these particles are detected one-by-one. The passage of each particle through the
detection zone reduces the incident light on the photo-detector and the voltage output of the
photo-detector is momentarily reduced. The changes in the voltage register as electrical
pulses that are converted by the instrument into the number of particles present. The method
is non-specific and measures particles regardless of their origin. Particle sizes monitored are
generally 10 um, and 25 um. In the case of HIAC, a stable pharmaceutical formulation
should exhibit no more than 6000 10µm particles per container (or unit), as compared to a
control sample. In certain embodiments, a stable pharmaceutical formulation should exhibit
no more than 5000, no more than 4000, no more than 3000, no more than 2000, no more
than 1000, 10µm particles per container (or unit) as compared to a control sample. In still
other embodiments, a stable pharmaceutical formulation should exhibit no more than 600
25µm particles per container (or unit) as compared to a control sample. In certain
embodiments, a stable pharmaceutical formulation should exhibit no more than 500, no more
than 400, no more than 300, no more than 200, no more than 100, no more than 50 25µm
particles per container (or unit) as compared to a control sample.
In certain embodiments, the pharmaceutical formulation stability is measured using
visual assessment. Visual assessment is a qualitative method used to describe the visible
physical characteristics of a sample. The sample is viewed against a black and/or white
background of an inspection booth, depending on the characteristic being evaluated (e.g.,
color, clarity, presence of particles or foreign matter). Samples are also viewed against an
opalescent reference standard and color reference standards. In the case of visual assessment,
a stable pharmaceutical formulation should exhibit no significant change in color, clarity,
presence of particles or foreign matter as compared to a control sample.
One aspect of the present invention is a pharmaceutical formulation which comprises:
(i) about 70 mg/ml to about 250 mg/ml of antigen binding protein to PCSK9; (ii) about 0.05
mM to about 40 mM of a buffer such as sodium acetate (“NaOAC”) serves as a buffering
agent; (iii) about 1% to about 5% proline, arginine, lysine, methionine, or taurine (also know
as 2-aminoethanesulfonic acid) and/or 0.5% to about 5% benzyl alcohol which serves as a
stabilizing agent; and (iv) about 0.004% to about 10% w/v of the formulation of a non-ionic
surfactant (including but not limited to Polysorbate-80, Polysorbate-60, Polysorbate-40, and
Polysorbate-20); wherein said formulation has a pH in the range of about 4.0 to 6.0. In
certain other embodiments, pharmaceutical formulations of this invention comprise (i) at least
about 70 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 150 mg/ml,
about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml of
an anti-PCSK9 antibody; (ii) about 10 mM NAOAC; (iii) about 0.01% polysorbate 80; and
(iv) between about 2%-3% proline (or about 170 mM to about 270 mM proline), wherein the
formulation has a pH of about 5. In certain other embodiments, pharmaceutical formulations
of this invention comprise (i) at least about 70 mg/ml, about 100 mg/ml, about 120 mg/ml,
about 140 mg/ml of the anti-PCSK9 antibody, 21B12, 26H5 and/or 31H4; (ii) about 10 mM
NAOAC; (iii) about 0.01% polysorbate 80; and (iv) between about 2%-3% proline (or about
170 mM to about 270 mM proline), wherein the formulation has a pH of about 5. In certain
other embodiments, pharmaceutical formulations of this invention comprise (i) at least about
150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about
200 mg/ml of the anti-PCSK9 antibody, 8A3, 11F1 and/or 8A1; (ii) about 10 mM NAOAC;
(iii) about 0.01% polysorbate 80; and (iv) between about 2%-3% proline (or about 170 mM to
about 270 mM proline), wherein the formulation has a pH of about 5.
One aspect of the present invention is a pharmaceutical formulation which comprises
(i) at least about 70 mg/ml to about 250 mg/ml of an anti-PCSK9 antibody; (ii) about 5 mM
to about 20 mM of a buffer, such as NAOAC; (iii) about 1% to about 10% w/v of the
formulation comprises a polyhydroxy hydrocarbon such as sorbitol, or a disaccharide such as
sucrose; and (iv) about 0.004% to about 10% w/v of the formulation of a surfactant, such as
polysorbate 20 or polysorbate 80; wherein said formulation has a pH in the range of about 4.8
to 5.8; and wherein the pharmaceutical formulation optionally comprises about 80 mM to
about 300 mM proline, arginine, lysine, methionine, or taurine and/or 0.5% to about 5%
benzyl alcohol which serves to reduce viscosity. In certain other embodiments,
pharmaceutical formulations of this invention comprise (i) at least about 70 mg/ml to about
250 mg/ml of the anti-PCSK9 antibody; (ii) about 10 mM NAOAC; (iii) about 9% sucrose;
and (iv) about 0.004% polysorbate 20, wherein the formulation has a pH of about 5.2. In
certain other embodiments, pharmaceutical formulations of this invention comprise (i) at least
about 70 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml,
about 180 mg/ml, about 200 mg/ml of an anti-PCSK9 antibody; (ii) about 15 mM NAOAC;
(iii) about 9% sucrose; and (iv) about 0.01% polysorbate 20, wherein the formulation has a
pH of about 5.2. In certain other embodiments, pharmaceutical formulations of this invention
comprise (i) at least about 70 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml,
about 160 mg/ml, about 180 mg/ml, about 200 mg/ml of an anti-PCSK9 antibody; (ii) about
mM NAOAC; (iii) about 9% sucrose; and (iv) about 0.01% polysorbate 20, wherein the
formulation has a pH of about 5.2. In certain other embodiments, pharmaceutical
formulations of this invention comprise (i) at least about 70 mg/ml, about 100 mg/ml, about
120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, about 200 mg/ml of an
anti-PCSK9 antibody; (ii) about 10 mM NAOAC; (iii) about 9% sucrose; (iv) about 0.01%
polysorbate 80; and (v) about 250 mM proline, wherein the formulation has a pH of about 5.
Pharmaceutical formulations of the invention can be administered in combination
therapy, i.e., combined with other agents. In certain embodiments, the combination therapy
comprises an antigen binding protein capable of binding PCSK9, in combination with at least
one anti-cholesterol agent. Agents include, but are not limited to, in vitro synthetically
prepared chemical formulations, antibodies, antigen binding regions, and combinations and
conjugates thereof. In certain embodiments, an agent can act as an agonist, antagonist,
allosteric modulator, or toxin. In certain embodiments, an agent can act to inhibit or
stimulate its target (e.g., receptor or enzyme activation or inhibition), and thereby promote
increased expression of LDLR or decrease serum cholesterol levels.
In certain embodiments, an antigen binding protein to PCSK9 can be administered
prior to, concurrent with, and subsequent to treatment with a cholesterol-lowering (serum
and/or total cholesterol) agent. In certain embodiments, an antigen binding protein to PCSK9
can be administered prophylacticly to prevent or mitigate the onset of hypercholesterolemia,
heart disease, diabetes, and/or any of the cholesterol related disorder. In certain
embodiments, an antigen binding protein to PCSK9 can be administered for the treatment of
an existing hypercholesterolemia condition. In some embodiments, the ABP delays the onset
of the disorder and/or symptoms associated with the disorder. In some embodiments, the
ABP is provided to a subject lacking any symptoms of any one of the cholesterol related
disorders or a subset thereof.
In certain embodiments, an antigen binding protein to PCSK9 is used with particular
therapeutic agents to treat various cholesterol related disorders, such as hypercholesterolemia.
In certain embodiments, in view of the condition and the desired level of treatment, two,
three, or more agents can be administered. In certain embodiments, such agents can be
provided together by inclusion in the same formulation. In certain embodiments, such
agent(s) and an antigen binding protein to PCSK9 can be provided together by inclusion in
the same formulation. In certain embodiments, such agents can be formulated separately and
provided together by inclusion in a treatment kit. In certain embodiments, such agents and an
antigen binding protein to PCSK9 can be formulated separately and provided together by
inclusion in a treatment kit. In certain embodiments, such agents can be provided separately.
In certain embodiments, a formulation comprising an antigen binding protein to
PCSK9, with or without at least one additional therapeutic agents, can be prepared for storage
by mixing the selected formulation having the desired degree of purity with optional
formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a
lyophilized cake or an aqueous solution. Further, in certain embodiments, a formulation
comprising an antigen binding protein to PCSK9, with or without at least one additional
therapeutic agent, can be formulated as a lyophilizate using appropriate excipients.
In certain embodiments, when parenteral administration is contemplated, a therapeutic
formulation can be in the form of a pyrogen-free, parenterally acceptable aqueous solution
comprising a desired antigen binding protein to PCSK9, with or without additional
therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, a
vehicle for parenteral injection is sterile distilled water in which an antigen binding protein to
PCSK9, with or without at least one additional therapeutic agent, is formulated as a sterile,
isotonic solution, properly preserved. In certain embodiments, the preparation can involve
the formulation of the desired molecule with an agent, such as injectable microspheres, bio-
erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads
or liposomes, that can provide for the controlled or sustained release of the product which can
then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be
used, and can have the effect of promoting sustained duration in the circulation. In certain
embodiments, implantable drug delivery devices can be used to introduce the desired
molecule.
In certain embodiments, a pharmaceutical formulation can be formulated for
inhalation. In certain embodiments, an antigen binding protein to PCSK9, with or without at
least one additional therapeutic agent, can be formulated as a dry powder for inhalation. In
certain embodiments, an inhalation solution comprising an antigen binding protein to PCSK9,
with or without at least one additional therapeutic agent, can be formulated with a propellant
for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary
administration is further described in PCT application no. PCT/US94/001875, which
describes pulmonary delivery of chemically modified proteins.
In certain embodiments, a pharmaceutical formulation can involve an effective
quantity of an antigen binding protein to PCSK9, with or without at least one additional
therapeutic agent, in a mixture with non-toxic excipients which are suitable for the
manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water, or
another appropriate vehicle, solutions can be prepared in unit-dose form. In certain
embodiments, suitable excipients include, but are not limited to, inert diluents, such as
calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or
binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium
stearate, stearic acid, or talc.
Additional pharmaceutical formulations will be evident to those skilled in the art,
including formulations involving antigen binding proteins to PCSK9, with or without at least
one additional therapeutic agent(s), in sustained- or controlled-delivery formulations. In
certain embodiments, techniques for formulating a variety of other sustained- or controlled-
delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and
depot injections, are also known to those skilled in the art. See for example, PCT Application
No. PCT/US93/00829 which describes the controlled release of porous polymeric
microparticles for the delivery of pharmaceutical formulations. In certain embodiments,
sustained-release preparations can include semi permeable polymer matrices in the form of
shaped articles, e.g. films, or microcapsules. Sustained release matrices can include
polyesters, hydrogels, polylactides (U.S. 3,773,919 and EP 058,481), copolymers of L-
glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556
(1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-
277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et
al., supra) or poly-D(-)hydroxybutyric acid (EP 133,988). In certain embodiments,
sustained release formulations can also include liposomes, which can be prepared by any of
several methods known in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA,
82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.
The pharmaceutical formulation to be used for in vivo administration typically is
sterile. In certain embodiments, this can be accomplished by filtration through sterile
filtration membranes. In certain embodiments, where the formulation is lyophilized,
sterilization using this method can be conducted either prior to or following lyophilization
and reconstitution. In certain embodiments, the formulation for parenteral administration can
be stored in lyophilized form or in a solution. In certain embodiments, parenteral
formulations generally are placed into a container having a sterile access port, for example,
an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection
needle.
In certain embodiments, once the pharmaceutical formulation has been formulated, it
can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated
or lyophilized powder. In certain embodiments, such formulations can be stored either in a
ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
In certain embodiments, once the pharmaceutical formulation has been formulated, it
can be stored in pre-filled syringes as a solution or suspension in a ready-to-use form
In certain embodiments, kits are provided for producing a single-dose administration
unit. In certain embodiments, the kit can contain both a first container having a dried protein
and a second container having an aqueous formulation. In certain embodiments, kits
containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes) are included.
In certain embodiments, the effective amount of a pharmaceutical formulation
comprising an antigen binding protein to PCSK9, with or without at least one additional
therapeutic agent, to be employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will appreciate that the appropriate
dosage levels for treatment, according to certain embodiments, will thus vary depending, in
part, upon the molecule delivered, the indication for which an antigen binding protein to
PCSK9, with or without at least one additional therapeutic agent, is being used, the route of
administration, and the size (body weight, body surface or organ size) and/or condition (the
age and general health) of the patient. In certain embodiments, the clinician can titer the
dosage and modify the route of administration to obtain the optimal therapeutic effect.
In certain embodiments, the formulation can be administered locally via implantation
of a membrane, sponge or another appropriate material onto which the desired molecule has
been absorbed or encapsulated. In certain embodiments, where an implantation device is
used, the device can be implanted into any suitable tissue or organ, and delivery of the
desired molecule can be via diffusion, timed-release bolus, or continuous administration.
Dosage and Dosing Regimens
Any of the antigen binding proteins to PCSK9 depicted in Table 2 and FIGs. 2 and/or
3 and/or A and 48B can be administered to a patient according to the methods of the
present invention. In some embodiments, the antigen binding proteins to PCSK9 include
21B12, 26H5, 31H4, 8A3, 11F1 or 8A1.
The amount of an antigen binding protein to PCSK9 (e.g., an anti-PCSK9 antibody)
administered to a patient according to the methods of the present invention is, generally, a
therapeutically effective amount. The amount of ABP may be expressed in terms of
milligrams of antibody (i.e., mg) or milligrams of antibody per kilogram of patient body
weight (i.e., mg/kg). In certain embodiments, a typical dosage of a PCSK9 antigen binding
protein can range from about 0.1 g/kg to up to about 100 mg/kg or more of antigen binding
protein to PCSK9,. In certain embodiments, the dosage can range from 0.1 g/kg up to about
100 mg/kg; or 1 g/kg up to about 100 mg/kg; or 5 g/kg up to about 100 mg/kg of antigen
binding protein to PCSK9; or 1 mg/kg to about 50 mg/kg of antigen binding protein to
PCSK9; or 2 mg/kg to about 20 mg/kg of antigen binding protein to PCSK9; or 2 mg/kg to
about 10 mg/kg of antigen binding protein to PCSK9 .
In certain embodiments, the amount (or dose) of antigen binding protein to PCSK9
can range from at least about 10 mg to at about 1400mg; or about 14 mg to about 1200 mg; or
about 14 mg to about 1000 mg; or about 14 mg to about 800 mg; or about 14 mg to about 700
mg; or about 14 mg to about 480 mg; or about 20 mg up to about 480 mg; or about 70 mg up
to about 480 mg; or about 80 mg to about 480 mg; or about 90 mg to about 480 mg; or about
100 mg to about 480 mg, or about 105 mg to about 480 mg; or about 110 mg to about 480
mg; or about 115 mg to about 480 mg; or about 120 mg to about 480 mg; or about 125 mg to
about 480 mg; or about 130 mg to about 480 mg; or about 135 mg to about 480 mg; or about
140 mg to about 480 mg; or about 145 mg to about 480 mg; or about 150 mg to about 480
mg; or about 160 mg to about 480 mg; or about 170 mg to about 480 mg; or about 180 mg to
about 480 mg or about 190 mg to about 480 mg or about 200 mg to about 480 mg; or about
210 mg to about 480 mg; or about 220 mg to about 480 mg; or about 230 mg to about 480
mg; or about 240 mg to about 480 mg; or about 250 mg to about 480 mg; or about 260 mg to
about 480 mg; or about 270 mg to about 480 mg; or about 280 mg to about 480 mg; or about
290 mg to about 480 mg; or about 300 mg to about 480 mg; or about 310 mg to about 480
mg; or about 320 mg to about 480 mg; or about 330 mg to about 480 mg; or about 340 mg to
about 480 mg; or about 350 mg to about 480 mg; or about 360 mg to about 480 mg; or about
370 mg to about 480 mg; or about 380 mg to about 480 mg; or about 390 mg to about 480
mg; or about 400 mg to about 480 mg; or about 410 mg to about 480 mg; or about 420 mg to
about 480 mg; or about 430 mg to about 480 mg; or about 440 mg to about 480 mg; or about
450 mg to about 480 mg; or about 460 mg to about 480 mg; or about 470 mg to about 480 mg
of antigen binding protein to PCSK9.
In certain embodiments, the frequency of dosing will take into account the
pharmacokinetic parameters of an antigen binding protein to PCSK9 and/or any additional
therapeutic agents in the formulation used. In certain embodiments, a clinician will
administer the formulation until a dosage is reached that achieves the desired effect. In
certain embodiments, the formulation can therefore be administered as a single dose, or as
two, three, four or more doses (which may or may not contain the same amount of the desired
molecule) over time, or as a continuous infusion via an implantation device or catheter. The
formulation can also be delivered subcutaneously or intravenously with a standard needle and
syringe. In addition, with respect to subcutaneious delivery, pen delivery devices, as well as
autoinjector delivery devices, have applications in delivering a pharmaceutical formulation of
the present invention. Further refinement of the appropriate dosage is routinely made by
those of ordinary skill in the art and is within the ambit of tasks routinely performed by them.
In certain embodiments, appropriate dosages can be ascertained through use of appropriate
dose-response data. In some embodiments, the amount and frequency of administration can
take into account the desired cholesterol level (serum and/or total) to be obtained and the
subject’s present cholesterol level, LDL level, and/or LDLR levels, all of which can be
obtained by methods that are well known to those of skill in the art.
In certain embodiments, a dose of at least about 10 mg; or up to about 14 mg; or up to
about 20 mg; or up to about 35 mg; or up to about 40 mg, or up to about 45 mg, or up to
about 50 mg; or up to about 70 mg of an antigen binding protein to PCSK9 is administered
once a week (QW) to a patient in need thereof.
In some other embodiments, a dose of at least about 70 mg, or up to about 100 mg; or
up to about 105 mg, or up to about 110 mg; or up to about 115 mg, or up to about 120 mg; or
up to about 140 mg; or up to about 160 mg; or up to about 200 mg; or up to about 250 mg; or
up to 280 mg; or up to 300 mg; or up to 350 mg; or up to 400 mg; or up to 420 mg of an
antigen binding protein to PCSK9 is administered once every other week, (or every two
weeks)(Q2W), to a patient in need thereof.
In certain other embodiments, a dose of at least about 250 mg; or up to about 280 mg;
or up to about 300 mg; or up to about 350 mg; or up to about 400 mg; or up to about 420 mg;
or up to about 450 mg; or up to 480 mg of a an antigen binding protein to PCSK9 is
administered once every four weeks, (or once a month), to a patient in need thereof.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
%, as compared to a predose serum LDL cholesterol level. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 20%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 25%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 30%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 40%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 50%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 55%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 60%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 65%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 70%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 80%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 85%. In some embodiments, the
serum LDL cholesterol level is reduced by at least about 90%.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
40%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
45%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
50%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
55%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
60%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
65%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
70%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
75%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
80%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
85%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
In some embodiments, the serum LDL cholesterol level is reduced by at least about
90%, as compared to a predose serum LDL cholesterol level, and the reduction is sustained
for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about
days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28
days, or at least about 31 days relative to a predose level.
Certain Therapeutic Applications
As will be appreciated by one of skill in the art, disorders that relate to, involve, or
can be influenced by varied cholesterol, LDL, LDLR, PCSK9, VLDL-C, apoprotein B
(“ApoB”), lipoprotein A (“Lp(a)”), triglycerides, HDL-C, non-HDL-C, and total cholesterol
levels can be addressed by the antigen binding proteins to PCSK9 described in the present
invention. In one aspect, antigen binding proteins to PCSK9 can be used in methods to treat
and/or prevent and/or reduce the risk of disorders that relate to elevated serum cholesterol
levels or in which elevated serum cholesterol levels are relevant. In one aspect, antigen
binding proteins to PCSK9 can be used in methods to treat and/or prevent and/or reduce the
risk of disorders that relate to elevated PCSK9 values or in which elevated PCSK9 values are
relevant. In one aspect, antigen binding proteins to PCSK9 can be used in methods to treat
and/or prevent and/or reduce the risk of disorders that relate to elevated total cholesterol
levels or in which elevated total cholesterol levels are relevant. In one aspect, antigen
binding proteins to PCSK9 can be used in methods to treat and/or prevent and/or reduce the
risk of disorders that relate to elevated non-HDL cholesterol levels or in which elevated non-
HDL cholesterol levels are relevant. In one aspect, antigen binding proteins to PCSK9 can be
used in methods to treat and/or prevent and/or reduce the risk of disorders that relate to
elevated ApoB levels or in which elevated ApoB levels are relevant. In one aspect, antigen
binding proteins to PCSK9 can be used in methods to treat and/or prevent and/or reduce the
risk of disorders that relate to elevated Lp(a) levels or in which elevated Lp(a) levels are
relevant. In one aspect, antigen binding proteins to PCSK9 can be used in methods to treat
and/or prevent and/or reduce the risk of disorders that relate to elevated triglyceride levels or
in which elevated triglyceride levels are relevant. In one aspect, antigen binding proteins to
PCSK9 can be used in methods to treat and/or prevent and/or reduce the risk of disorders that
relate to elevated VLDL-C levels or in which elevated VLDL-C levels are relevant.
In one aspect, an antigen binding protein to PCSK9 is used to modulate serum LDL
cholesterol levels in a patient. In some embodiments, the antigen binding protein to PCSK9
is used to decrease the amount of serum LDL cholesterol from an abnormally high level or
even a normal level. In certain embodiments, the serum LDL cholesterol level is reduced by
at least about 30%. In certain embodiments, the serum LDL cholesterol level is reduced by at
least about 35%. In certain embodiments, the serum LDL cholesterol level is reduced by at
least about 40%. In certain embodiments, the serum LDL cholesterol level is reduced by at
least about 45%. In certain embodiments, the serum LDL cholesterol level is reduced by at
least about 50%. In certain embodiments, the serum LDL cholesterol level is reduced by at
least about 55%. In some embodiments, the serum LDL cholesterol level is reduced by at
least about 60%. In some embodiments, the serum LDL cholesterol level is reduced by at
least about 65%. In some embodiments, the serum LDL cholesterol level is reduced by at
least about 70%. In some embodiments, the serum LDL cholesterol level is reduced by at
least about 75%. In some embodiments, the serum LDL cholesterol level is reduced by at
least about 80%. In some embodiments, the serum LDL cholesterol level is reduced by at
least about 85%. In some embodiments, the serum LDL cholesterol level is reduced by at
least about 90%.
In one aspect, an antigen binding protein to PCSK9 is used to modulate serum PCSK9
values in a patient. In certain embodiments, the antigen binding protein to PCSK9 is
neutralizing. In some embodiments, the antigen binding protein to PCSK9 is used to
decrease PCSK9 values from an abnormally high level or even a normal level. In some
embodiments, the serum PCSK9 value is reduced by at least about 60%. In some
embodiments, the serum PCSK9 value is reduced by at least about 65%. In some
embodiments, the serum PCSK9 value is reduced by at least about 70%. In some
embodiments, the serum PCSK9 value is reduced by at least about 75%. In some
embodiments, the serum PCSK9 value is reduced by at least about 80%. In some
embodiments, the serum PCSK9 value is reduced by at least about 85%. In some
embodiments, the serum PCSK9 value is reduced by at least about 90%.
In one aspect, an antigen binding protein to PCSK9 is used to modulate total
cholesterol level in a patient. In certain embodiments, the antigen binding protein to PCSK9
is neutralizing. In some embodiments, the antigen binding protein to PCSK9 is used to
decrease the amount of total cholesterol from an abnormally high level or even a normal
level. In some embodiments, the total cholesterol level is reduced by at least about 20%. In
some embodiments, the total cholesterol level is reduced by at least about 25%. In some
embodiments, the total cholesterol level is reduced by at least about 30%. In some
embodiments, the total cholesterol level is reduced by at least about 35%. In some
embodiments, the total cholesterol level is reduced by at least about 40%. In some
embodiments, the total cholesterol level is reduced by at least about 45%. In some
embodiments, the total cholesterol level is reduced by at least about 50%. In some
embodiments, the total cholesterol level is reduced by at least about 55%. In some
embodiments, the total cholesterol level is reduced by at least about 60%.
In one aspect, an antigen binding protein to PCSK9 is used to modulate the non-HDL
cholesterol level in a patient. In certain embodiments, the antigen binding protein to PCSK9
is neutralizing. In some embodiments, the antigen binding protein to PCSK9 is used to
decrease the non-HDL cholesterol from an abnormally high level or even a normal level. In
some embodiments, the non-HDL cholesterol level is reduced by at least about 30%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 35%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 40%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 50%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 55%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 60%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 65%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 70%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 75%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 80%. In some
embodiments, the non-HDL cholesterol level is reduced by at least about 85%.
In one aspect, an antigen binding protein to PCSK9 is used to modulate the ApoB
levels in a patient. In certain embodiments, the antigen binding protein to PCSK9 is
neutralizing. In some embodiments, the antigen binding protein to PCSK9 is used to
decrease the amount of ApoB from an abnormally high level or even a normal level. In some
embodiments, the ApoB level is reduced by at least about 25%. In some embodiments, the
ApoB level is reduced by at least about 30%. In some embodiments, the ApoB level is
reduced by at least about 35%. In some embodiments, the ApoB level is reduced by at least
about 40%. In some embodiments, the ApoB level is reduced by at least about 45%. In some
embodiments, the ApoB level is reduced by at least about 50%. In some embodiments, the
ApoB level is reduced by at least about 55%. In some embodiments, the ApoB level is
reduced by at least about 60%. In some embodiments, the ApoB level is reduced by at least
about 65%. In some embodiments, the ApoB level is reduced by at least about 70%. In some
embodiments, the ApoB level is reduced by at least about 75%.
In one aspect, an antigen binding protein to PCSK9 is used to modulate the Lp(a)
levels in a patient. In certain embodiments, the antigen binding protein to PCSK9 is
neutralizing. In some embodiments, the antigen binding protein to PCSK9 is used to
decrease the amount of Lp(a) from an abnormally high level or even a normal level. In
some embodiments, the Lp(a) level is reduced by at least about 5%. In some embodiments,
the Lp(a) level is reduced by at least about 10%. In some embodiments, the Lp(a) level is
reduced by at least about 15%. In some embodiments, the Lp(a) level is reduced by at least
about 20%. In some embodiments, the Lp(a) level is reduced by at least about 25%. In some
embodiments, the Lp(a) level is reduced by at least about 30%. In some embodiments, the
Lp(a) level is reduced by at least about 35%. In some embodiments, the Lp(a) level is
reduced by at least about 40%. In some embodiments, the Lp(a) level is reduced by at least
about 45%. In some embodiments, the Lp(a) level is reduced by at least about 50%. In some
embodiments, the Lp(a) level is reduced by at least about 55%. In some embodiments, the
Lp(a) level is reduced by at least about 60%. In some embodiments, the Lp(a) level is
reduced by at least about 65%.
As will be appreciated by one of skill in the art, the antigen binding proteins to
PCSK9 of the present invention can be therapeutically useful in treating and/or preventing
cholesterol related disorders. In some embodiments, a “cholesterol related disorder” (which
includes “serum cholesterol related disorders”) includes any one or more of the following:
familial hypercholesterolemia, non-familial hypercholesterolemia, hyperlipidemia, heart
disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular
diseases, Alzheimer’s disease and generally dyslipidemias, which can be manifested, for
example, by an elevated total serum cholesterol, elevated LDL, elevated triglycerides,
elevated VLDL, and/or low HDL. Some non-limiting examples of primary and secondary
dyslipidemias that can be treated using an ABP, either alone, or in combination with one or
more other agents include the metabolic syndrome, diabetes mellitus, familial combined
hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemias, including
heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial defective
apoplipoprotein B-100; polygenic hypercholesterolemia; remnant removal disease, hepatic
lipase deficiency; dyslipidemia secondary to any of the following: dietary indiscretion,
hypothyroidism, drugs including estrogen and progestin therapy, beta-blockers, and thiazide
diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary
cirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly, insulinoma, isolated
growth hormone deficiency, and alcohol-induced hypertriglyceridemia. ABP can also be
useful in preventing or treating atherosclerotic diseases, such as, for example, cardiovascular
death, non-cardiovascular or all-cause death, coronary heart disease, coronary artery disease,
peripheral arterial disease, stroke (ischaemic and hemorrhagic), angina pectoris, or
cerebrovascular disease and acute coronary syndrome, myocardial infarction and untable
angina. In some embodiments, the ABP is useful in reducing the risk of: fatal and nonfatal
heart attacks, fatal and non-fatal strokes, certain types of heart surgery, hospitalization for
heart failure, chest pain in patients with heart disease, and/or cardiovascular events because
of established heart disease such as prior heart attack, prior heart surgery, and/or chest pain
with evidence of clogged arteries and/or transplant-related vascular disease. In some
embodiments, the ABP is useful in preventing or reducing the cardiovascular risk due to
elevated CRP or hsCRP. In some embodiments, the ABP and methods can be used to reduce
the risk of recurrent cardiovascular events.
As will be appreciated by one of skill in the art, diseases or disorders that are
generally addressable (either treatable or preventable) through the use of statins can also
benefit from the application of the instant antigen binding proteins. In addition, in some
embodiments, disorders or disease that can benefit from the prevention of cholesterol
synthesis or increased LDLR expression can also be treated by various embodiments of the
antigen binding proteins. In addition, as will be appreciated by one of skill in the art, the use
of the anti-PCSK9 antibodies can be especially useful in the treatment of diabetes. Not only
is diabetes a risk factor for coronary heart disease, but insulin increases the expression of
PCSK9. That is, people with Diabetes have elevated plasma lipid levels (which can be
related to high PCSK9 levels) and can benefit from lowering those levels. This is generally
discussed in more detail in Costet et al. (“Hepatic PCSK9 Expression is Regulated by
Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1C”, J. Biol.
Chem., 281: 6211-6218, 2006), the entirety of which is incorporated herein by reference.
In some embodiments, the antigen binding protein is administered to those who have
diabetes mellitus, abdominal aortic aneurysm, atherosclerosis and/or peripheral vascular
disease in order to decrease their serum cholesterol levels to a safer range. In some
embodiments, the antigen binding protein is administered to patients at risk of developing any
of the herein described disorders. In some embodiments, the ABPs are administered to
subjects that smoke, or used to smoke (i.e., former smokers),, have hypertension or a familial
history of early heart attacks.
In some embodiments, a subject is administered an ABP if they are at a moderate risk
or higher on the 2004 NCEP treatment goals. In some embodiments, the ABP is
administered to a subject if the subject’s LDL cholesterol level is greater than 160 mg/dl. In
some embodiments, the ABP is administered if the subjects LDL cholesterol level is greater
than 130 (and they have a moderate or moderately high risk according to the 2004 NCEP
treatment goals). In some embodiments, the ABP is administered if the subjects LDL
cholesterol level is greater than 100 (and they have a high or very high risk according to the
2004 NCEP treatment goals). In some embodiments, the ABP is administered if the subjects
LDL cholesterol level is greater than 80mg/dL. In some embodiments, the ABP is
administered if the subjects LDL cholesterol level is greater than 70 mg/dL.
A physician will be able to select appropriate treatment indications and target lipid
levels depending on the individual profile of a particular patient. One well-accepted standard
for guiding treatment of hyperlipidemia is the Third Report of the National Cholesterol
Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of the
High Blood Cholesterol in Adults (Adult Treatment Panel III) Final Report, National
Institutes of Health, NIH Publication No. 02-5215 (2002), the printed publication of which is
hereby incorporated by reference in its entirety.
In some embodiments, antigen binding proteins to PCSK9 are used to treat or prevent
hypercholesterolemia, hyperlipidemia or dyslipidemia and/or in the preparation of
medicaments therefore and/or for other cholesterol related disorders (such as those noted
herein). In certain embodiments, an antigen binding protein to PCSK9 is used to treat or
prevent conditions such as hypercholesterolemia in which PCSK9 activity is normal. In such
conditions, for example, reduction of PCSK9 activity to below normal can provide a
therapeutic effect.
Combination Therapies
In certain embodiments, methods are provided of treating a cholesterol
related disorder, such as hypercholesterolemia, hyperlipidemia or dyslipidemia, comprising
administering a therapeutically effective amount of one or more antigen binding proteins to
PCSK9 and another therapeutic agent. In certain embodiments, an antigen binding protein to
PCSK9 is administered prior to the administration of at least one other therapeutic agent. In
certain embodiments, an antigen binding protein to PCSK9 is administered concurrent with
the administration of at least one other therapeutic agent. In certain embodiments, an antigen
binding protein to PCSK9 is administered subsequent to the administration of at least one
other therapeutic agent.
Therapeutic agents (apart from the antigen binding protein), include, but
are not limited to, at least one other cholesterol-lowering (serum and/or total body
cholesterol) agent. In some embodiments, the agent increases the expression of LDLR, have
been observed to increase serum HDL levels, lower LDL levels or lower triglyceride levels.
Exemplary agents include, but are not limited to, statins (atorvastatin, cerivastatin,
fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin),
Nicotinic acid (Niacin) (NIACOR, NIASPAN (slow release niacin), SLO-NIACIN (slow
release niacin), CORDAPTIVE (laropiprant)), Fibric acid (LOPID (Gemfibrozil), TRICOR
(fenofibrate), Bile acid sequestrants (QUESTRAN (cholestyramine), colesevelam
(WELCHOL), COLESTID (colestipol)), Cholesterol absorption inhibitors (ZETIA
(ezetimibe)), Combining nicotinic acid with statin (ADVICOR (LOVASTATIN and
NIASPAN), Combining a statin with an absorption inhibitor (VYTORIN (ZOCOR and
ZETIA) and/or lipid modifying agents. In some embodiments, the ABP is combined with
PPAR gamma agonsits, PPAR alpha/gamma agonists, squalene synthase inhibitors, CETP
inhibitors, anti-hypertensives, anti-diabetic agents (such as sulphonyl ureas, insulin, GLP-1
analogs, DDPIV inhibitors, e.g., metaformin), ApoB modulators, such as mipomersan, MTP
inhibitoris and /or arteriosclerosis obliterans treatments. In some embodiments, the ABP is
combined with an agent that increases the level of LDLR protein in a subject, such as statins,
certain cytokines like oncostatin M, estrogen, and/or certain herbal ingredients such as
berberine. In some embodiments, the ABP is combined with an agent that increases serum
cholesterol levels in a subject (such as certain anti-psycotic agents, certain HIV protease
inhibitors, dietary factors such as high fructose, sucrose, cholesterol or certain fatty acids and
certain nuclear receptor agonists and antagonists for RXR, RAR, LXR, FXR). In some
embodiments, the ABP is combined with an agent that increases the level of PCSK9 in a
subject, such as statins and/or insulin. The combination of the two can allow for the
undesirable side-effects of other agents to be mitigated by the ABP.
In certain embodiments, an antigen binding protein to PCSK9 can be used with at
least one therapeutic agent for inflammation. In certain embodiments, an antigen binding
protein to PCSK9 can be used with at least one therapeutic agent for an immune disorder.
Exemplary therapeutic agents for inflammation and immune disorders include, but are not
limited to cyclooxygenase type 1 (COX-1) and cyclooxygenase type 2 (COX-2 ) inhibitors
small molecule modulators of 38 kDa mitogen-activated protein kinase (p38-MAPK); small
molecule modulators of intracellular molecules involved in inflammation pathways, wherein
such intracellular molecules include, but are not limited to, jnk, IKK, NF- κB, ZAP70, and
lck. Certain exemplary therapeutic agents for inflammation are described, e.g., in C.A.
Dinarello & L.L. Moldawer Proinflammatory and Anti-Inflammatory Cytokines in
Rheumatoid Arthritis: A Primer for Clinicians Third Edition (2001) Amgen Inc. Thousand
Oaks, CA.
Diagnostic Applications
In some embodiments, the ABP is used as a diagnostic tool. The ABP can be used to
assay the amount of PCSK9 present in a sample and/or subject. As will be appreciated by
one of skill in the art, such ABPs need not be neutralizing ABPs. In some embodiments, the
diagnostic ABP is not a neutralizing ABP. In some embodiments, the diagnostic ABP binds
to a different epitope than the neutralizing ABP binds to. In some embodiments, the two
ABPs do not compete with one another.
In some embodiments, the ABPs disclosed herein are used or provided in an assay kit
and/or method for the detection of PCSK9 in mammalian tissues or cells in order to
screen/diagnose for a disease or disorder associated with changes in levels of PCSK9. The
kit comprises an ABP that binds PCSK9 and means for indicating the binding of the ABP
with PCSK9, if present, and optionally PCSK9 protein levels. Various means for indicating
the presence of an ABP can be used. For example, fluorophores, other molecular probes, or
enzymes can be linked to the ABP and the presence of the ABP can be observed in a variety
of ways. The method for screening for such disorders can involve the use of the kit, or
simply the use of one of the disclosed ABPs and the determination of whether the ABP binds
to PCSK9 in a sample. As will be appreciated by one of skill in the art, high or elevated
levels of PCSK9 will result in larger amounts of the ABP binding to PCSK9 in the sample.
Thus, degree of ABP binding can be used to determine how much PCSK9 is in a sample.
Subjects or samples with an amount of PCSK9 that is greater than a predetermined amount
(e.g., an amount or range that a person without a PCSK9 related disorder would have) can be
characterized as having a PCSK9 mediated disorder. In some embodiments, the ABP is
administered to a subject taking a statin, in order to determine if the statin has increased the
amount of PCSK9 in the subject.
In some embodiments, the ABP is a non-neutralizing ABP and is used to determine
the amount of PCSK9 in a subject receiving an ABP and/or statin treatment.
EXAMPLES
The following examples, including the experiments conducted and results achieved,
are provided for illustrative purposes only and are not to be construed as limiting the present
invention.
EXAMPLE 1
Immunization and Titering
Generation of Anti-PCSK9 Antibodies and Hybridomas
Antibodies to the mature form of PCSK9 (depicted as the sequence in , with
the pro-domain underlined), were raised in XenoMouse mice (Abgenix, Fremont, CA),
which are mice containing human immunoglobulin genes. Two groups of XenoMouse
mice, group 1 and 2, were used to produce antibodies to PCSK9. Group 1 included mice of
the XenoMouse strain XMG2-KL, which produces fully human IgG2 and
IgG2 antibodies. Group 1 mice were immunized with human PCSK9. PCSK9 was
prepared using standard recombinant techniques using the GenBank sequence as reference
(NM_174936). Group 2 involved mice of the XenoMouse strain XMG4-KL, which produce
fully human IgG4 and IgG4 antibodies. Group 2 mice were also immunized with human
PCSK9.
The mice of both groups were injected with antigen eleven times, according to the
schedule in Table 3. In the initial immunizations, each mouse was injected with a total of 10
g of antigen delivered intraperitoneally into the abdomen. Subsequent boosts are 5ug doses
and injection method is staggered between intraperitoneal injections into the abdomen and
sub-cutaneous injections at the base of the tail. For intraperitoneal injections antigen is
prepared as an emulsion with TiterMax Gold (Sigma, Cat # T2684) and for subcutaneous
injections antigen is mixed with Alum (aluminum phosphate) and CpG oligos. In injections 2
through 8 and 10, each mouse was injected with a total of 5 g of antigen in the adjuvant
alum gel. A final injection of 5 g of antigen per mouse is delivered in Phospho buffered
saline and delivered into 2 sites 50% IP into the abdomen and 50% SQ at the base of tail.
The immunization programs are summarized in Table 3, shown below.
TABLE 3
Group 1 2
mouse strain XMG2/kl XMG4/kl
# of animals 10 10
immunogen PCSK9-V5/His PCSK9-V5/His
1st boost IP injection IP injection
10ug each 10ug each
Titermax Gold Titermax Gold
2nd boost tail injection tail injection
5ug each 5ug each
Alum/CpG ODN Alum/CpG ODN
3rd boost
IP injection IP injection
5ug each 5ug each
Titermax Gold Titermax Gold
4th boost
tail injection tail injection
5ug each 5ug each
Alum/CpG ODN Alum/CpG ODN
5th boost IP injection IP injection
5ug each 5ug each
Titermax Gold Titermax Gold
6th boost tail injection tail injection
5ug each 5ug each
Alum/CpG ODN Alum/CpG ODN
7th boost
IP injection IP injection
5ug each 5ug each
Titermax Gold Titermax Gold
8th boost
tail injection tail injection
5ug each 5ug each
Alum/CpG ODN Alum/CpG ODN
bleed
9th boost IP injection IP injection
5ug each 5ug each
Titermax Gold Titermax Gold
10th boost
tail injection tail injection
5ug each 5ug each
Alum/CpG ODN Alum/CpG ODN
11th boost
BIP BIP
5ug each 5ug each
PBS PBS
harvest
The protocol used to titer the XenoMouse animals was as follows: Costar 3368
medium binding plates were coated with neutravadin @ 8ug/ml (50ul/well) and incubated at
4ºC in 1XPBS/0.05% azide overnight. They were washed using TiterTek 3-cycle wash with
RO water. Plates were blocked using 250ul of 1XPBS/1%milk and incubated for at least 30
minutes at RT. Block was washed off using TiterTek 3-cycle wash with RO water. One then
captured b-human PCSK9 @ 2ug/ml in 1XPBS/1%milk/10mM Ca2+ (assay diluent)
50ul/well and incubated for 1hr at RT. One then washed using TiterTek 3-cycle wash with
RO water. For the primary antibody, sera were titrated 1:3 in duplicate from 1:100. This was
done in assay diluent 50ul/well and incubated for 1hr at RT. One then washed using TiterTek
3-cycle wash with RO water. The secondary antibody was goat anti Human IgG Fc HRP @
400 ng/ml in assay diluent at 50ul/well. This was incubated for 1hr at RT. This was then
washed using TiterTek 3-cycle wash with RO water and patted dry on paper towels. For the
substrate, one-step TMB solution (Neogen, Lexington, Kentucky) was used (50ul/well) and it
was allowed to develop for 30 min at RT.
The protocols followed in the ELISA assays were as follows: For samples comprising
b-PCSK9 with no V5His tag the following protocol was employed: Costar 3368 medium
binding plates (Corning Life Sciences) were employed. The plates were coated with
neutravadin at 8 g/ml in 1XPBS/0.05%Azide, (50 l/well). The plates were incubated at
4ºC overnight. The plates were then washed using a Titertek M384 plate washer (Titertek,
Huntsville, AL). A 3-cycle wash was performed. The plates were blocked with 250 l of
1XPBS/1% milk and incubated approximately 30 minutes at room temperature. The plates
were then washed using the M384 plate washer. A 3-cycle wash was performed. The
capture was b-hu PCSK9, without a V5 tag, and was added at 2 g/ml in
1XPBS/1%milk/10mM Ca (40 l/well). The plates were then incubated for 1 hour at room
temperature. A 3-cycle wash was performed. Sera were titrated 1:3 in duplicate from 1:100,
and row H was blank for sera. The titration was done in assay diluent, at a volume of 50
l/well. The plates were incubated for 1 hour at room temperature. Next, a 3-cycle wash
was performed. Goat anti Human IgG Fc HRP at 100 ng/ml (1:4000) in
1XPBS/1%milk/10mM Ca (50 l/well) was added to the plate and was incubated 1 hour at
room temperature. The plates were washed once again, using a 3-cycle wash. The plates
were then patted dry with paper towel. Finally, 1 step TMB (Neogen, Lexington, Kentucky)
(50 l/well) was added to the plate and was quenched with 1N hydrochloric acid (50 l/well)
after 30 minutes at room temperature. OD's were read immediately at 450 nm using a
Titertek plate reader.
Positive controls to detect plate bound PCSK9 were soluble LDL receptor (R&D
Systems, Cat #2148LD/CF) and a polyclonal rabbit anti-PCSK9 antibody (Caymen Chemical
#10007185) titrated 1:3 in duplicate from 3 g/ml in assay diluent. LDLR was detected with
goat anti LDLR (R&D Systems, Cat #AF2148) and rabbit anti goat IgGFc HRP at a
concentration of 400 ng/ml; the rabbit polyclonal was detected with goat anti-rabbit IgG Fc at
a concentration of 400 ng/ml in assay diluent. Negative control was naive XMG2-KL and
XMG4-KL sera titrated 1:3 in duplicate from 1:100 in assay diluent.
For samples comprising b-PCSK9 with a V5His tag the following protocol was
employed: Costar 3368 medium binding plates (Corning Life Sciences) were employed. The
plates were coated with neutravadin at 8 g/ml in 1XPBS/0.05%Azide, (50 l/well). The
plates were incubated at 4ºC overnight. The plates were then washed using a Titertek M384
plate washer (Titertek, Huntsville, AL). A 3-cycle wash was performed. The plates were
blocked with 250 l of 1XPBS/1% milk and incubated approximately 30 minutes at room
temperature. The plates were then washed using the M384 plate washer. A 3-cycle wash
was performed. The capture was b-hu PCSK9, with a V5 tag, and was added at 2 g/ml in
1XPBS/1%milk/10mM Ca (40 l/well). The plates were then incubated for 1 hour at room
temperature. A 3-cycle wash was performed. Sera were titrated 1:3 in duplicate from 1:100,
and row H was blank for sera. The titration was done in assay diluent, at a volume of 50
l/well. The plates were incubated for 1 hour at room temperature. Next, the plates were
washed using the M384 plate washer operated using a 3-cycle wash. Goat anti Human IgG
Fc HRP at 400 ng/ml in 1XPBS/1%milk/10mM Ca was added at 50 l/well to the plate
and the plate was incubated 1 hour at room temperature. The plates were washed once again,
using a 3-cycle wash. The plates were then patted dry with paper towel. Finally, 1 step TMB
(Neogen, Lexington, Kentucky) (50 l/well) was added to the plate and the plate was
quenched with 1N hydrochloric acid (50 l/well) after 30 minutes at room temperature. OD's
were read immediately at 450 nm using a Titertek plate reader.
Positive control was LDLR, rabbit anti-PCSK9 titrated 1:3 in duplicate from 3 g/ml
in assay diluent. LDLR detect with goat anti-LDLR (R&D Systems, Cat #AF2148) and
rabbit anti-goat IgG Fc HRP at a concentration of 400 ng/ml; rabbit poly detected with goat
anti-rabbit IgG Fc at a concentration of 400 ng/ml in assay diluent. Human anti-His 1.2,3 and
anti-V5 1.7.1 titrated 1:3 in duplicate from 1 g/ml in assay diluent; both detected with goat
anti-human IgG Fc HRP at a concentration of 400 ng/ml in assay diluent. Negative control
was naive XMG2-KL and XMG4-KL sera titrated 1:3 in duplicate from 1:100 in assay
diluent.
Titers of the antibody against human PCSK9 were tested by ELISA assay for mice
immunized with soluble antigen as described. Table 4 summarizes the ELISA data and
indicates that there were some mice which appeared to be specific for PCSK9. See, e.g.,
Table 4. Therefore, at the end of the immunization program, 10 mice (in bold in Table 4)
were selected for harvest, and splenocytes and lymphocytes were isolated from the spleens
and lymph nodes respectively, as described herein.
TABLE 4
Summary of ELISA Results
Titer Titer
b-hu PCSK9 b-hu PCSK9 @
Animal
(V5His) @ 2ug/ml 2ug/ml
P175807 >72900 @ OD 2.2 68359
P175808 >72900 @ OD 2.3 >72900 @ OD 2.5
P175818 >72900 @ OD 3.2 >72900 @ OD 3.0
>72900 @ OD 3.4
P175819 >72900 @ OD 3.2
Group 1 -
P175820 >72900 @ OD 2.4 >72900 @ OD 2.5
P175821 >72900 @ OD 3.4 >72900 @ OD 3.0
IgG2k/l
P175830 >72900 @ OD 2.6 >72900 @ OD 2.5
>72900 @ OD 3.1
P175831 >72900 @ OD 3.1
>72900 @ OD 3.8
P175832 >72900 @ OD 3.6
P175833 >72900 @ OD 2.6 >72900 @ OD 2.3
P174501 19369 17109
P174503 31616 23548
48472
P174508 30996
P174509 23380 21628
Group 2 -
P174510 15120 9673
P175773 19407 15973
IgG4k/l
54580
P175774 44424
P175775 60713 55667
P175776 30871 22899
P175777 16068 12532
Naïve
G2 < 100 @ OD 0.54 < 100 @ OD 0.48
Naïve
G4 < 100 @ OD 1.57 < 100 @ OD 1.32
EXAMPLE 2
Recovery of Lymphocytes, B-cell Isolations, Fusions
and Generation of Hybridomas
This example outlines how the immune cells were recovered and the hybridomas were
generated. Selected immunized mice were sacrificed by cervical dislocation and the draining
lymph nodes were harvested and pooled from each cohort. The B cells were dissociated from
lymphoid tissue by grinding in DMEM to release the cells from the tissues, and the cells were
suspended in DMEM. The cells were counted, and 0.9 ml DMEM per 100 million
lymphocytes was added to the cell pellet to resuspend the cells gently but completely.
Lymphocytes were mixed with nonsecretory myeloma P3X63Ag8.653 cells
purchased from ATCC, cat.# CRL 1580 (Kearney et al., (1979) J. Immunol. 123, 1548-1550)
at a ratio of 1:4. The cell mixture was gently pelleted by centrifugation at 400 x g 4 min.
After decanting of the supernatant, the cells were gently mixed using a 1 ml pipette.
Preheated PEG/DMSO solution from Sigma (cat# P7306) (1 ml per million of B-cells) was
slowly added with gentle agitation over 1 min followed by 1 min of mixing. Preheated
IDMEM (2 ml per million of B cells) (DMEM without glutamine, L-glutamine, pen/strep,
MEM non-essential amino acids (all from Invitrogen), was then added over 2 minutes with
gentle agitation. Finally preheated IDMEM (8 ml per 10 B-cells) was added over 3 minutes.
The fused cells were spun down 400 x g 6 min and resuspended in 20 ml selection
media (DMEM (Invitrogen), 15 % FBS (Hyclone), supplemented with L-glutamine,
pen/strep, MEM Non-essential amino acids, Sodium Pyruvate, 2-Mercaptoethanol (all from
Invitrogen), HA-Azaserine Hypoxanthine and OPI (oxaloacetate, pyruvate, bovine insulin)
(both from Sigma) and IL-6 (Boehringer Mannheim)) per million B-cells. Cells were
incubated for 20-30 min at 37C and then resuspended in 200 ml selection media and cultured
for 3-4 days in T175 flask prior to 96 well plating. Thus, hybridomas that produced antigen
binding proteins to PCSK9 were produced.
EXAMPLE 3
Selection of PCSK9 Antibodies
The present example outlines how the various PCSK9 antigen binding proteins were
characterized and selected. The binding of secreted antibodies (produced from the
hybridomas produced in Examples 1 and 2) to PCSK9 was assessed. Selection of antibodies
was based on binding data and inhibition of PCSK9 binding to LDLR and affinity. Binding
to soluble PCSK9 was analyzed by ELISA, as described below. BIAcore (surface plasmon
resonance) was used to quantify binding affinity.
Primary Screen
A primary screen for antibodies which bind to wild-type PCSK9 was performed. The
primary screen was performed on two harvests. The primary screen comprised an ELISA
assay and was performed using the following protocol:
Costar 3702 medium binding 384 well plates (Corning Life Sciences) were employed.
The plates were coated with neutravadin at a concentration of 4 g/ml in
1XPBS/0.05%Azide, at a volume of 40 l/well. The plates were incubated at 4ºC overnight.
The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). A 3-
cycle wash was performed. The plates were blocked with 90 l of 1XPBS/1%milk and
incubated approximately 30 minutes at room temperature. The plates were then washed.
Again, a 3-cycle wash was performed. The capture sample was biotinylated-PCSK9, without
a V5 tag, and was added at 0.9 g/ml in 1XPBS/1%milk/10mM Ca at a volume of 40
l/well. The plates were then incubated for 1 hour at room temperature. Next, the plates
were washed using the Titertek plate washer operated using a 3-cycle wash. 10 l of
supernatant was transferred into 40 l of 1XPBS/1%milk/10mM Ca and incubated 1.5
hours at room temperature. Again the plates were washed using the Titertek plate washer
operated using a 3-cycle wash. 40 l/well of Goat anti-Human IgG Fc POD at a
concentration of 100 ng/ml (1:4000) in 1XPBS/1%milk/10mM Ca was added to the plate
and was incubated 1 hour at room temperature. The plates were washed once again, using a
3-cycle wash. Finally, 40 l/well of One-step TMB (Neogen, Lexington, Kentucky) was
added to the plate and quenching with 40 l/well of 1N hydrochloric acid was performed
after 30 minutes at room temperature. OD’s were read immediately at 450 nm using a
Titertek plate reader.
The primary screen resulted in a total of 3104 antigen specific hybridomas being
identified from the two harvests. Based on highest ELISA OD, 1500 hybridomas per harvest
were advanced for a total of 3000 positives.
Confirmatory Screen
The 3000 positives were then rescreened for binding to wild-type PCSK9 to confirm
stable hybridomas were established. The screen was performed as follows: Costar 3702
medium binding 384 well plates (Corning Life Sciences) were employed. The plates were
coated with neutravadin at 3 g/ml in 1XPBS/0.05%Azide at a volume of 40 l/well. The
plates were incubated at 4ºC overnight. The plates were then washed using a Titertek plate
washer (Titertek, Huntsville, AL). A 3-cycle wash was performed. The plates were blocked
with 90 l of 1XPBS/1%milk and incubated approximately 30 minutes at room temperature.
The plates were then washed using the M384 plate washer. A 3-cycle wash was performed.
The capture sample was b-PCSK9, without a V5 tag, and was added at 0.9 g/ml in
1XPBS/1%milk/10mM Ca at a volume of 40 l/well. The plates were then incubated for 1
hour at room temperature. Next, the plates were washed using a 3-cycle wash. 10 l of
supernatant was transferred into 40 l of 1XPBS/1%milk/10mM Ca and incubated 1.5
hours at room temperature. Again the plates were washed using the Titertek plate washer
operated using a 3-cycle wash. 40 l/well of Goat anti-Human IgG Fc POD at a
concentration of 100 ng/ml (1:4000) in 1XPBS/1%milk/10mM Ca was added to the plate,
and the plate was incubated 1 hour at room temperature. The plates were washed once again,
using the Titertek plate washer operated using a 3-cycle wash. Finally, 40 l/well of One-
step TMB (Neogen, Lexington, Kentucky) was added to the plate and was quenched with 40
l/well of 1N hydrochloric acid after 30 minutes at room temperature. OD’s were read
immediately at 450 nm using a Titertek plate reader. A total of 2441 positives repeated in the
second screen. These antibodies were then used in the subsequent screenings.
Mouse Cross-reactivity Screen
The panel of hybridomas was then screened for cross-reactivity to mouse PCSK9 to
make certain that the antibodies could bind to both human and mouse PCSK9. The following
protocol was employed in the cross-reactivity screen: Costar 3702 medium binding 384 well
plates (Corning Life Sciences) were employed. The plates were coated with neutravadin at 3
g/ml in 1XPBS/0.05%Azide at a volume of 40 l/well. The plates were incubated at 4ºC
overnight. The plates were then washed using a Titertek plate washer (Titertek, Huntsville,
AL). A 3-cycle wash was performed. The plates were blocked with 90 l of 1XPBS/1%milk
and incubated approximately 30 minutes at room temperature. The plates were then washed
using the Titertek plate washer. A 3-cycle wash was performed. The capture sample was
biotinylated-mouse PCSK9, and was added at 1 g/ml in 1XPBS/1%milk/10mM Ca at a
volume of 40 l/well. The plates were then incubated for 1 hour at room temperature. Next,
the plates were washed using the Titertek plate washer operated using a 3-cycle wash. 50 l
of supernatant was transferred to the plates and incubated 1 hour at room temperature. Again
the plates were washed using a 3-cycle wash. 40 l/well of Goat anti-Human IgG Fc POD at
a concentration of 100 ng/ml (1:4000) in 1XPBS/1%milk/10mM Ca was added to the plate
and the plate was incubated 1 hour at room temperature. The plates were washed once again,
using a 3-cycle wash. Finally, 40 l/well One-step TMB (Neogen, Lexington, Kentucky)
was added to the plate and was quenched with 40 l/well of 1N hydrochloric acid after 30
minutes at room temperature. OD’s were read immediately at 450 nm using a Titertek plate
reader. 579 antibodies were observed to cross-react with mouse PCSK9. These antibodies
were then used in the subsequent screenings.
D374Y Mutant Binding Screen
The D374Y mutation in PCSK9 has been documented in the human population (e.g.,
Timms KM et al, “A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia
in a Utah pedigree”, Hum. Genet. 114: 349-353, 2004). In order to determine if the
antibodies were specific for the wild type or also bound to the D374Y form of PCSK9, the
samples were then screened for binding to the mutant PCSK9 sequence comprising the
mutation D374Y. The protocol for the screen was as follows: Costar 3702 medium binding
384 well plates (Corning Life Sciences) were employed in the screen. The plates were coated
with neutravadin at 4 g/ml in 1XPBS/0.05% Azide at a volume of 40 l/well. The plates
were incubated at 4ºC overnight. The plates were then washed using a Titertek plate washer
(Titertek, Huntsville, AL). A 3-cycle wash was performed. The plates were blocked with 90
l of 1XPBS/1%milk and incubated approximately 30 minutes at room temperature. The
plates were then washed using the Titertek plate washer. A 3-cycle wash was performed.
The plates were coated with biotinylated human PCSK9 D374Y at a concentration of 1 g/ml
in 1XPBS/1%milk/10mMCa and incubated for 1 hour at room temperature. The plates
were then washed using a Titertek plate washer. A 3-cycle wash was performed. Late
exhaust hybridoma culture supernatant was diluted 1:5 in PBS/milk/Ca (10 ml plus 40 ml)
and incubated for 1 hour at room temperature. Next, 40 l/well of rabbit anti-human PCSK9
(Cayman Chemical) and human anti-His 1.2.3 1:2 at 1ug/ml in 1XPBS/1%milk/10mMCa
was titrated onto the plates, which were then incubated for 1 hour at room temperature. The
plates were then washed using a Titertek plate washer. A 3-cycle wash was performed. 40
l/well of Goat anti-Human IgG Fc HRP at a concentration of 100 ng/ml (1:4000) in
1XPBS/1%milk/10mM Ca was added to the plate and the plate was incubated 1 hour at
room temperature. 40 l/well of Goat anti-rabbit IgG Fc HRP at a concentration of 100
ng/ml (1:4000) in 1XPBS/1%milk/10mM Ca was added to the plate and the plate was
incubated 1 hour at room temperature. The plates were then washed using a Titertek plate
washer. A 3-cycle wash was performed. Finally, 40 l/well of One-step TMB (Neogen,
Lexington, Kentucky) was added to the plate and was quenched with 40 l/well of 1N
hydrochloric acid after 30 minutes at room temperature. OD’s were read immediately at 450
nm using a Titertek plate reader. Over 96% of the positive hits on the wild-type PCSK9 also
bound mutant PCSK9.
Large Scale Receptor Ligand Blocking Screen
To screen for the antibodies that block PCSK9 binding to LDLR an assay was
developed using the D374Y PCSK9 mutant. The mutant was used for this assay because it
has a higher binding affinity to LDLR allowing a more sensitive receptor ligand blocking
assay to be developed. The following protocol was employed in the receptor ligand blocking
screen: Costar 3702 medium binding 384 well plates (Corning Life Sciences) were employed
in the screen. The plates were coated with goat anti-LDLR (R&D Cat #AF2148) at 2 g/ml
in 1XPBS/0.05%Azide at a volume of 40 l/well. The plates were incubated at 4ºC
overnight. The plates were then washed using a Titertek plate washer (Titertek, Huntsville,
AL). A 3-cycle wash was performed. The plates were blocked with 90 l of 1XPBS/1%
milk and incubated approximately 30 minutes at room temperature. The plates were then
washed using the Titertek plate washer. A 3-cycle wash was performed. The capture sample
was LDLR (R&D, Cat #2148LD/CF), and was added at 0.4 g/ml in 1XPBS/1%milk/10mM
Ca at a volume of 40 l/well. The plates were then incubated for 1 hour and 10 minutes at
room temperature. Contemporaneously, 20 ng/ml of biotinylated human D374Y PCSK9 was
incubated with 15 micro liters of hybridoma exhaust supernatant in Nunc polypropylene
plates and the exhaust supernatant concentration was diluted 1:5. The plates were then pre-
incubated for about 1 hour and 30 minutes at room temperature. Next, the plates were
washed using the Titertek plate washer operated using a 3-cycle wash. 50 l/well of the pre-
incubated mixture was transferred onto the LDLR coated ELISA plates and incubated for 1
hour at room temperature. To detect LDLR-bound b-PCSK9, 40 l/well streptavidin HRP at
500 ng/ml in assay diluent was added to the plates. The plates were incubated for 1 hour at
room temperature. The plates were again washed using a Titertek plate washer. A 3-cycle
wash was performed. Finally, 40 l/well of One-step TMB (Neogen, Lexington, Kentucky)
was added to the plate and was quenched with 40 l/well of 1N hydrochloric acid after 30
minutes at room temperature. OD’s were read immediately at 450 nm using a Titertek plate
reader. The screen identified 384 antibodies that blocked the interaction between PCSK9 and
the LDLR well, 100 antibodies blocked the interaction strongly (OD < 0.3). These antibodies
inhibited the binding interaction of PCSK9 and LDLR greater than 90% (greater than 90%
inhibition).
Receptor Ligand Binding Assay on Blocker Subset
The receptor ligand assay was then repeated using the mutant enzyme on the 384
member subset of neutralizers identified in the first large scale receptor ligand inhibition
assay. The same protocol was employed in the screen of the 384 member blocker subset
assay as was done in the large scale receptor ligand blocking screen. This repeat screen
confirmed the initial screening data.
This screen of the 384 member subset identified 85 antibodies that blocked interaction
between the PCSK9 mutant enzyme and the LDLR greater than 90%.
Receptor Ligand Binding Assay of Blockers that Bind the Wild Type PCSK9 but not the
D374Y Mutant
In the initial panel of 3000 sups there were 86 antibodies shown to specifically bind to
the wild-type PCSK9 and not to the huPCSK9(D374Y) mutant. These 86 sups were tested
for the ability to block wild-type PCSK9 binding to the LDLR receptor. The following
protocol was employed: Costar 3702 medium binding 384 well plates (Corning Life
Sciences) were employed in the screen. The plates were coated with anti-His 1.2.3 at 10
g/ml in 1XPBS/0.05% Azide at a volume of 40 l/well. The plates were incubated at 4ºC
overnight. The plates were then washed using a Titertek plate washer (Titertek, Huntsville,
AL). A 3-cycle wash was performed. The plates were blocked with 90 l of 1XPBS/1%milk
and incubated approximately 30 minutes at room temperature. The plates were then washed
using the Titertek plate washer. A 3-cycle wash was performed. LDLR (R&D Systems,
#2148LD/CF or R&D Systems, #2148LD) was added at 5 g/ml in 1XPBS/1%milk/10mM
Ca at a volume of 40 l/well. The plates were then incubated for 1 hour at room
temperature. Next, the plates were washed using the Titertek plate washer operated using a
3-cycle wash. Contemporaneously, biotinylated human wild-type PCSK9 was pre-incubated
with hybridoma exhaust supernatant in Nunc polypropylene plates. 22 l of hybridoma sup
was transferred into 33ul of b-PCSK9 at a concentration of 583 ng/ml in
1XPBS/1%milk/10mMCa2+, giving a final b-PCSK9 concentration = 350 ng/ml and the
exhaust supernatant at a final dilution of 1:2.5. The plates were pre-incubated for
approximately 1 hour and 30 minutes at room temperature. 50 l/well of the preincubated
mixture was transferred onto LDLR captured ELISA plates and incubated for 1 hour at room
temperature. The plates were then washed using the Titertek plate washer. A 3-cycle wash
was performed. 40 l/well streptavidin HRP at 500 ng/ml in assay diluent was added to the
plates. The plates were incubated for 1 hour at room temperature. The plates were then
washed using a Titertek plate washer. A 3-cycle wash was performed. Finally, 40 l/well of
One-step TMB (Neogen, Lexington, Kentucky) was added to the plate and was quenched
with 40 l/well of 1N hydrochloric acid after 30 minutes at room temperature. OD’s were
read immediately at 450 nm using a Titertek plate reader.
Screening Results
Based on the results of the assays described, several hybridoma lines were identified
as producing antibodies with desired interactions with PCSK9. Limiting dilution was used to
isolate a manageable number of clones from each line. The clones were designated by
hybridoma line number (e.g. 21B12) and clone number (e.g. 21B12.1). In general, no
difference among the different clones of a particular line was detected by the functional
assays described herein. In a few cases, clones were identified from a particular line that
behaved differently in the functional assays, for example, 25A7.1 was found not to block
PCSK9/LDLR but 25A7.3 (referred to herein as 25A7) was neutralizing. The isolated clones
were each expanded in 50-100 ml of hybridoma media and allowed to grow to exhaustion,
(i.e., less than about 10% cell viability). The concentration and potency of the antibodies to
PCSK9 in the supernatants of those cultures were determined by ELISA and by in vitro
functional testing, as described herein. As a result of the screening described herein, the
hybridomas with the highest titer of antibodies to PCSK9 were identified. The selected
hybridomas are shown in FIGS 2A-3D and Table 2.
EXAMPLE 4.1
Production of Human 31H4 IgG4 Antibodies from Hybridomas
This example generally describes how one of the antigen binding proteins was
produced from a hybridoma line. The production work used 50ml exhaust supernatant
generation followed by protein A purification. Integra production was for scale up and was
performed later. Hybridoma line 31H4 was grown in T75 flasks in 20 ml of media (Integra
Media, Table 5). When the hybridoma was nearly confluent in the T75 flasks, it was
transferred to an Integra flask (Integra Biosciences, Integra CL1000, cat# 90 005).
The Integra flask is a cell culture flask that is divided by a membrane into two
chambers, a small chamber and a large chamber. A volume of 20-30 ml hybridoma cells at a
minimum cell density of 1x10 cells per ml from the 31H4 hybridoma line was placed into
the small chamber of an Integra flask in Integra media (see Table 5 for components of Integra
media). Integra media alone (1L) was placed in the large chambers of the Integra flasks. The
membrane separating the two chambers is permeable to small molecular weight nutrients but
is impermeable to hybridoma cells and to antibodies produced by those cells. Thus, the
hybridoma cells and the antibodies produced by those hybridoma cells were retained in the
small chamber.
After one week, media was removed from both chambers of the Integra flask and was
replaced with fresh Integra media. The collected media from the small chambers was
separately retained. After a second week of growth, the media from the small chamber was
again collected. The collected media from week 1 from the hybridoma line was combined
with the collected media from week 2 from the hybridoma line. The resulting collected
media sample from the hybridoma line was spun to remove cells and debris (15 minutes at
3000rpm) and the resulting supernatant was filtered (0.22µm). Clarified conditioned media
was loaded onto a Protein A-Sepharose column. Optionally, the media can be first
concentrated and then loaded onto a Protein A Sepharose column. Non-specific bindings
were removed by an extensive PBS wash. Bound antibody proteins on the Protein A column
were recovered by standard acidic antibody elution from Protein A columns (such as 50 mM
Citrate, pH 3.0). Aggregated antibody proteins in the Protein A Sepharose pool were
removed by size exclusion chromatography or binding ion exchange chromatography on
anion exchanger resin such as Q Sepharose resin. The specific IEX conditions for the 31H4
proteins are Q-Sepharose HP at pH 7.8-8.0. Antibody was eluted with a NaCl gradient of 10
mM-500 mM in 25 column volumes.
TABLE 5
Composition of Media
INTEGRA MEDIA
HSFM
% Ultra Low IgG serum
2mmol/L L-glutamine
1% NEAA
4g/L glucose
EXAMPLE 4.2
Production of Recombinant 31H4 Human IgG2
Antibodies From Transfected Cells
The present example outlines how 31H4 IgG2 antibodies were produced from
transfected cells. 293 cells for transient expression and CHO cells for stable expression were
transfected with plasmids that encode 31H4 heavy and light chains. Conditioned media from
transfected cells was recovered by removing cells and cell debris. Clarified conditioned
media was loaded onto a Protein A-Sepharose column. Optionally, the media can first be
concentrated and then loaded onto a Protein A Sepharose column. Non-specific bindings
were removed by extensive PBS wash. Bound antibody proteins on the Protein A column
were recovered by standard acidic antibody elution from Protein A columns (such as 50 mM
citrate, pH 3.0). Aggregated antibody proteins in the Protein A Sepharose pool were
removed by size exclusion chromatography or binding ion exchange chromatography on
anion exchanger resin such as Q Sepharose resin. The specific IEX conditions for the 31H4
proteins are Q-Sepharose HP at pH 7.8-8.0. The antibody was eluted with a NaCl gradient of
mM-500 mM in 25 column volumes.
EXAMPLE 5
Production of Human 21B12 IgG4 Antibodies from Hybridomas
The present example outlines how antibody 21B12 IgG4 was produced from
hybridomas. Hybridoma line 21B12 was grown in T75 flasks in media (Integra Media, Table
). When the hybridomas were nearly confluent in the T75 flasks, they were transferred to
Integra flasks (Integra Biosciences, Integra CL1000, cat# 90 005).
The Integra flask is a cell culture flask that is divided by a membrane into two
chambers, a small chamber and a large chamber. A volume of 20-30 ml hybridoma cells at a
minimum cell density of 1x10 cells per ml from the 31H4 hybridoma line was placed into
the small chamber of an Integra flask in Integra media (see Table 5 for components of Integra
media). Integra media alone (1L) was placed in the large chambers of the Integra flasks. The
membrane separating the two chambers is permeable to small molecular weight nutrients but
is impermeable to hybridoma cells and to antibodies produced by those cells. Thus, the
hybridoma cells and the antibodies produced by those hybridoma cells were retained in the
small chamber.
After one week, media was removed from both chambers of the Integra flask and was
replaced with fresh Integra media. The collected media from the small chambers was
separately retained. After a second week of growth, the media from the small chamber was
again collected. The collected media from week 1 from the hybridoma line was combined
with the collected media from week 2 from the hybridoma line. The resulting collected
media sample from the hybridoma line was spun to remove cells and debris (15 minutes at
3000 rpm) and the resulting supernatant was filtered (0.22 m). Clarified conditioned media
were loaded onto a Protein A Sepharose column. Optionally, the media are first concentrated
and then loaded onto a Protein A Sepharose column. Non-specific bindings were removed by
an extensive PBS wash. Bound antibody proteins on the Protein A column were recovered
by standard acidic antibody elution from Protein A columns (such as 50 mM Citrate, pH
3.0). Aggregated antibody proteins in the Protein A Sepharose pool were removed by size
exclusion chromatography or binding ion exchange chromatography on anion exchanger
resin such as Q Sepharose resin. The specific IEX conditions for the 21B12 proteins are Q-
Sepharose HP at pH 7.8-8.0. The antibody was eluted with a NaCl gradient of 10 mM-500
mM in 25 column volumes.
EXAMPLE 6
Production of Human 21B12 IgG2 Antibodies
From Transfected Cells
The present example outlines how 21B12 IgG2 antibodies were produced from
transfected cells. Cells (293 cells for transient expression and CHO cells for stable
expression) were transfected with plasmids that encode 21B12 heavy and light chains.
Conditioned media from hybridoma cells were recovered by removing cells and cell debris.
Clarified conditioned media were loaded onto a Protein A-Sepharose column. Optionally, the
media can first be concentrated and then loaded onto a Protein A Sepharose column. Non-
specific bindings were removed by extensive PBS wash. Bound antibody proteins on the
Protein A column were recovered by standard acidic antibody elution from Protein A
columns (50 mM Citrate, pH 3.0). Aggregated antibody proteins in the Protein A Sepharose
pool were removed by size exclusion chromatography or binding ion exchange
chromatography on cation exchanger resin such as SP-Sepharose resin. The specific IEX
conditions for the 21B12 proteins were SP-Sepharose HP at pH 5.2. Antibodies were eluted
with 25 column volumes of buffer that contains a NaCl gradient of 10 mM-500 mM in 20
mM sodium acetate buffer.
EXAMPLE 7
Sequence Analysis of Antibody Heavy and Light Chains
The nucleic acid and amino acid sequences for the light and heavy chains of the above
antibodies were then determined by Sanger (dideoxy) nucleotide sequencing. Amino acid
sequences were then deduced for the nucleic acid sequences. The nucleic acid sequences for
the variable domains are depicted in FIG.s 3E-3JJ.
The cDNA sequences for the lambda light chain variable regions of 31H4, 21B12,
and 16F12 were determined and are disclosed as SEQ ID NOs: 153, 95, and 105 respectively.
The cDNA sequences for the heavy chain variable regions of 31H4, 21B12, and
16F12 were determined and are disclosed as SEQ ID NOs: 152, 94, and 104 respectively.
The lambda light chain constant region (SEQ ID NO: 156), and the IgG2 and IgG4
heavy chain constant regions (SEQ ID NOs: 154 and 155) are shown in K.
The polypeptide sequences predicted from each of those cDNA sequences were
determined. The predicted polypeptide sequences for the lambda light chain variable regions
of 31H4, 21B12, and 16F12 were predicted and are disclosed as SEQ ID NOs: 12, 23, and 35
respectively, the lambda light chain constant region (SEQ ID NO: 156), the heavy chain
variable regions of 31H4, 21B12, and 16F12 were predicted and are disclosed as (SEQ. ID
NOs. 67, 49, and 79 respectively. The IgG2 and IgG4 heavy chain constant regions (SEQ ID
NOs: 154 and 155).
The FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 divisions are shown in FIG 2A-3D.
Based on the sequence data, the germline genes from which each heavy chain or light
chain variable region was derived was determined. The identity of the germline genes are
indicated next to the corresponding hybridoma line in FIGs. 2A-3D and each is represented
by a unique SEQ ID NO. FIGs. 2A-3D also depict the determined amino acid sequences for
additional antibodies that were characterized.
EXAMPLE 8
Characterization of Binding of Antibodies to PCSK9
Having identified a number of antibodies that bind to PCSK9, several approaches
were employed to quantify and further characterize the nature of the binding. In one aspect
of the study, a Biacore affinity analysis was performed. In another aspect of the study a
KinExA affinity analysis was performed. The samples and buffers employed in these
studies are presented in Table 6 below.
TABLE 6
[sample] [sample]
sample mg/ml Buffer µm
hPCSK9 1.26 PBS 16.6
mPCSK9-8xHIS 1.44 PBS 18.9
cPCSK9-V5-6xHIS 0.22 PBS 2.9
20mM NaOAC, pH
16F12, anti-PCSK9 huIgG4 4.6 31.9
.2, 50mM NaCl
10mM NAOAC, pH
21B12, anti-PCSK9 huIgG4 3.84 5.2, 9% Sucrose 27.0
10mM NAOAC, pH
31H4, anti-PCSK9 huIgG4 3.3 22.9
.2, 9% Sucrose
BIAcore Affinity Measurements
A BIAcore (surface plasmon resonance device, Biacore, Inc., Piscataway, NJ)
affinity analysis of the 21B12 antibodies to PCSK9 described in this Example was performed
according to the manufacturer’s instructions.
Briefly, the surface plasmon resonance experiments were performed using Biacore
2000 optical biosensors (Biacore, GE Healthcare, Piscataway, NJ). Each individual anti-
PCSK9 antibody was immobilized to a research-grade CM5 biosensor chip by amine-
coupling at levels that gave a maximum analyte binding response (Rmax) of no more than
200 resonance units (RU). The concentration of PCSK9 protein was varied at 2 fold intervals
(the analyte) and was injected over the immobilized antibody surface (at a flow rate of 100
μl/min for 1.5 minutes). Fresh HBS-P buffer (pH 7.4, 0.01 M Hepes, 0.15 M NaCl, 0.005%
surfactant P-20, Biacore) supplemented with 0.01% BSA was used as binding buffer.
Binding affinities of each anti-PCSK9 antibody were measured in separate experiments
against each of the human, mouse, and cynomolgus monkey PCSK9 proteins at pH 7.4 (the
concentrations used were 100, 50, 25, 12.5, 6.25, 3.125, and 0 nM).
In addition, the binding affinities of antibody to human PCSK9 were also measured at
pH 6.0 with the pH 6.0 HBS-P buffer (pH 6.0, 0.01 M Hepes, 0.15 M NaCl, 0.005%
surfactant P-20, Biacore) supplemented with 0.01% BSA. The binding signal obtained was
proportional to the free PCSK9 in solution. The dissociation equilibrium constant (K ) was
obtained from nonlinear regression analysis of the competition curves using a dual-curve one-
site homogeneous binding model (KinExA software, Sapidyne Instruments Inc., Boise, ID)
(n=1 for the 6.0 pH runs). Interestingly, the antibodies appeared to display a tighter binding
affinity at the lower pH (where the Kd was 12.5, 7.3, and 29 pM for 31H4, 21B12, and 16F12
respectively).
Antibody binding kinetic parameters including k (association rate constant), k
(dissociation rate constant), and K (dissociation equilibrium constant) were determined
using the BIA evaluation 3.1 computer program (BIAcore, Inc. Piscataway, NJ). Lower
dissociation equilibrium constants indicate greater affinity of the antibody for PCSK9. The
K values determined by the BIAcore affinity analysis are presented in Table 7.1, shown
below.
TABLE 7.1
Antibody hPCSK9 CynoPCSK9 mPCSK9
31H4 210 pM 190 pM 6 nM
21B12 190 pM 360 pM 460 nM
16F12 470 pM 870 pM 6.4 nM
Table 7.2 depicts the k and k rates.
on off
TABLE 7.2
K (M-1 s-1) K (s-1) K
on off D
31H4.1, pH 7.4 2.45 e+5 5.348 e-5 210 pM
31H4.1, pH 6 5.536 e+6 6.936 e-5 12.5 pM
21B12.1, pH 7.4 3.4918 e+4 6.634 e-6 190 pM
21B12.1, pH 6 2.291 e+6 1.676 e-5 7.3 pM
16F12.1, pH 7.4 1.064 e+5 4.983 e-5 470 pM
16F12.1, pH 6 2.392 e+6 7.007 e-5 29 pM
KinExA Affinity Measurements
A KinExA (Sapidyne Instruments, Inc., Boise, ID) affinity analysis of 16F12 and
31H4 was performed according to the manufacturer’s instructions. Briefly, Reacti-Gel™
(6x) (Pierce) was pre-coated with one of human, V5-tagged cyno or His-tagged mouse
PCSK9 proteins and blocked with BSA. 10 or 100 pM of antibody 31H4 and one of the
PCSK9 proteins was then incubated with various concentrations (0.1 pM – 25 nM) of PCSK9
proteins at room temperature for 8 hours before being passed through the PCSK9-coated
beads. The amount of the bead-bound 31H4 was quantified by fluorescently (Cy5) labeled
goat anti-human IgG (H+L) antibody (Jackson Immuno Research). The binding signal is
proportional to the concentration of free 31H4 at binding equilibrium. Equilibrium
dissociation constant (K ) were obtained from nonlinear regression of the two sets of
competition curves using a one-site homogeneous binding model. The KinExA Pro
software was employed in the analysis. Binding curves generated in this analysis are
presented as FIGs. 4A-4F.
Both the 16F12 and 31H4 antibodies showed similar affinity to human and cyno
PCSK9, but approximately 10-250 fold lower affinity to mouse PCSK9. Of the two
antibodies tested using the KinExA system, antibody 31H4 showed higher affinity to both
human and cyno PCSK9 with 3 and 2 pM K respectively. 16F12 showed slightly weaker
affinity at 15pM K to human PCSK9 and 16 pM K to cyno PCSK9.
The results of the KinExA affinity analysis are summarized in Table 8.1, shown
below.
TABLE 8.1
hPCSK9 cPCSK mPCSK
KD (pM) KD (pM) KD (pM)
Sample 95% CI 95% CI 95% CI
3 2 500
31H4.1 1~5 1~3 400~620
In addition, a SDS PAGE was run to check the quality and quantity of the samples
and is shown in . cPCSK9 showed around 50% less on the gel and also from the
active binding concentration calculated from KinExA assay. Therefore, the K of the mAbs
to cPCSK9 was adjusted as 50% of the active cPCSK9 in the present.
A BIAcore solution equilibrium binding assay was used to measure the Kd values for
ABP 21B12. 21B12.1 showed little signal using KinExA assay, therefore, biacore solution
equilibrium assay was applied. Since no significant binding was observed on binding of
antibodies to immobilized PCSK9 surface, 21B12 antibody was immobilized on the flow cell
4 of a CM5 chip using amine coupling with density around 7000 RU. Flow cell 3 was used
as a background control. 0.3, 1, and 3 nM of human PCSK9 or cyno PCSK9 were mixed with
a serial dilutions of 21B12.1 antibody samples (ranged from 0.001 ~ 25 nM) in PBS plus
0.1mg/ml BSA, 0.005% P20. Binding of the free PCSK9 in the mixed solutions were
measured by injecting over the 21B12.1 antibody surface. 100% PCSK9 binding signal on
21B12.1 surface was determined in the absence of mAb in the solution. A decreased PCSK9
binding response with increasing concentrations of mAb indicated that PCSK9 binding to
mAb in solution, which blocked PCSK9 from binding to the immobilized peptibody surface.
Plotting the PCSK9 binding signal versus mAb concentrations, K was calculated from three
sets of curves (0.3, 1 and 3nM fixed PCSK9 concentration) using a one-site homogeneous
binding model in KinExA Pro™ software. Although cPCSK9 has lower protein
concentration observed from KinExA assay and SDS-gel, its concentration was not adjusted
here since the concentration of cPCSK9 was not used for calculation of K . The results are
displayed in Table 8.2 below and in FIGs. 5B-5D. depicts the results from the
solution equilibrium assay at three different hPCSK9 concentrations for hPCSK9.
depicts a similar set of results for mPCSK9. depicts the results from the above
biacore capture assay.
TABLE 8.2
hPCSK9 cPCSK mPCSK
Sample KD (pM) 95% CI KD (pM) 95% CI KD (pM) 95% CI
21B12.1 15 9~23 11 7~16 17000 -
EXAMPLE 9
Efficacy of 31H4 and 21B12 for Blocking D374Y PCSK9/LDLR Binding
This example provides the IC50 values for two of the antibodies in blocking PCSK9
D374Y’s ability to bind to LDLR. Clear 384 well plates (Costar) were coated with 2
micrograms/ml of goat anti-LDL receptor antibody (R&D Systems) diluted in buffer A (100
mM sodium cacodylate, pH 7.4). Plates were washed thoroughly with buffer A and then
blocked for 2 hours with buffer B (1% milk in buffer A). After washing, plates were
incubated for 1.5 hours with 0.4 micrograms/ml of LDL receptor (R&D Systems) diluted in
buffer C (buffer B supplemented with 10 mM CaCl2). Concurrent with this incubation, 20
ng/ml of biotinylated D374Y PCSK9 was incubated with various concentrations of the 31H4
IgG2, 31H4 IgG4, 21B12 IgG2 or 21B12 IgG4 antibody, which was diluted in buffer A, or
buffer A alone (control). The LDL receptor containing plates were washed and the
biotinylated D374Y PCSK9/antibody mixture was transferred to them and incubated for 1
hour at room temperature. Binding of the biotinylated D374Y to the LDL receptor was
detected by incubation with streptavidin-HRP (Biosource) at 500 ng/ml in buffer C followed
by TMB substrate (KPL). The signal was quenched with 1N HCl and the absorbance read at
450 nm.
The results of this binding study are shown in FIGs. 6A-6D. Summarily, IC values
were determined for each antibody and found to be 199 pM for 31H4 IgG2 (), 156
pM for 31H4 IgG4 (), 170 pM for 21B12 IgG2 (), and 169 pM for 21B12
IgG4 ().
The antibodies also blocked the binding of wild-type PCSK9 to the LDLR in this
assay.
EXAMPLE 10
Cell LDL Uptake Assay
This example demonstrates the ability of various antigen binding proteins to reduce
LDL uptake by cells. Human HepG2 cells were seeded in black, clear bottom 96-well plates
(Costar) at a concentration of 5x10 cells per well in DMEM medium (Mediatech, Inc)
supplemented with 10% FBS and incubated at 37ºC (5% CO2) overnight. To form the
PCSK9 and antibody complex, 2 g/ml of D374Y human PCSK9 was incubated with various
concentrations of antibody diluted in uptake buffer (DMEM with 1% FBS) or uptake buffer
alone (control) for 1 hour at room temperature. After washing the cells with PBS, the D374Y
PCSK9/antibody mixture was transferred to the cells, followed by LDL-BODIPY
(Invitrogen) diluted in uptake buffer at a final concentration of 6 g/ml. After incubation for
3 hours at 37ºC (5% CO2), cells were washed thoroughly with PBS and the cell fluorescence
signal was detected by Safire™ (TECAN) at 480-520nm (excitation) and 520-600nm
(emission).
The results of the cellular uptake assay are shown in FIGs. 7A-7D. Summarily, IC
values were determined for each antibody and found to be 16.7 nM for 31H4 IgG2 (),
13.3 nM for 31H4 IgG4 (), 13.3 nM for 21B12 IgG2 (), and 18 nM for
21B12 IgG4 (). These results demonstrate that the applied antigen binding proteins
can reduce the effect of PCSK9 (D374Y) to block LDL uptake by cells The antibodies also
blocked the effect of wild-type PCSK9 in this assay.
EXAMPLE 11
Serum cholesterol Lowering Effect of the 31H4 Antibody in 6 Day Study
In order to assess total serum cholesterol (TC) lowering in wild type (WT) mice via
antibody therapy against PCSK9 protein, the following procedure was performed.
Male WT mice (C57BL/6 strain, aged 9-10 weeks, 17-27 g) obtained from Jackson
Laboratory (Bar Harbor, ME) were fed a normal chow (Harland-Teklad, Diet 2918) through
out the duration of the experiment. Mice were administered either anti-PCSK9 antibody
31H4 (2 mg/ml in PBS) or control IgG (2 mg/ml in PBS) at a level of 10mg/kg through the
mouse’s tail vein at T=0. Naïve mice were also set aside as a naïve control group. Dosing
groups and time of sacrifice are shown in Table 9.
TABLE 9
Group Treatment Time point after dosing Number
1 IgG 8 hr 7
2 31H4 8 hr 7
3 IgG 24 hr 7
4 31H4 24 hr 7
IgG 72 hr 7
6 31H4 72 hr 7
7 IgG 144 hr 7
8 31H4 144 hr 7
9 Naïve n/a 7
Mice were sacrificed with CO2 asphyxiation at the pre-determined time points shown
in Table 9. Blood was collected via vena cava into eppendorf tubes and was allowed to clot
at room temperature for 30 minutes. The samples were then spun down in a table top
centrifuge at 12,000xg for 10 minutes to separate the serum. Serum total cholesterol and
HDL-C were measured using Hitachi 912 clinical analyzer and Roche/Hitachi TC and HDL-
C kits.
The results of the experiment are shown in FIGs. 8A-8D. Summarily, mice to which
antibody 31H4 was administered showed decreased serum cholesterol levels over the course
of the experiment ( and ). In addition, it is noted that the mice also showed
decreased HDL levels ( and ). For and , the percentage
change is in relation to the control IgG at the same time point (*P<0.01, # P<0.05). For and FIG 8D, the percentage change is in relation to total serum cholesterol and HDL
levels measured in naïve animals at t=0 hrs (*P<0.01, # P<0.05).
In respect to the lowered HDL levels, it is noted that one of skill in the art will
appreciate that the decrease in HDL in mice is not indicative that an HDL decrease will occur
in humans and merely further reflects that the serum cholesterol level in the organism has
decreased. It is noted that mice transport the majority of serum cholesterol in high density
lipoprotein (HDL) particles which is different to humans who carry most serum cholesterol
on LDL particles. In mice the measurement of total serum cholesterol most closely
resembles the level of serum HDL-C. Mouse HDL contains apolipoprotein E (apoE) which is
a ligand for the LDL receptor (LDLR) and allows it to be cleared by the LDLR. Thus,
examining HDL is an appropriate indicator for the present example, in mice (with the
understanding that a decrease in HDL is not expected for humans). For example, human
HDL, in contrast, does not contain apoE and is not a ligand for the LDLR. As PCSK9
antibodies increase LDLR expression in mouse, the liver can clear more HDL and therefore
lowers serum HDL-C levels.
EXAMPLE 12
Effect of Antibody 31H4 on LDLR Levels in a 6 Day Study
The present example demonstrates that an antigen binding protein alters the level of
LDLR in a subject, as predicted, over time. A Western blot analysis was performed in order
to ascertain the effect of antibody 31H4 on LDLR levels. 50-100 mg of liver tissue obtained
from the sacrificed mice described in Example 13 was homogenized in 0.3 ml of RIPA buffer
(Santa Cruz Biotechnology Inc.) containing complete protease inhibitor (Roche). The
homogenate was incubated on ice for 30 minutes and centrifuged to pellet cellular debris.
Protein concentration in the supernatant was measured using BioRad protein assay reagents
(BioRad laboratories). 100µg of protein was denatured at 70ºC for 10 minutes and separated
on 4-12% Bis-Tris SDS gradient gel (Invitrogen). Proteins were transferred to a 0.45 µm
PVDF membrane (Invitrogen) and blocked in washing buffer (50mM Tris PH7.5, 150mM
NaCL, 2mM CaCl and 0.05% Tween 20) containing 5% non-fat milk for 1 hour at room
temperature. The blot was then probed with goat anti-mouse LDLR antibody (R&D system)
1:2000 or anti-ß actin (sigma) 1:2000 for 1 hour at room temperature. The blot was washed
briefly and incubated with bovine anti-goat IgG-HRP (Santa Cruz Biotechnology Inc.)
1:2000 or goat anti-mouse IgG-HRP (Upstate) 1:2000. After a 1 hour incubation at room
temperature, the blot was washed thoroughly and immunoreactive bands were detected using
ECL plus kit (Amersham biosciences). The Western blot showed an increase in LDLR
protein levels in the presence of antibody 31H4, as depicted in
EXAMPLE 13
Serum cholesterol Lowering Effect of Antibody 31H4 in a 13 Day Study
In order to assess total serum cholesterol (TC) lowering in wild type (WT) mice via
antibody therapy against PCSK9 protein in a 13 day study, the following procedure was
performed.
Male WT mice (C57BL/6 strain, aged 9-10 weeks, 17-27 g) obtained from Jackson
Laboratory (Bar Harbor, ME) were fed a normal chow (Harland-Teklad, Diet 2918) through
out the duration of the experiment. Mice were administered either anti-PCSK9 antibody
31H4 (2 mg/ml in PBS) or control IgG (2 mg/ml in PBS) at a level of 10 mg/kg through the
mouse’s tail vein at T=0. Naïve mice were also set aside as naïve control group.
Dosing groups and time of sacrifice are shown in Table 10. Animals were sacrificed
and livers were extracted and prepared as in Example 13.
TABLE 10
Group Treatment Time point after dosing Number Dose
1 IgG 72 hr 6 10mg/kg
2 31H4 72 hr 6 10mg/kg
3 31H4 72 hr 6 1mg/kg
4 IgG 144 hr 6 10mg/kg
31H4 144 hr 6 10mg/kg
6 31H4 144 hr 6 1mg/kg
7 IgG 192 hr 6 10mg/kg
8 31H4 192 hr 6 10mg/kg
9 31H4 192 hr 6 1mg/kg
IgG 240 hr 6 10mg/kg
11 31H4 240hr 6 10mg/kg
12 31H4 240hr 6 1mg/kg
13 IgG 312 hr 6 10mg/kg
14 31H4 312 hr 6 10mg/kg
31H4 312 hr 6 1mg/kg
16 Naive n/a 6 n/a
When the 6 day experiment was extended to a 13 day study, the same serum
cholesterol lowering effect observed in the 6 day study was also observed in the 13 day study.
More specifically, animals dosed at 10 mg/kg demonstrated a 31% decrease in serum
cholesterol on day 3, which gradually returned to pre-dosing levels by day 13. A
depicts the results of this experiment. C depicts the results of repeating the above
procedure with the 10mg/kg dose of 31H4, and with another antibody, 16F12, also at
10mg/kg. Dosing groups and time of sacrifice are shown in Table 11.
TABLE 11
Group Treatment Time point after dosing Number Dose
1 IgG 24 hr 6 10mg/kg
2 16F12 24 hr 6 10mg/kg
3 31H4 24 hr 6 10mg/kg
4 IgG 72 hr 6 10mg/kg
16F12 72 hr 6 10mg/kg
6 31H4 72 hr 6 10mg/kg
7 IgG 144 hr 6 10mg/kg
8 16F12 144 hr 6 10mg/kg
9 31H4 144 hr 6 10mg/kg
IgG 192 hr 6 10mg/kg
11 16F12 192 hr 6 10mg/kg
12 31H4 192 hr 6 10mg/kg
13 IgG2 240 hr 6 10mg/kg
14 16F12 240hr 6 10mg/kg
31H4 240hr 6 10mg/kg
16 IgG2 312 hr 6 10mg/kg
17 16F12 312 hr 6 10mg/kg
18 31H4 312 hr 6 10mg/kg
19 Naive n/a 6 10mg/kg
As shown in C both 16F12 and 31H4 resulted in significant and substantial
decreases in total serum cholesterol after just a single dose and provided benefits for over a
week (10 days or more). The results of the repeated 13 day study were consistent with the
results of the first 13 day study, with a decrease in serum cholesterol levels of 26% on day 3
being observed. For A and B, the percentage change is in relation to the
control IgG at the same time point (*P<0.01). For C, the percentage change is in
relation to the control IgG at the same time point (*P<0.05).
EXAMPLE 14
Effect of Antibody 31H4 on HDL Levels in a 13 Day Study
The HDL levels for the animals in Example 15 were also examined. HDL levels
decreased in the mice. More specifically, animals dosed at 10 mg/kg demonstrated a 33%
decrease in HDL levels on day 3, which gradually returned to pre-dosing levels by day 13.
B depicts the results of the experiment. There was a decrease in HDL levels of 34%
on day 3. B depicts the results of the repeated 13 day experiment.
As will be appreciated by one of skill in the art, while the antibodies will lower mouse
HDL, this is not expected to occur in humans because of the differences in HDL in humans
and other organisms (such as mice). Thus, the decrease in mouse HDL is not indicative of a
decrease in human HDL.
EXAMPLE 15
Repeated Administration of Antibodies Produce Continued Benefits
of Antigen Binding Peptides
In order to verify that the results obtained in the Examples above can be prolonged for
further benefits with additional doses, the Experiments in Examples 15 and 16 were repeated
with the dosing schedule depicted in A. The results are displayed in B. As
can be seen in the graph in B, while both sets of mice displayed a significant decrease
in total serum cholesterol because all of the mice received an initial injection of the 31H4
antigen binding protein, the mice that received additional injections of the 31H4 ABP
displayed a continued reduction in total serum cholesterol, while those mice that only
received the control injection eventually displayed an increase in their total serum
cholesterol. For , the percentage change is in relation to the naïve animals at t=0
hours (*P<0.01, **P<0.001).
The results from this example demonstrate that, unlike other cholesterol treatment
methods, in which repeated applications lead to a reduction in efficacy because of biological
adjustments in the subject, the present approach does not seem to suffer from this issue over
the time period examined. Moreover, this suggests that the return of total serum cholesterol
or HDL cholesterol levels to baseline, observed in the previous examples is not due to some
resistance to the treatment being developed by the subject, but rather the depletion of the
antibody availability in the subject.
EXAMPLE 16
Uses of PCSK9 Antibodies for the Treatment of
Cholesterol Related Disorders
A human patient exhibiting a Cholesterol Related Disorder (in which a reduction in
cholesterol (such as serum cholesterol) can be beneficial) is administered a therapeutically
effective amount of PCSK9 antibody, 31H4 (or, for example, 21B12). At periodic times
during the treatment, the patient is monitored to determine whether the symptoms of the
disorder have subsided. Following treatment, it is found that patients undergoing treatment
with the PCSK9 antibody have reduced serum cholesterol levels, in comparison to patients
that are not treated.
EXAMPLE 17
Uses of PCSK9 Antibodies for the Treatment of Hypercholesterolemia
A human patient exhibiting symptoms of hypercholesterolemia is administered a
therapeutcially effective amount of PCSK9 antibody, such as 31H4 (or, for example, 21B12).
At periodic times during the treatment, the human patient is monitored to determine whether
the serum cholesterol level has declined. Following treatment, it is found that the patient
receiving the treatment with the PCSK9 antibodies has reduced serum cholesterol levels in
comparison to arthritis patients not receiving the treatment.
EXAMPLE 18
Uses of PCSK9 Antibodies for the Prevention of
Coronary Heart Disease and/or Recurrent Cardiovascular Events
A human patient at risk of developing coronary heart disease is identified. The
patient is administered a therapeutically effective amount of PCSK9 antibody, such as 31H4
(or, for example, 21B12), either alone, concurrently or sequentially with a statin, e.g.,
simvastatin. At periodic times during the treatment, the human patient is monitored to
determine whether the patient’s total serum cholesterol level changes. Throughout the
preventative treatment, it is found that the patient receiving the treatment with the PCSK9
antibodies has reduced serum cholesterol thereby reducing their risk to coronary heart
diseases or recurrent cardiovascular events in comparison to patients not receiving the
treatment.
EXAMPLE 19
Use of PCSK9 Antigen Binding Protein for the
Prevention of Hypercholesterolemia
A human patient exhibiting a risk of developing hypercholesterolemia is identified via
family history analysis and/or lifestyle, and/or current cholesterol levels. The subject is
regularly administered (e.g., one time weekly) a therapeutically effective amount of PCSK9
antibody, 31H4 (or, for example, 21B12). At periodic times during the treatment, the patient
is monitored to determine whether serum cholesterol levels have decreased. Following
treatment, it is found that subjects undergoing preventative treatment with the PCSK9
antibody have lowered serum cholesterol levels, in comparison to subjects that are not
treated.
EXAMPLE 20
A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Ascending
Single Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics and
Pharmacodynamics of a Human Anti-PCSK9 Antibody in Healthy Subjects
This Study was a randomized, double-blind, placebo-controlled, ascending-single-
dose study to evaluate the safety, tolerability, PK, pharmacodynamics (PD) (LDL-C), and
immunogenicity of a human anti-PCSK9 antibody (monoclonal antibody 21B12) in healthy
subjects. Subjects were randomized in a 3:1 ratio (21B12:placebo; 8 subjects per dose cohort
for a total of 56 subjects in 7 cohorts) to receive 21B12 at doses of 7, 21, 70, 210, or 420 mg
SC, or corresponding placebo; or 21B12 at doses of 21 or 420 mg IV, or corresponding
placebo.
Fifty-six subjects were randomized and received investigational product (42 21B12,
14 placebo); 40 subjects (30 21B12, 10 placebo) received investigational product by the SC
route of administration, and 16 subjects (12 21B12, 4 placebo) received investigational
product by the IV route. Fifty-three of the 56 subjects (95%) who received investigational
product completed the study. Three subjects who received 21B12 withdrew full consent and
did not complete the study.
The study population was primarily composed of men (54 [96%]) and had a mean age
of 31.2 (range: 20 to 45) years. Eighty-six percent of subjects were white, followed by 9%
Hispanic/Latino, 4% black and 1% other. Mean baseline LDL-C values were similar
between treatment groups and ranged from 113 to 143 mg/dL.
In this study, 21B12 reduced LDL-C by an average of 55% to 60% at single doses
≥ 70 mg SC with the duration of effect being dose dependent. The LDL-C nadir was
observed within 2 weeks of dosing. Complete suppression of PCSK9 was observed at single
doses ≥ 70 mg SC, which correlated well with the effects seen on circulating LDL-C.
PK analyses demonstrated that 21B12 exhibited nonlinear (concentration-dependent)
elimination. The mean t ranged from 4 to 6 days. As expected, the highest median
maximum observed concentration (C ) and area under the concentration-time curve from
time 0 to infinity (AUC ) occurred in the 420 mg IV group and were 139 µg/mL and 1550
0-inf
day•µg/mL, respectively.
Treatment-emergent adverse events were reported for 29 of the 42 subjects (69%)
who received 21B12 at any dose, and for 10 of the 14 subjects (71%) who received placebo.
No relationship was apparent between the subject incidence of adverse events and the dose of
21B12, or between the subject incidence of adverse events and the route of administration of
21B12 (SC versus IV).
No adverse events were reported as serious, and no subjects discontinued study due to
an adverse event. There were no deaths on study.
Treatment-related adverse events were reported for 18 of the 42 subjects (43%)
who received 21B12 and for 10 of the 14 subjects (71%) who received placebo. No
relationship was apparent between the subject incidence of treatment related adverse events
and the dose of 21B12, or between the subject incidence of treatment-related adverse events
and the route of administration of 21B12 (SC versus IV).
There were no trends indicative of clinically important effects of 21B12 on selected
laboratory variables, electrocardiograms (ECGs), or vital signs.
In this study, 21B12 appeared to be well tolerated at single SC and IV doses up to
420 mg.
Serum samples from subjects enrolled in this study were tested for the presence
(baseline) or development (post-treatment) of anti-21B12 antibodies. Samples from all 42 of
the subjects who received 21B12 were negative for anti-21B12 antibodies.
EXAMPLE 21
A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Ascending
Multiple Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics and
Pharmacodynamics of a Human Anti-PCSK9 Antibody in Subjects with Hyperlipidemia on
Stable Doses of a Statin
This Study is a phase 1b, randomized, double-blind, placebo controlled, ascending,
multiple-dose study using a human anti-PCSK9 antibody (monoclonal antibody 21B12) in
hyperlipidemic (e.g., hypercholesterolemic) subjects currently on stable doses of a statin.
The study had seven cohorts. Objectives for all cohorts included characterization of the
safety, tolerability, and immunogenicity of 21B12, and characterization of the PK and PD
(LDL-C and PCSK9). Cohorts 1 to 5 of the study represented the 21B12 dose-escalation
portion, in hypercholesterolemic subjects on stable low to moderate doses of a statin.
Subjects in cohorts 1 to 5 (n = 8 per cohort) with LDL-C (70-200 mg/dL) on stable daily
rosuvastatin <40 mg, atorvastatin <80 mg or simvastatin 20–80 mg for ≥1 month were
randomized in a 3:1 ratio to receive 1 of 5 SC dosages of 21B12 (14 or 35 mg QW 6 times; or
140 mg or 280 mg Q2W 3 times; or 420 mg Q4W 2 times) or corresponding placebo,
respectively. Cohort 6 was conducted in hypercholesterolemic subjects on high doses of a
statin (atorvastatin 80 mg or rosuvastatin 40 mg). Subjects in this cohort (n=12) were on
either rosuvastatin 40 mg or atorvastatin 80 mg and were randomized in a 3:1 ratio to receive
21B12 (140 mg SC Q2W 3 times) or corresponding placebo, respectively. Cohort 7 was
conducted in subjects with heterozygous familial hypercholesterolemia (identified using
WHO criteria); subjects in this cohort (n = 6) were randomized in a 2:1 ratio to receive
21B12 (140 mg SC Q2W 3 times) or corresponding placebo, respectively. For clarity,
Cohort 1 received SC doses of 14 mg 21B12 once a week, 6 times. Cohort 2 received SC
doses of 35 mg 21B12 once a week, 6 times. Cohort 3 received SC doses of 140 mg 21B12
once every other week, 3 times. Cohort 4 received SC doses of 280 mg 21B12 once every
other week, 3 times. Cohort 5 received SC doses of 420 mg 21B12every 4 weeks, 2 times.
Preliminary results were obtained from 40 subjects who had been enrolled and
randomized to 21B12 or placebo. Of these 40 subjects, 28 subjects had received ≥ 1 dose of
investigational product (21B12 or placebo) and therefore represented the preliminary safety
analysis set (blinded to treatment). Preliminary blinded safety data were available for these
28 subjects, all of whom were from cohorts 1 to 4. No deaths, serious adverse events, or
early withdrawals due to adverse events had been reported. Overall, at least 1 adverse event
had been reported for 15 of the 28 subjects (54%) who had received ≥ 1 dose of
investigational product. Most adverse events (blinded to treatment) were reported for single
subjects, with the exception of fatigue, arthralgia, constipation, and viral upper respiratory
tract infection, each of which was reported for 2 of the 28 subjects (7%).
Preliminary pharmacodynamics results (blinded to treatment) were available for
cohorts 1, 2, and 3. 21B12-dose-dependent reduction in circulating LDL-C was observed, in
subjects on stable moderate doses of statins. The LDL-C nadir was observed within 2 weeks
of initial dosing and was in the range of 60% to 80% reduction in cohort 3 (140 mg Q2W SC
3 times). Near-complete suppression of PCSK9 was observed in cohort 3, which correlated
well with the effects seen on circulating LDL-C.
In the final results, subjects (N=51) in cohorts 1-6 were randomized to receive 21B12
(N=39) or placebo (N=12); 26 subjects (51%) were male; mean (SD) age was 58 (7) years.
No deaths or serious adverse events (AEs) were reported and no subjects discontinued the
study due to an AE. No neutralizing antibodies to 21B12 were detected.
Subjects in cohorts 1-5 on low to moderate doses of statins had mean LDL-C
reductions of up to 81% vs placebo at maximal reduction and 75% vs placebo at the end of
the dosing interval (i.e., at week 6) after 3 biweekly SC doses of 21B12, and 66% at the end
of the dosing interval (i.e., at week 8) after 2, every 4 week SC doses. Subjects in cohorts 1-5
on low to moderate doses of statins had maximum LDL-C reductions of up to 81% vs
placebo at maximal reduction and 75% vs placebo at the end of the dosing interval (Figure
14). The magnitude and duration of effect were dose-dependent. Plasma PCSK9 was
undetectable at higher doses. Similarly, at the end of the dosing interval after 3 biweekly
doses, subjects on high-dose statins (cohort 6) had a mean reduction in LDL-C of 63% vs
placebo, and a maximum reduction in LDL-C of 73% versus placebo (Figure 15).
These data show that repeated SC doses of 21B12 over 6 weeks decreased circulating
LDL-C up to 81% vs placebo, depending on dosing regimen, in subjects on either low-to-
moderate or high-dose statins, with no serious AEs. The LDL-C-lowering effect of 21B12
was comparable between the high dose statin and low-to-moderate statin dose groups.
Subjects in cohorts 1-5 on low to moderate doses of statins had mean reduction of
PCSK9 levels of up to 94% vs placebo at the end of the dosing interval, data not shown.
Subjects in cohorts 1-5 on low-to-moderate doses of statins had mean ApoB reductions of up
to 54% vs placebo at the end of the dosing interval, and maximum reductions ranging from
48% (35 mg QW) to 59% (140 mg and 280 mg Q2W and 420 mg Q4W) during the study
(p < 0.001)(Figure 16). In addition, Subjects in cohorts 1-6 on low-to-moderate and high-
doses of statins had mean Lp(a) reductions of up to 43% vs placebo at the end of the dosing
interval (Figure 17).
Subjects in cohort 7 with heFH had a mean reduction in LDL-C of 65% vs placebo at
the end of the dosing interval (i.e., week 6, 2 weeks after the third biweekly SC dose of
21B12), and a maximum LDL-C reduction of 70% versus placebo (Figure 18). LDL-C
reductions during the dosing interval were comparable to those observed in subjects without
heFH. After 21B12 treatment, circulating PCSK9 was undetectable in heFH subjects.
Subjects in cohort 7 with heFH had a mean reduction in serum PCSK9 values of 78%
vs placebo at the end of the dosing interval (i.e., week 6, 2 weeks after the third biweekly SC
dose of 21B12)(Figure 19). Subjects in cohort 7 with heFH had a mean reduction in total
cholesterol of up to 42% vs placebo at the end of the dosing interval (i.e., week 6, 2 weeks
after the third biweekly SC dose of 21B12), and a maximum total cholesterol reduction of
47% versus placebo (Figure 20). Subjects in cohort 7 with heFH had a mean reduction in
non-HDL cholesterol of 61% vs placebo at the end of the dosing interval (i.e., week 6, 2
weeks after the third biweekly SC dose of 21B12), and a maximum reduction of non-HDL
cholesterol of 67% versus placebo (Figure 21). Subjects in cohort 7 with heFH had a mean
reduction in ApoB levels of up to 47% vs placebo at the end of the dosing interval (i.e., week
6, 2 weeks after the third biweekly SC dose of 21B12), and a maximum reduction of ApoB of
57% versus placebo (Figure 22). Subjects in cohort 7 with heFH had a mean reduction in
lipoprotein a (Lp(a)) of 50% vs placebo at the end of the dosing interval (i.e., week 6, 2
weeks after the third biweekly SC dose of 21B12) (Figure 23).
In cohort 7, 21B12 decreased unbound PCSK9 levels and substantially lowered
circulating LDL-C levels in subjects with heFH and hyperlipidemia who were receiving
standard-of-care therapy. The bi-weekly dose tested provided LDL-C reductions in heFH
subjects that were comparable to those in non-heFH subjects. No serious AEs were reported.
EXAMPLE 22
A Double-blind, Randomized, Placebo-controlled Study to Evaluate Tolerability and
Efficacy of a Human Anti-PCSK9 Antibody in Patients with Heterozygous Familial
Hypercholesterolemia
The objective of this study is to evaluate the effect of 12 weeks of subcutaneous (SC)
human, anti-PCSK9 antibody (monoclonal antibody 21B12) compared with placebo, on
percent change from baseline in low-density lipoprotein cholesterol (LDL-C) in subjects with
heterozygous familial hypercholesterolemia (HeFH).
This study is a double-blind, randomized, stratified, placebo-controlled clinical trial
evaluating the safety, tolerability, and efficacy of monoclonal antibody 21B12 in subjects
having a diagnosis of HeFH. A total enrollment of 150 subjects is planned. Subjects who
meet all inclusion/exclusion criteria will be randomized with equal allocation into 3 treatment
groups: monoclonal antibody, 21B12 at 350 mg or 420 mg Q4W SC (once every 4 weeks,
subcutaneous) or placebo Q4W SC. Randomization will be stratified by screening LDL-C
level (< 130 mg/dL [3.4 mmol/L] vs ≥ 130 mg/dL) and ezetimibe use at baseline (yes vs no).
Randomization should occur within 5 – 10 days of the screening LDL-C evaluation used to
determine eligibility. Monoclonal antibody, 21B12, and placebo will be blinded. Study visits
are at weeks 2, 4, 8, and 12. Final administration of monoclonal antibody, 21B12, or placebo
is at week 8. The end-of-study (EOS) visit and the last evaluation of lipids is at week 12.
Males and females, ≥ 18 to ≤ 75 years of age, and with a diagnosis of heterozygous
familial hypercholesterolemia by the diagnostic criteria of the Simon Broome Register Group
(SBRG), are eligible for this study. For enrollment, subjects must be on an approved statin,
with stable dose(s) for all allowed (eg, ezetimibe, bile-acid sequestering resin, stanols, or
regulatory-approved and marketed niacin (eg, Niaspan or Niacor)) lipid-regulating drugs for
at least 4 weeks before LDL-C screening and, in the opinion of the investigator, not requiring
uptitration. Fasting LDL-C must be ≥ 100 mg/dL (2.6 mmol/L) and fasting triglycerides ≤
400 mg/dL (4.5 mmol/L) by central laboratory at screening.
Preliminary data (data not shown) demonstrated that subjects treated with 350 mg
21B12 had a least squares (LS) mean percent reduction from baseline in LDL-C of 38.46% at
the end of the dosing interval, and subjects treated with 420 mg 21B12 had a LS mean
percent reduction from baseline in LDL-C of 45.68%. Subjects treated with 350 mg 21B12
had a LS mean percent reduction from baseline in Lp(a) of 21.69% at the end of the dosing
interval, and subjects treated with 420 mg 21B12 had a LS mean percent reduction from
baseline in Lp(a) of 28.23%. Subjects treated with 350 mg 21B12 had a LS mean percent
increase from baseline in HDL-C of 15.39% at the end of the dosing interval, and subjects
treated with 420 mg 21B12 had a LS mean percent increase from baseline in HDL-C of
6.77%. Subjects treated with 350 mg 21B12 had a LS mean percent reduction from baseline
in VLDL-C of 17.16% at the end of the dosing interval, and subjects treated with 420 mg
21B12 had a LSmean percent reduction from baseline in VLDL-C of 18.49%. Subjects
treated with 350 mg 21B12 had a LS mean percent reduction from baseline in triglycerides of
17.24% at the end of the dosing interval, and subjects treated with 420 mg 21B12 had a LS
mean percent reduction from baseline in triglycerides 4.56%. Subjects treated with 350 mg
21B12 had a LS mean percent reduction from baseline in non-HDL cholesterol of 36.16% at
the end of the dosing interval, and subjects treated with 420 mg 21B12 had a LS mean
percent reduction from baseline in non-HDL cholesterol of 41.81%. Finally, subjects treated
with 350 mg 21B12 had a LS mean percent reduction from baseline in total cholesterol of
24.82% at the end of the dosing interval, and subjects treated with 420 mg 21B12 had a LS
mean percent reduction from baseline in total cholesterol of 29.45%. (data not shown)
Figure 24 is a graph representing the LDL-C reduction data for following doses of
21B12: 70 mg, 105 mg and 140 mg (Q2W or once every two weeks dosing) and 280 mg,
350 mg and 420 (Q4W or once a month dosing). This data is the aggregate data from the
studies described in Examples 22-25). In brief, the aggregate data shows that 140 mg Q2W
results in an approximate 60% reduction from baseline in LDL-C at week 12 and smooth
maintenance of LDL-C reduction. In addition, this data shows that the 420 mg Q4W results
in an approximate 56% reduction from baseline in LDL-C at week 12 and less LDL-C
rebound at end of dosing interval.
Figures 25A-25D are bar graphs showing the beneficial effects of doses of 21B12 on
Lp(a), HDL-C, triglycerides and VLDL-C, respectively, derived from the aggregate data
from the studies described in Examples 22-25 . In addition, dose dependent reductions from
baseline were observed for total cholesterol (25-37%, p values <0.001), non-HDL-C (36-
53%, p values <0.001), and ApoB (36-53%, p values < 0.001) (data not shown).
EXAMPLE 23
A Randomized Study to Evaluate Tolerability and Efficacy of a Human Anti-PCSK9
Antibody on LDL-C Compared with Ezetimibe in Hypercholesterolemic Patients Unable to
Tolerate an Effective Dose of a HMG-Co-A Reductase Inhibitor
The objective of this study is to evaluate the effect of 12 weeks of subcutaneous (SC)
human, anti-PCSK9 antibody (monoclonal antibody 21B12) compared with ezetimibe, on
percent change from baseline in low-density lipoprotein cholesterol (LDL-C) in
hypercholesterolemic subjects unable to tolerate an effective dose of an HMG-CoA reductase
inhibitor.
This study is a randomized, stratified, parallel group clinical trial for the human anti-
PCSK9 antibody, monoclonal antibody, 21B12. It is planned to enroll 150 subjects. Subjects
who meet all inclusion/exclusion criteria will be randomized with equal allocation into 5
treatment groups: monoclonal antibody, 21B12 at 280 mg, 350 mg or 420 mg Q4W SC (once
every 4 weeks, subcutaneous); ezetimibe at 10 mg daily (QD) oral (PO) with monoclonal
antibody, 21B12 at 420 mg Q4W SC; or ezetimibe 10 mg QD PO with placebo Q4W SC.
Randomization will be stratified by screening LDL-C level (< 130 mg/dL [3.4 mmol/L] vs ≥
130 mg/dL) and statin use at baseline (yes vs no). Randomization should occur within 5 –10
days of the screening LDL-C evaluation used to determine eligibility. Monoclonal antibody,
21B12, and placebo will be blinded. Ezetimibe is not blinded. Study visits are at weeks 2, 4,
8, and 12. Final administration of monoclonal antibody, 21B12, or placebo is at week 8. The
end-of-study visit and the last evaluation of lipids is at week 12.
Males and females, ≥ 18 to ≤ 75 years of age, are eligible for this study. Subject must
have tried at least 1 statin and have been unable to tolerate any dose or an increase in statin
dose above the following total weekly maximum doses due to myalgia or myopathy:
atorvastatin ≤ 70 mg, simvastatin ≤ 140 mg, pravastatin ≤ 140 mg, rosuvastatin ≤ 35 mg,
lovastatin ≤ 140 mg, fluvastatin ≤ 280 mg. For unlisted statins, the maximal total weekly
dose should not exceed 7 times the smallest available tablet size. Symptoms must have
resolved when statin was discontinued or the dose reduced. If receiving statin (not exceeding
the maximal dose defined above), bile-acid sequestering resin, and/or stanol therapy, the
dose(s) must be stable for at least 4 weeks prior to LDL-C screening. If the subject is on
ezetimibe at start of screening, ezetimibe must be discontinued for ≥ 4 weeks before LDL –C
screening. Depending on their risk category (based on NCEP ATP III treatment goals)
subjects must meet the following fasting LDL-C (by central laboratory) criteria at screening:
≥ 100 mg/dL (2.6 mmol/L) for subjects with diagnosed coronary heart disease (CHD) or
CHD risk equivalent; ≥ 130 mg/dL (3.4 mmol/L) for subjects without diagnosed CHD or risk
equivalent and 2 or more risk factors; ≥ 160 mg/dL (4.1 mmol/L) for subjects without
diagnosed CHD or risk equivalent and with 1 or no risk factors. Fasting triglycerides must be
≤ 400 mg/dL (4.5 mmol/L) as determined by the central laboratory analysis at screening.
Preliminary data (data not shown) demonstrated that subjects treated with 280 mg
21B12 had a LS mean percent reduction from baseline in LDL-C of 38.79% at the end of the
dosing interval; subjects treated with 350 mg 21B12 had a LS mean percent reduction from
baseline in LDL-C of 40.01% at the end of the dosing interval; and subjects treated with 420
mg 21B12 had a LS mean percent reduction from baseline in LDL-C of 50.63% Preliminary
data demonstrated that subjects treated with 280 mg 21B12 had a LS mean percent reduction
from baseline in Lp(a) of 27.38% at the end of the dosing interval; subjects treated with 350
mg 21B12 had a LS mean percent reduction from baseline in Lp(a) of 16.04% at the end of
the dosing interval; and subjects treated with 420 mg 21B12 had a LS mean percent reduction
from baseline in Lp(a) of 23.84%. Preliminary data demonstrated that subjects treated with
280 mg 21B12 had a LS mean percent increase from baseline in HDL-C of 8.62% at the end
of the dosing interval; subjects treated with 350 mg 21B12 had a LS mean percent increase
from baseline in HDL-C of 4.62% at the end of the dosing interval; and subjects treated with
420 mg 21B12 had a LS mean percent increase from baseline in HDL-C of 7.55%.
Preliminary data demonstrated that subjects treated with 280 mg 21B12 had a LS mean
percent reduction from baseline in VLDL-C of 31.02% at the end of the dosing interval;
subjects treated with 350 mg 21B12 had a LS mean percent reduction from baseline in
VLDL-C of 38.14% at the end of the dosing interval; and subjects treated with 420 mg
21B12 had a LS mean percent reduction from baseline in VLDL-C of 37.27%. Preliminary
data demonstrated that subjects treated with 280 mg 21B12 had a LS mean percent reduction
from baseline in triglycerides of 15.35% at the end of the dosing interval; subjects treated
with 350 mg 21B12 had a LS mean percent reduction from baseline in triglycerides of
19.22% at the end of the dosing interval; and subjects treated with 420 mg 21B12 had a LS
mean percent reduction from baseline in triglycerides of 19.55%. Preliminary data
demonstrated that subjects treated with 280 mg 21B12 had a LS mean percent reduction from
baseline in total cholesterol of 31.03% at the end of the dosing interval; subjects treated with
350 mg 21B12 had a LS mean percent reduction from baseline in total cholesterol of 34.46%
at the end of the dosing interval; and subjects treated with 420 mg 21B12 had a LS mean
percent reduction from baseline in total cholesterol of 42.23%. Preliminary data
demonstrated that subjects treated with 280 mg 21B12 had a LS mean percent reduction from
baseline in non-HDL-C of 39.92% at the end of the dosing interval; subjects treated with 350
mg 21B12 had a LS mean percent reduction from baseline in non-HDL-C of 42.86% at the
end of the dosing interval; and subjects treated with 420 mg 21B12 had a LS mean percent
reduction from baseline in non-HDL-C of 53.49%.
EXAMPLE 24
A Randomized, Placebo and Ezetimibe-Controlled, Dose-ranging Study to Evaluate
Tolerability and Efficacy of a Human Anti-PCSK9 Antibody on LDL-C in
Hypercholesterolemic Patients with a 10 Year Framingham Risk Score of 10% or Less
The objective of this study was to evaluate the effect of 12 weeks of subcutaneous
(SC) human, anti-PCSK9 antibody (monoclonal antibody 21B12) every 2 weeks (Q2W) or
every 4 weeks (Q4W), compared with placebo, on percent change from baseline in low-
density lipoprotein cholesterol (LDL-C) when used as monotherapy in hypercholesterolemic
subjects with a 10 year Framingham risk score of 10% or less.
This study was a randomized, stratified, placebo and ezetimibe controlled, parallel
group dose ranging clinical trial for the human anti-PCSK9 antibody, monoclonal antibody,
21B12, enrolling 411 subjects. Subjects who meet all inclusion/exclusion criteria were
randomized with equal allocation into 9 treatment groups: 1 of 6 dose regimens of
monoclonal antibody, 21B12 (70 mg, 105 mg, or 140 mg Q2W SC, or 280 mg, 350 mg or
420 mg Q4W SC (once every 4 weeks, subcutaneous), placebo with either Q2W or Q4W SC
administration, or ezetimibe with daily (QD) oral (PO) administration. Randomization was
stratified by screening LDL-C level (<130 mg/dL [3.4 mmol/L] vs > 130 mg/dL).
Randomization occurred within 5 –10 days of the screening LDL-C evaluation used to
determine eligibility. Study visits were every 2 weeks, irrespective whether the subject
receives Q2W SC or Q4W treatment or ezetimibe. The 3 Q2W dose groups of monoclonal
antibody, 21B12, and 1 Q2W placebo group was blinded against each other, and the 3 Q4W
dose groups and 1 Q4W placebo group was blinded against each other. Ezetimibe was not
blinded. The end-of-study visit and the last estimation of lipids was at week 12 for subjects
on Q4W IP schedule or on ezetimibe and week 14 for subjects on Q2W IP schedule.
Males and females, ≥ 18 to ≤ 75 years of age, were eligible for this study. Fasting
LDL-C was ≥ 100 mg/dL (2.6 mmol/L) and < 190 mg/dL (4.9 mmol/L) and fasting
triglycerides ≤ 400 mg/dL (4.5 mmol/L) by central laboratory at screening. Subjects had a
National Cholesterol Education Panel Adult Treatment Panel III (NCEP ATP III)
Framingham risk score of 10% or less.
The primary endpoint was the percent change from baseline in LDL-C at week 12.
Secondary endpoints included percent changes in apolipoprotein B (ApoB), lipoprotein (a)
(Lp(a)), and in the ratio of total cholesterol to high-density lipoprotein (HDL)-C. Tolerability
and safety were also evaluated.
Preliminary data demonstrated that subjects treated with 70 mg 21B12 (Q2W) had a
mean percent reduction from baseline in LDL-C of 41.21% at the end of the dosing interval;
subjects treated with 105 mg 21B12 (Q2W) had a mean percent reduction from baseline in
LDL-C of 45.44% at the end of the dosing interval; and subjects treated with 140 mg 21B12
(Q2W) had a mean percent reduction from baseline in LDL-C of 51.56% (data not shown).
Preliminary data demonstrated that subjects treated with 280 mg 21B12 (Q4W) had a
mean percent reduction from baseline in LDL-C of 37.53% at the end of the dosing interval;
subjects treated with 350 mg 21B12 had a mean percent reduction from baseline in LDL-C of
42.16% at the end of the dosing interval; and subjects treated with 420 mg 21B12 had a mean
percent reduction from baseline in LDL-C of 47.52% (data not shown).
Final data demonstrated that at week 12, subjects receiving 21B12 had a least-squares
(LS) mean percent reducton from baseline in LDL-C of up to 51% (Table 12); the percent
change from baseline for ezetimibe was 14%. The change from baseline to week 12 was up to
72 mg/dL greater with 21B12 than with placebo. Subjects receiving 21B12 had LDL-C
reductions from baseline 37%– 53% greater than placebo and 37% greater than ezetimibe.
Mean reductions from baseline for ApoB (up to 44%), Lp(a) (up to 29%) and total
cholesterol/HDL ratio (up to 38%) were greater with 21B12 than with placebo.
TABLE 12:
Week 12 Percent Change from Baseline in LDL-C: SC 21B12 vs Ezetimibe or Placebo
Q2W Q4W
Ezetimibe
Placebo 70 mg 105 mg 140 mg Placebo 280 mg 350 mg 420 mg QD
(N=45) (N=45) (N=46) (N=45) (N=45) (N=45) (N=45) (N=45) (N=45)
Least squares -3.71 -40.98 -43.87 -50.93 4.54 -39.02 -43.20 -47.98 -14.26
mean percent
change from
baseline (%)
Treatment - -37.27* -40.17* -47.23* - -43.57* -47.74* -52.53* -
difference vs
placebo (%)
Treatment - -26.73* -29.62* -36.68* - -25.17* -29.34* -34.14* -
difference vs
ezetimibe (%)
SC: subcutaneous Q2W: every 2 weeks; Q4W: every 4 weeks or once a month; QD: daily
* P < 0.001
EXAMPLE 25
A Double-blind, Randomized, Placebo-controlled, Dose-ranging Study to Evaluate
Tolerability and Efficacy of a Human Anti-PCSK9 Antibody on LDL-C in Combination with
HMG-Co-A Reductase Inhibitors in Hypercholesterolemic Patients
The objective of this study is to evaluate the effect of 12 weeks of subcutaneous (SC)
human, anti-PCSK9 antibody (monoclonal antibody 21B12) every 2 weeks (Q2W) or every 4
weeks (Q4W), compared with placebo, on percent change from baseline in low-density
lipoprotein cholesterol (LDL-C) when used in addition to HMG-Co-A reductase inhibitor
(e.g., a statin) in subjects with hypercholesterolemia.
This study is a double-blind, randomized, stratified, placebo controlled, parallel group
dose ranging clinical trial for the human anti-PCSK9 antibody, monoclonal antibody, 21B12,
enrolling 631 subjects. Subjects who are on stable dose(s) for at least 4 weeks of statin
therapy with or without ezetimibe and who meet all inclusion/exclusion criteria will be
randomized with equal allocation into 8 treatment groups: monoclonal antibody, 21B12
subcutaneous (SC) (70 mg Q2W, 105 mg Q2W, 140 mg Q2W, 280 mg Q4W, 350 mg Q4W,
and 420 mg Q4W, placebo Q2W SC, or placebo Q4W SC). Randomization will be stratified
by screening LDL-C level (<130 mg/dL [3.4 mmol/L] vs > 130 mg/dL) and ezetimibe use at
baseline (yes vs no). Randomization should occur within 5 –10 days of the screening LDL-C
evaluation used to determine eligibility. Study visits are every 2 weeks, irrespective whether
the subject receives Q2W SC or Q4W treatment. The 3 Q2W dose groups of monoclonal
antibody, 21B12, and 1 Q2W placebo group will be blinded against each other, and the 3
Q4W dose groups and 1 Q4W placebo group will be blinded against each other. The end-of-
study visit and the last estimation of lipids is at week 12 for subjects on Q4W IP schedule and
week 14 for subjects on Q2W IP schedule.
Males and females, ≥ 18 to ≤ 80 years of age, are eligible for this study. For
enrollment, subjects must be on a statin, with or without ezetimibe, with stable dose(s) for at
least 4 weeks before LDL-C screening and not requiring uptitration. Fasting LDL-C at
screening must be ≥ 85 mg/dL (2.2 mmol/L). Enrollment of subjects with screening fasting
LDL-C between ≥ 85 mg/dL (2.2 mmol/L) and < 100 mg/dL (2.6 mmol/L) will be limited to
no more than approximately 20% of total planned enrollment. Fasting triglycerides must be ≤
400 mg/dL (4.5 mmol/L) as determined by the central laboratory analysis at screening.
Preliminary data demonstrated that subjects treated with 70 mg 21B12 (Q2W) had a
LS mean percent reduction from baseline in LDL-C of 39.22% at the end of the dosing
interval; subjects treated with 105 mg 21B12 (Q2W) had a LS mean percent reduction from
baseline in LDL-C of 56.38% at the end of the dosing interval; and subjects treated with 140
mg 21B12 (Q2W) had a LS mean percent reduction from baseline in LDL-C of 68.76% (data
not shown). Preliminary data demonstrated that subjects treated with 70 mg 21B12 (Q2W)
had a LS mean percent reduction from baseline in Lp(a) of 21.17% at the end of the dosing
interval; subjects treated with 105 mg 21B12 (Q2W) had a LS mean percent reduction from
baseline in Lp(a) of 33.41% at the end of the dosing interval; and subjects treated with 140
mg 21B12 (Q2W) had a LS mean percent reduction from baseline in Lp(a) of 33.87% (data
not shown). Preliminary data demonstrated that subjects treated with 70 mg 21B12 (Q2W)
had a LS mean percent increase from baseline in HDL-C of 21.17% at the end of the dosing
interval; subjects treated with 105 mg 21B12 (Q2W) had a LS mean percent increase from
baseline in HDL-C of 6.80% at the end of the dosing interval; and subjects treated with 140
mg 21B12 (Q2W) had a LS mean percent increase from baseline in HDL-C of 8.43% (data
not shown). Preliminary data demonstrated that subjects treated with 70 mg 21B12 (Q2W)
had a LS mean percent reduction from baseline in VLDL-C of 14.84% at the end of the
dosing interval; subjects treated with 105 mg 21B12 (Q2W) had a LS mean percent reduction
from baseline in VLDL-C of 12.75% at the end of the dosing interval; and subjects treated
with 140 mg 21B12 (Q2W) had a LS mean percent reduction from baseline in VLDL-C of
45.14% (data not shown). Preliminary data demonstrated that subjects treated with 70 mg
21B12 (Q2W) had a LS mean percent reduction from baseline in triglycerides of 7.20% at the
end of the dosing interval; subjects treated with 105 mg 21B12 (Q2W) had a LS mean
percent reduction from baseline in triglycerides of 5.65% at the end of the dosing interval;
and subjects treated with 140 mg 21B12 (Q2W) had a LS mean percent reduction from
baseline in triglycerides of 17.60% (data not shown). Preliminary data demonstrated that
subjects treated with 70 mg 21B12 (Q2W) had a LS mean percent reduction from baseline in
non-HDL-C of 36.20% at the end of the dosing interval; subjects treated with 105 mg 21B12
(Q2W) had a LS mean percent reduction from baseline in non-HDL-C of 51.20% at the end
of the dosing interval; and subjects treated with 140 mg 21B12 (Q2W) had a LS mean
percent reduction from baseline in non-HDL-C of 64.61% (data not shown). Preliminary
data demonstrated that subjects treated with 70 mg 21B12 (Q2W) had a LS mean percent
reduction from baseline in total cholesterol of 26.33% at the end of the dosing interval;
subjects treated with 105 mg 21B12 (Q2W) had a LS mean percent reduction from baseline
in total cholesterol of 36.91% at the end of the dosing interval; and subjects treated with 140
mg 21B12 (Q2W) had a LS mean percent reduction from baseline in total cholesterol of
46.17% (data not shown).
Preliminary data demonstrated that subjects treated with 280 mg 21B12 (Q4W) had a
LS mean percent reduction from baseline in LDL-C of 42.62% at the end of the dosing
interval; subjects treated with 350 mg 21B12 had a LS mean percent reduction from baseline
in LDL-C of 56.84% at the end of the dosing interval; and subjects treated with 420 mg
21B12 had a LS mean percent reduction from baseline in LDL-C of 52.19% (data not
shown). Preliminary data demonstrated that subjects treated with 280 mg 21B12 (Q2W) had
a LS mean percent reduction from baseline in Lp(a) of 22.54% at the end of the dosing
interval; subjects treated with 350 mg 21B12 (Q2W) had a LS mean percent reduction from
baseline in Lp(a) of 29.43% at the end of the dosing interval; and subjects treated with 420
mg 21B12 (Q2W) had a LS mean percent reduction from baseline in Lp(a) of 23.29% (data
not shown). Preliminary data demonstrated that subjects treated with 280 mg 21B12 (Q2W)
had a LS mean percent increase from baseline in HDL-C of 2.17% at the end of the dosing
interval; subjects treated with 350 mg 21B12 (Q2W) had a LS mean percent increase from
baseline in HDL-C of 6.92% at the end of the dosing interval; and subjects treated with 420
mg 21B12 (Q2W) had a LS mean percent increase from baseline in HDL-C of 7.42% (data
not shown). Preliminary data demonstrated that subjects treated with 280 mg 21B12 (Q2W)
had a LS mean percent reduction from baseline in VLDL-C of 18.12% at the end of the
dosing interval; subjects treated with 350 mg 21B12 (Q2W) had a LS mean percent reduction
from baseline in VLDL-C of 20.89% at the end of the dosing interval; and subjects treated
with 420 mg 21B12 (Q2W) had a LS mean percent reduction from baseline in VLDL-C of
28.66% (data not shown). Preliminary data demonstrated that subjects treated with 280 mg
21B12 (Q2W) had a LS mean percent reduction from baseline in triglycerides of 6.75% at the
end of the dosing interval; subjects treated with 350 mg 21B12 (Q2W) had a LS mean
percent reduction from baseline in triglycerides of 9.17% at the end of the dosing interval;
and subjects treated with 420 mg 21B12 (Q2W) had a LS mean percent reduction from
baseline in triglycerides of 11.13% (data not shown). Preliminary data demonstrated that
subjects treated with 280 mg 21B12 (Q2W) had a LS mean percent reduction from baseline
in non-HDL-C of 38.89% at the end of the dosing interval; subjects treated with 350 mg
21B12 (Q2W) had a LS mean percent reduction from baseline in non-HDL-C of 50.83% at
the end of the dosing interval; and subjects treated with 420 mg 21B12 (Q2W) had a LS
mean percent reduction from baseline in non-HDL-C of 48.54% (data not shown).
Preliminary data demonstrated that subjects treated with 280 mg 21B12 (Q2W) had a LS
mean percent reduction from baseline in total cholesterol of 28.08% at the end of the dosing
interval; subjects treated with 350 mg 21B12 (Q2W) had a LS mean percent reduction from
baseline in total cholesterol of 36.04% at the end of the dosing interval; and subjects treated
with 420 mg 21B12 (Q2W) had a LS mean percent reduction from baseline in total
cholesterol of 42.76% (data not shown).
EXAMPLE 26
PCSK9 ABPs Further Upregulated LDLR in the Presence of Statins
This example demonstrates that ABPs to PCSK9 produced further increases in LDLR
availability when used in the presence of statins, demonstrating that further benefits can be
achieved by the combined use of the two.
HepG2 cells were seeded in DMEM with 10% fetal bovine serum (FBS) and grown to
~90% confluence. The cells were treated with indicated amounts of mevinolin (a statin,
Sigma) and PCSK9 ABPs (FIGs. 12A-12C) in DMEM with 3% FBS for 48 hours. Total cell
lysates were prepared. 50 mg of total proteins were separated by gel electrophoresis and
transferred to PVDF membrane. Immunoblots were performed using rabbit anti-human LDL
receptor antibody (Fitzgerald) or rabbit anti-human b-actin antibody. The enhanced
chemiluminescent results are shown in the top panels of FIGs. 12A-12C. The intensity of the
bands were quantified by ImageJ software and normalized by b-actin. The relative levels of
LDLR are shown in the lower panels of FIGs. 12A-12C. ABPs 21B12 and 31H4 are PCSK9
neutralizing antibodies, while 25A7.1 is a non-neutralizing antibody.
HepG2-PCSK9 cells were also created. These were stable HepG2 cell line
transfected with human PCSK9. The cells were seeded in DMEM with 10% fetal bovine
serum (FBS) and grew to ~90% confluence. The cells were treated with indicated amounts of
mevinolin (Sigma) and PCSK9 ABPs (FIGs. 12D-12F) in DMEM with 3% FBS for 48 hours.
Total cell lysates were prepared. 50 mg of total proteins were separated by gel electrophoresis
and transferred to PVDF membrane. Immunoblots were performed using rabbit anti-human
LDL receptor antibody (Fitzgerald) or rabbit anti-human b-actin antibody. The enhanced
chemiluminescent results are shown in the top panels. The intensity of the bands were
quantified by ImageJ software and normalized by b-actin.
As can be seen in the results depicted in FIGs. 12A-12F, increasing amounts of the
neutralizing antibody and increasing amounts of the statin generally resulted in increases in
the level of LDLR. This increase in effectiveness for increasing levels of the ABP is
especially evident in FIGs. 12D-12F, in which the cells were also transfected with PCSK9,
allowing the ABPs to demonstrate their effectiveness to a greater extent.
Interestingly, as demonstrated by the results in the comparison of FIGs. 12D-12F to
12A-12C, the influence of the ABP concentrations on LDLR levels increased dramatically
when PCSK9 was being produced by the cells. In addition, it is clear that the neutralizing
ABPs (21B12 and 31H4) resulted in a greater increase in LDLR levels, even in the presence
of statins, than the 25A7.1 ABP (a non-neutralizer), demonstrating that additional benefits
can be achieved by the use of both statins and ABPs to PCSK9.
EXAMPLE 27
Consensus Sequences
Consensus sequences were determined using standard phylogenic analyses of the
CDRs corresponding to the V and V of anti-PCSK9 ABPs. The consensus sequences were
determined by keeping the CDRs contiguous within the same sequence corresponding to a V
or V . Briefly, amino acid sequences corresponding to the entire variable domains of either
V or V were converted to FASTA formatting for ease in processing comparative
alignments and inferring phylogenies. Next, framework regions of these sequences were
replaced with an artificial linker sequence (“bbbbbbbbbb” placeholders, non-specific nucleic
acid construct) so that examination of the CDRs alone could be performed without
introducing any amino acid position weighting bias due to coincident events (e.g., such as
unrelated antibodies that serendipitously share a common germline framework heritage)
while still keeping CDRs contiguous within the same sequence corresponding to a V or V .
V or V sequences of this format were then subjected to sequence similarity alignment
interrogation using a program that employs a standard ClutalW-like algorithm (see,
Thompson et al., 1994, Nucleic Acids Res. 22:4673-4680). A gap creation penalty of 8.0 was
employed along with a gap extension penalty of 2.0. This program likewise generated
phylograms (phylogenic tree illustrations) based on sequence similarity alignments using
either UPGMA (unweighted pair group method using arithmetic averages) or Neighbor-
Joining methods (see, Saitou and Nei, 1987, Molecular Biology and Evolution 4:406-425) to
construct and illustrate similarity and distinction of sequence groups via branch length
comparison and grouping. Both methods produced similar results but UPGMA-derived trees
were ultimately used as the method employs a simpler and more conservative set of
assumptions. UPGMA-derived trees were generated where similar groups of sequences were
defined as having fewer than 15 substitutions per 100 residues (see, legend in tree
illustrations for scale) amongst individual sequences within the group and were used to define
consensus sequence collections. The results of the comparisons are depicted in FIGs. 13A-
13J and FIGs. 48-49 In E, the groups were chosen so that sequences in the light chain
that clade are also a clade in the heavy chain and have fewer than 15 substitutions.
EXAMPLE 28
Preparation of PCSK9 ABP Formulations
UF/DF – Ultrafiltration/Diafiltration Methodology
Drug substance, e.g., antibody 21B12 and antibody 11F1, was buffer exchanged into
formulation buffer, including stabilizer, with a bench scale Millipore TFF UF/DF system
using a Millipore Pellicon XL Filter, 50 cm size (regenerated cellulose, 30,000 Molecular
Weight Cut-Off) membrane. The diafiltration step was performed until at least ten volumes
of diafiltration buffer were exchanged. Once the diafiltration step was completed, the UF/DF
system was switched to ultrafiltration mode and each formulation was concentrated to the
target concentration levels. After the UF/DF step was completed, the appropriate amount of
polysorbate 20 or 80 was added to each formulation from a 1.0% (w/w) freshly prepared
polysorbate (“PS”) stock solution to reach the desired polysorbate concentration.
Prior to filling primary containers, each formulation was filtered aseptically under a
laminar flow hood and using a 0.2 micron filter. Filling was also performed aseptically and
was performed manually or automatically using the appropriate filling instrumentation.
Example 29
High Concentration PCSK9 ABP Formulations with Lowed Viscosity
To evaluate the effects of different excipients on viscosity of high protein
concentrations, a viscosity, stability and solubility screening assay was used to explore
excipient viscosity modulators for high concentration protein formulations. Specifically, all
sample preparation, e.g., antibody 21B12 sample, was done aseptically under a laminar-flow
hood. Lyophilization of the samples to be tested allowed a simple method for achieving high
protein concentrations. 1.5 mL of 70mg/mL protein (e.g., 21B12) was pipetted into 3cc glass
vials for lyophilization. Lyophilization was performed using a generic Lyophilization cycle
on a VirTis Lab Scale Lyophilizer. The lyophilization buffer was 10mM L-glutamate with
1.0% sucrose, pH 4.8. Lyophilized samples (e.g., lyophilized 21B12 sample) were
reconstituted individually with approximately 0.65 mL of the excipient buffers, shown in
Table 13 below, to a final protein concentration of 150-200 mg/mL. Reconstituted samples
sat overnight to allow complete dissolution. Viscosity was then measured as described
below.
TABLE 13
Excipient Type Excipient Level Adjusted pH
150 mM L-Alanine pH 4.5
150 mM L-Glycine pH 4.2
75 mM L-Lysine pH 4.2
150 mM L-Methionine pH 4.5
Amino Acids
150 mM L-Proline pH 4.2
150 mM L-Serine pH 4.2
70 mM L-Arginine pH 4.5
150 mM L-Serine pH 4.4
mM Magnesium chloride pH 4.2
70 mM Sodium chloride pH 4.2
Salts 30 mM Calcium chloride pH 4.4
50 mM Sodium sulfate pH 4.1
30mM Zinc chloride pH 4.7
150 mM Glycerol pH 4.5
Polyols
150 mM Sucrose pH 4.2
150 mM Carnitine pH 4.8
Other 150 mM Creatinine pH 5.0
150 mM Taurine pH 4.4
Results from the viscosity, stability, solubility screen showed changes in 21B12
viscosity after addition of various excipients (Figure 26). Not all excipients used in for
screening purposes resulted in a lowering of solution viscosity; L-alanine, glycerol, sodium
sulfate, sucrose, and zinc chloride addition resulted in a much higher viscosity as compared to
the control sample. Several excipients used in the screen appeared to be good viscosity
modulating candidates, for example, L-arginine, carnitine, creatinine, L-methionine, and
taurine.
To evaluate the effects of different formulations on viscosity of a specific PCSK9
ABP, compositions of 21B12 were formulated in six different formulations shown in Table
29.2 below. The concentration of 21B12 in all formulations was 134 mg/ml. Compositions
were filled to a final volume of 1.0 ml in vials. Compositions were incubated at room
temperature (i.e., 25ºC) .
Dialysis and Concentration of 21B12
Sucrose removal from 21B12 originally in 10 mM Sodium acetate, 9.0% (w/v)
sucrose was achieved via dialysis by adding approximately 10 mL 21B12 to Pierce Slide-A-
Lyzer (Rockford, IL) dialysis cassettes and dialyzing against 2 L buffer at 4°C for 3 cycles (2
hours x 2 and 16 hours x 1) for complete buffer exchange. Buffer for dialysis contained 10
mM sodium acetate (made from acetic acid) at pH 5.0. All samples were subsequently
concentrated using Millipore Amicon UltraPrep Devices (Billerica, MA) in a Beckman
Coulter Allegra 6R Centrifuge (Fullerton, California) spun at 3000 rpm until the sample
volume was slightly below the volume required for the desired concentration.
Concentration determination was then carried out by measuring absorbance at A280
using an Agilent 8453 Spectrophotometer (Santa Clara, California). Protein concentration
was calculated using the appropriate extinction coefficient. The appropriate amount of buffer
was then added to the sample to dilute it back down to the desired concentration and another
A280 was performed to obtain the final concentration for the experiment.
Addition of stabilizers that may also act to lower viscosity:
Excipients, such as proline, benzyl alcohol, creatinine, methionine, taurine, etc.,were
tested in an attempt to lower viscosity. These excipients were added individually to the
21B12 formulation samples from high concentration stock solutions.
Viscosity Measurements
Viscosity was measured using Brookfield LV-DVII cone and plate viscometer
(Middleboro, Massachusetts) with a CPE-40 spindle with matching sample cup temperature
regulated by a circulating water bath at constant 25C. 500 ul of sample was added to sample
cup with positive displacement pipettor. After sample cup was secured the rotational speed
of the spindle was gradually increased until about 80% torque was achieved. At this point the
rotational speed was stopped and a viscosity reading was generated by Rheocalc software.
TABLE 14
Stabilizer/Excipients Added to Lower Viscosity
Buffer Stabilizer
Viscosity (cP)
mM Na acetate 42.4
mM Na acetate 9.0 % sucrose 2% L-Proline (174 mM) 20.3
mM Na acetate 9.0 % sucrose 3% L-Proline (261 mM) 17.9
mM Na acetate 9.0 % sucrose 3% Benzyl alcohol 17.8
mM Na acetate 9.0 % sucrose 150 mM Creatinine 11.97
mM Na acetate 9.0 % sucrose 150 mM L-Methionine 16.0
mM Na acetate 9.0 % sucrose 150 mM L-Taurine 16.8
The results show that L-proline, benzyl alcohol, creatinine, methionine and taurine all
had a significant viscosity lowering effect in high concentrations of PCSK9 ABP, 21B12 (see
Table 14) .
To further evaluate the effects of different formulations on a specific PCSK9 ABP,
compositions of 21B12 were formulated in different formulations shown in Table 15 below.
The formulations fall into three groups: (1) a set of various concentrations of 21B12 in 10 mM
sodium acetate buffer, pH 5.2, (2) a set of various concentrations of 21B12 in 10 mM sodium
acetate buffer, pH 5.2 with 3% (approximately 261 mM) L-Proline spiked into each sample,
and (3) a set of 21B12 samples concentrated at about 117-134 mg/mL in 10 mM sodium acetate
buffer at different pH levels (4.0 to 5.5) plus two samples in 10 mM sodium acetate buffer, pH
.2 with either NaCl or a L-Methionine/Benzyl alcohol combination added.
TABLE 15
21B12 Formulation Additional Viscosity Viscosity Osmolality
conc. Excipients (Cp) @ 25°C (Cp) @ 40°C (mOsmol/kg)
(mg/mL)
76 10mM Na acetate, pH 5.2 N/A 2.84 53
104 10mM Na acetate, pH 5.2 N/A 7.1 57
126 10mM Na acetate, pH 5.2 N/A 16 8.9 58
154 10mM Na acetate, pH 5.2 N/A 101 49 Did not freeze
73 10mM Na acetate, pH 5.2 + 3% proline 2.6 253
104 10mM Na acetate, pH 5.2 + 3% proline 5 252
122 10mM Na acetate, pH 5.2 + 3% proline 8.8 274
148 10mM Na acetate, pH 5.2 + 3% proline 24.4 9.5 301
125 10mM Na acetate, pH 5.2 + 150 mM 11 6.6 346
NaCl
134 10mM Na acetate, pH 4 N/A 13.3 8.87 59
117 10mM Na acetate, pH 4.5 N/A 10.8 6 59
130 10mM Na acetate, pH 5 N/A 16.2 7.1 59
133 10mM Na acetate, pH 5.5 N/A 23 12.6 64
134 10mM Na acetate, pH 5.5 + 150 mM 6.5 520
methionine
and 3% benzyl
alcohol
The results showed that L-Proline had a significant viscosity lowering effect in high
concentrations of PCSK9 ABP, 21B12 (See Figure 27).
To still further evaluate the effects of different formulations on a specific PCSK9 ABP,
compositions of 21B12 were formulated in different formulations shown in Table 16 below.
TABLE 16
21B12
Viscosity (cP) Osmolality
conc. Formulation Excipients
@ 25°C (mOsmol/kg)
(mg/mL)
mM sodium acetate,
116 N/A 10.4 72
pH 4.8
mM sodium acetate, 50 mM methionine + 2% benzyl
116 7 329
pH 4.8 alcohol
mM sodium acetate,
116 150 mM arginine 3.7 241
pH 4.8
mM sodium acetate,
116 2% proline + 1% benzyl alcohol 7 313
pH 4.8
mM sodium acetate,
116 1.5% proline + 1% benzyl alcohol 7.3 277
pH 4.8
The results show that 21B12 formulations formulated with 1.5% or 2.0% proline
(approximately 131 nM – 174 mM proline) and 1% benzyl alcohol had a significant viscosity
lowering effect in high concentrations of PCSK9 ABP, 21B12.
To still further evaluate the effects of different formulations on a specific PCSK9 ABP,
compositions of 21B12 were formulated in different formulations shown in Table 17 below.
TABLE 17:
Final Ave A280
Viscosity (cP) @ Shear Stress Shear Rate
Final Excipient Buffers 21B12 Conc
25C (Pa) @ 25C (1/sec) @ 25C
(mg/mL)
79 540
3.43 18.50
96 4.97 18.60 375
mM sodium acetate, 9%
110 7.68 18.44 240
Sucrose pH 5.2
166 223.19 18.40 8.25
89 4.80 18.00 375
105 5.97 18.30 307.5
mM sodium acetate, 125 122 9.10 18.40 202.5
mM Arginine, 3% Sucrose
150 19.31 18.80 97.5
pH 5.0
167 40.10 18.10 45
195 193.80 18.90 9.75
85 562.5
3.20 18.00
106 375
4.89 18.30
122 7.85 18.90 240
mM sodium acetate, 100
mM Methionine, 4% Sucrose
139 13.55 18.30 135
pH 5.0
168 121.22 18.20 15
193 309.56 18.60 6
85 3.20 18.00 562.5
108 4.57 18.85 412.5
125 7.61 18.27 240
mM sodium acetate, 250
139 13.54 18.30 135
mM Proline pH 5.0
180 14.3
133.73 19.00
203 6
323.35 19.40
The results show the ability to attain high concentrations of 21B12 protein having
reduced viscosity with formulations having specific stabilizers/excipients (See Figures 28A-
28D). Specifically, Figure 28A is a graph showing the viscosity of various concentrations of
anti-PCSK9 antibody, 21B12, in a formulation comprising 10 mM sodium acetate, and 9%
Sucrose pH 5.2 at 25ºC and 40ºC.
Figure 28B is a graph showing the viscosity of various concentrations of anti-PCSK9
antibody, 21B12, in a formulation comprising 10 mM sodium acetate, and 9% Sucrose pH
.2 at 25ºC and 40ºC, as compared to a formulation comprising 10 mM sodium acetate, 125
mM arginine, and 3% Sucrose pH 5.0 at 25ºC and 40ºC.
Figure 28C is a graph showing the viscosity of various concentrations of anti-PCSK9
antibody, 21B12, in a formulation comprising 10 mM sodium acetate, and 9% Sucrose pH
.2 at 25ºC and 40ºC, as compared to a formulation comprising 10 mM sodium acetate, 100
mM methionine, and 4% Sucrose pH 5.0 at 25ºC and 40ºC.
Figure 28D is a graph showing the viscosity of various concentrations of anti-PCSK9
antibody, 21B12, in a formulation comprising 10 mM sodium acetate, and 9% Sucrose pH
.2 at 25ºC and 40ºC, as compared to a formulation comprising 10 mM sodium acetate and
250 mM proline, pH 5.0 at 25ºC and 40ºC.
EXAMPLE 30
High Concentration 11F1 Viscosity Studies
Table 30 shows the viscosity of the 11F1 antibody at 25 degrees Celsius at various
antibody concentrations and in various formulations.
High concentration stock solution of 11F1 was prepared similarly as described for
21B12 in Example 29 above. Concentration determination was then carried out by measuring
absorbance at A280 using an Agilent 8453 Spectrophotometer (Santa Clara, California).
Protein concentration was calculated using the appropriate extinction coefficient. The
appropriate amount of buffer was then added to the sample to dilute it back down to the
desired concentration and another A280 was performed to obtain the final concentration for
the experiment. Excipients were added individually to the 11F1 formulations samples
derived from the high concentration stock solutions.
Viscosity was measured using Brookfield LV-DVII cone and plate viscometer
(Middleboro, Massachusetts) with a CPE-40 spindle with matching sample cup temperature
regulated by a circulating water bath at constant 25°C. 500 µL of sample was added to
sample cup with positive displacement pipettor. After sample cup was secured the rotational
speed of the spindle was gradually increased until about 80% torque was achieved. At this
point the rotational speed was stopped and a viscosity reading was generated by Rheocalc
software.
High concentration protein formulations were sometimes measured using a different
type of viscometer, an Anton Paar Physica Model MCR300 with a CP50-1 spindle. A 600 uL
sample is used in this instrument and Rheoplus software version 3.4 was use to calculate
solution viscosity. There was not a large difference in measurements using either viscometer.
TABLE 30
Final Ave A280 11F1 Viscosity (cP)
Final Excipient Buffers
Conc (mg/mL) @ 25C
mM sodium acetate, 9% Sucrose 0.01% Poly 14
Sorbate (“PS”) 20,pH 5.2
191 53
224 133
147 13
mM sodium acetate, 150 mM Methionine, 3% 31
Sucrose, 0.01% PS 20, pH 5.2
mM sodium acetate, 250 mM Proline , 0.01% 196 36
PS 20, pH 5.0
mM sodium acetate, 9% Sucrose, 100 mM
211 26
Arginine, pH 5.2
mM sodium acetate, 9% Sucrose, 150 mM
211 62
sodium chloride, pH 5.2
mM sodium acetate, 9% Sucrose, 150 mM
211 45
Glycine, pH 5.2
mM sodium acetate, 9% Sucrose, 150 mM
211 48
Serine, pH 5.2
mM sodium acetate, 9% Sucrose, 150 mM
211 43
Alanine, pH 5.2
mM sodium acetate, 9% Sucrose, pH 5.2 211 73
mM sodium acetate, pH 5.2 211 58
The results shown in Table 30 demonstrate the ability to attain high concentrations of
the 11F1 antibody with relatively low viscosity in formulations having specific
stabilizers/excipients. Formulations comprising the stabilizers methionine, proline, arginine,
glycine, serine and alanine exhibited particularly lower viscosity.
EXAMPLE 31
Stability Study of High Concentration PCSK9 ABP Formulations
To evaluate the effects of stability on high protein PCSK9 ABP formulations,
compositions of 21B12 were formulated in different formulations shown in Table 31.1 below.
Formulations were incubated in the indicated containers at -30 °C or 4°C for 0 weeks, 1 month,
2 months, 3 months, and 6 months, and 1 year. For each formulation at each time point, a
sample was removed from each package for monitoring of antibody monomer by native Size
Exclusion HPLC (SEC-HPLC) and Subvisible Particle Detection by Light Obscuration
(HIAC).
Table 31.1
21B12 Fill
Polysorbate Target
Formulations Conc Vol. Package Buffer Excipients
80 pH
(mg/mL) (mL)
mM Na
1 110 3.0 5cc Vial 9.0% Sucrose 0.010% 5.2
acetate
100 mM
mM Na
0.010%
2 120 3.0 5cc Vial Methionine, 5.0
acetate
4% Sucrose
mM Na 250 mM
0.010%
3 120 3.0 5cc Vial 5.0
acetate Proline
BD Glass 10 mM Na
0.010%
4 110 1.0 9.0% Sucrose 5.2
Syringe acetate
100 mM
BD Glass 10 mM Na
0.010%
120 1.0 Methionine, 5.0
Syringe acetate
4% Sucrose
BD Glass 10 mM Na 250 mM
0.010%
6 120 1.0 5.0
Syringe acetate Proline
CZ Plastic 10 mM Na
0.010%
7 110 1.2 9.0% Sucrose 5.2
Syringe acetate
100 mM
CZ Plastic 10 mM Na
0.010%
8 120 1.2 Methionine, 5.0
Syringe acetate
4% Sucrose
CZ Plastic 10 mM Na 250 mM
0.010%
120 1.2 5.0
Syringe acetate Proline
SEC-HPLC:
SEC-HPLC separates proteins based on differences in their hydrodynamic volumes.
Molecules with larger hydrodynamic proteins volumes elute earlier than molecules with
smaller volumes. Native SEC-HPLC was performed using a TSK-GEL G3000SWXL 7.8 mm
x 300 mm column (Tosoh Bioscience), with 5 m particle size, on an Agilent HPLC with a
Variable Wavelength Detector. The mobile phase was 100 mM Sodium Phosphate, 250 mM
Sodium Chloride, pH 6.8 0.1. The flow rate was 0.5 mL/minute. The column eluate was
monitored at 280 nm. Integrated peak areas in the chromatograms were used to quantify the
amounts of monomer and high molecular weight species.
Table 31.2:
%HMW at -30C %HMW at 4C
Formulations T=0 T=1M T=6M T=1Y T=0 T=1M T=2M T=3M
T=6M T=1Y
1 0.03 0.03 0.04 0.04 0.03 0.04 0.01 0.03 0.06 0.07
2 0.06 0.15 0.12 0.15 0.06 0.06 0.03 0.05 0.06 0.06
3 0.03 0.03 0.04 0.04 0.03 0.03 0.01 0.02 0.02 0.07
4 0.04 0.05 0.09 0.05 0.04 0.05 0.01 0.04 0.06 0.09
0.06 0.20 0.24 0.21 0.06 0.06 0.03 0.05 0.01 0.07
6 0.04 0.04 0.1 0.05 0.04 0.03 0.01 0.03 0.1 0.07
7 0.04 0.04 0.09 0.06 0.04 0.05 0.01 0.03 0.07 0.09
8 0.06 0.18 0.17 0.06 0.06 0.03 0.05
0.19 0.1 0.06
9 0.04 0.04 0.02 0.05 0.04 0.04 0.01 0.03 0.06 0.08
Table 31.2 shows the results of native SEC-HPLC analysis of 21B12 formulations
listed in Table 31.1 incubated at XºC for 0 weeks, 1 month, 2 months, 3 months, and 6
months. “% HMW” reflects the quantity of high molecular weight 21B12 monomer in a
sample. These results indicate that no formulation issues were observed after 6 months;
however some high molecular weight species did increase in the methionine formulation (i.e.,
formulations 2, 5 and 8).
Subvisible Particle Detection by Light Obscuration (HIAC):
An electronic, liquid-borne particle-counting system (HIAC/Royco 9703 or
equivalent) containing a light-obscuration sensor (HIAC/Royco HRLD-150 or equivalent)
with a liquid sampler quantifies the number of particles and their size range in a given test
sample. When particles in a liquid pass between the light source and the detector they
diminish or “obscure” the beam of light that falls on the detector. When the concentration of
particles lies within the normal range of the sensor, these particles are detected one-by-one.
The passage of each particle through the detection zone reduces the incident light on the
photo-detector and the voltage output of the photo-detector is momentarily reduced. The
changes in the voltage register as electrical pulses that are converted by the instrument into
the number of particles present. The method is non-specific and measures particles regardless
of their origin. The particle sizes that were monitored were 10 µm, and 25 µm.
In this example, HIAC analysis was performed using samples that had been stored at
4°C. Specifically, samples of 21B12 formulations in Table 31.1 were subject to vacuum
(also called “degassing”) in order to remove air bubbles that could be detected as particles in
the particle-counting system. For the 21B12 samples, the method was to subject the samples
to vacuum at 75 torr for 1 to 2 hours. Particle counting was performed within 2 hours of
completing the degassing process.
Figure 29A and 29B shows the results of the HIAC assays for the above-identified
formulations incubated in containers for 0 weeks, 1 month, 2 months, 3 months, and 6
months. 10 µm, and 25 µm particles were counted. Figures 29A and 29B demonstrate that
all of the formulations of 21B12 were stable as measured with HIAC. Although the
formulations in glass syringes, i.e., formulations 4-6, showed higher levels of particles across
protein concentration and formulation, those particle counts are below USP limits for each
particle size (10 µm and 25 µm). USP limits for 10 µm particles is 6000 per container and
for 25 µm particles, 600 per container.
EXAMPLE 32
11F1 Stability Studies
To study high concentration formulations (150 mg/mL) of 11F1, several formulations
were made using candidate excipients as indicated in Table 32A below. The formulations
were stored in the indicated containers at -30 °C or 4°C for at least six months.
Table 32A: Formulations Studied
Target Conc Polysorbate Final
Formulation Name Container Buffer Target Excipients
(mg/mL) 20 pH
mM Na
1 150 5cc Glass Vial 9.0% Sucrose 0.010% 5.2
acetate
BD Glass 10 mM Na
2 150 9.0% Sucrose 0.010% 5.2
Syringe acetate
150 mM
BD Glass 10 mM Na
3 150 Methionine, 0.010% 5.2
Syringe acetate
3% Sucrose
BD Glass 10 mM Na
4 150 250 mM Proline 0.010% 5.2
Syringe acetate
CZ Plastic 10 mM Na
150 9.0% Sucrose 0.010% 5.2
Syringe acetate
150 mM
CZ Plastic 10 mM Na
6 150 Methionine, 0.010% 5.2
Syringe acetate
3% Sucrose
CZ Plastic 10 mM Na
7 150 250 mM Proline 0.010% 5.2
Syringe acetate
%HMW species was assessed by size exclusion HPLC after storage at -30°C and 4°C
at the time points indicated in Table 32B below. Briefly, size exclusion HPLC separates
proteins based on differences in their hydrodynamic volumes. Molecules with larger
hydrodynamic proteins volumes elute earlier than molecules with smaller volumes. Native
SEC-HPLC was performed using a TSK-GEL G3000SWXL 7.8 mm x 300 mm column
(Tosoh Bioscience), with 5 µm particle size, on an Agilent HPLC with a Variable
Wavelength Detector. The mobile phase was 100 mM Sodium Phosphate, 250 mM Sodium
Chloride, pH 6.8 +/- 0.1. The flow rate was 0.5 mL/minute. The column eluate was
monitored at 280 nm. Integrated peak areas in the chromatograms were used to quantify the
amounts of monomer and high molecular weight species.
TABLE 32 B
%HMW at -30°C %HMW at 4°C
Formulations T=0 T=4M T=0 T=2M T=4M T=6M
0.05 0.05 0.05 0.06 0.05 0.05
0.05 0.05 0.05 0.06 0.04 0.02
0.07 0.26 0.07 0.07 0.07 0.06
0.06 0.07 0.06 0.07 0.06 0.08
0.05 0.04 0.05 0.05 0.04 0.06
0.06 0.32 0.06 0.06 0.06 0.06
0.08 0.07 0.08 0.06 0.07 0.08
Table 32B shows the results of native SEC-HPLC analysis of 11F1 formulations listed
in Table 32A incubated at 4°C or -30°C for 0 weeks, 2 months, 4 months, or 6 months. “%
HMW” reflects the quantity of high molecular weight 11F1 in a sample. These results indicate
that no formulation issues were observed up to 6 months, however some high molecular weight
species did increase in the methionine formulations stored at -30°C (i.e. formulations 3, and 6).
The stability of additional high concentration 11F1 formulations was assessed by
preparing the formulations in the primary containers as indicated in Table 32C below:
Table 32 C
Buffer Final pH
11F1
Primary 0.010%
Formulation Conc Excipients
Container Polysorbate
(mg/mL)
mM Na
150 Glass Vials 9.0% Sucrose PS 20
acetate
mM Na
150 Glass Vials 9.0% Sucrose PS 80 5.2
acetate
BD Glass 150mM Methionine, 10 mM Na
180 PS 20
Syringe 3% Sucrose acetate
BD Glass 150mM MET, 3% 10 mM Na
40 180 PS 80 5.2
acetate
Syringe Sucrose
BD Glass 10 mM Na
50 180 250mM Prolinee PS 20 5.2
acetate
Syringe
BD Glass 10 mM Na
60 180 250mM Proline PS 80
acetate
Syringe
CZ Plastic 150mM Methionine,
mM Na
70 180 PS 20 5.2
acetate
Syringe 3% Sucrose
CZ Plastic 150mM Methionine, 10 mM Na
80 180 PS 80
acetate
Syringe 3% Sucrose
CZ Plastic 10 mM Na
90 180 250mM Proline PS 20 5.2
acetate
Syringe
CZ Plastic 10 mM Na
100 180 250mM Proline PS 80 5.2
acetate
Syringe
The formulations were incubated at 4 ° Celsius for one year. At the time points
indicated in the Table 32D below, a sample was removed from each container and analyzed
by SEC-HPLC as described for Table 32B above..
Table 32D
Size exclusion %HMW forms after 1 year storage at 4°C
4°C % HMW
Formulations T=0 T=2wk T=4wk T=6wk T=6M T=6.5M T=1Yr Change
0.03
0.04 0.07 0.08 0.06 0.07 N/A 0.07
0.01
0.05 0.07 0.07 0.07 0.06 N/A 0.06
-0.03
0.08 0.14 N/A N/A N/A N/A 0.05
-0.03
40 0.09 0.15 0 N/A N/A N/A 0.06
-0.01
50 0.08 0.15 0 0 N/A N/A 0.07
0.01
60 0.07 0.16 0 0 N/A N/A 0.08
-0.02
70 0.08 0.14 0 0 N/A 0.09 0.06
0.00
80 0.07 0.14 0 0 N/A N/A 0.07
-0.04
90 0.09 0.15 0 0 N/A 0.09 0.05
0.00
100 0.08 0.15 0 0 N/A N/A 0.08
At the time points indicated in the Table 32E below, a sample was removed from each
container analyzed by cation-exchange HPLC (CEX-HPLC). Cation-exchange HPLC
separates proteins based on differences in their surface charge. At a set pH, charged isoforms
of 11F1 are separated on a cation-exchange column and eluted using a salt gradient. The
eluent is monitored by UV absorbance. The charged isoform distribution is evaluated by
determining the peak area of each isoform as a percent of the total peak area..
Native CEX-HPLC was performed using a Dionex G3000SWXL 4.0 mm ID x 250
mm column (Tosoh Bioscience), with 10 µm particle size, on an Agilent HPLC with a
Variable Wavelength Detector. The mobile phase was a linear gradient of 20 mM MES, pH
6.0 +/-0.1 and the same buffer with 500 mM Sodium Chloride. The flow rate was 0.6
mL/minute. The column eluate was monitored at 280 nm. Integrated peak areas in the
chromatograms were used to quantify the amounts of differently charged isoforms.
Table 32E
Cation exchange HPLC % Main Isoform Peak after 1 year storage at 4°C
4°C % Main Isoform Peak
Formulation T=0 2W 4W 6W 1Y % Change
76.0 75.9 75.7 75.6 76.2
76.0 76.4 75.7 75.6 76.4 0.5
76.0 N/A N/A N/A 76.3 0.4
40 75.8 N/A N/A N/A 76.0 0.2
50 76.0 N/A N/A N/A 76.3 0.4
60 75.8 N/A N/A N/A 75.8 0.1
70 75.9 N/A N/A N/A 76.2 0.5
80 76.1 N/A N/A N/A 76.3 0.3
90 76.0 N/A N/A N/A 76.0 0.0
100 75.8 N/A N/A N/A 75.9 0.0
Both tables 32D and 32E demonstrate that the described 11F1 formulations exhibited
less than 5% increase in %HMW (SEC-HPLC) or less than a 3-5 % variation in the Main
Isoform Peak (CATION HPLC) up to 1 year storage at 4°C. In fact changes in both
parameters were very low which is indicative of highly stable formulations.
Subvisible Particle Detection by Light Obscuration (HIAC):
An electronic, liquid-borne particle-counting system (HIAC/Royco 9703 or
equivalent) containing a light-obscuration sensor (HIAC/Royco HRLD-150 or equivalent)
with a liquid sampler quantifies the number of particles and their size range in a given test
sample. When particles in a liquid pass between the light source and the detector they
diminish or “obscure” the beam of light that falls on the detector. When the concentration of
particles lies within the normal range of the sensor, these particles are detected one-by-one.
The passage of each particle through the detection zone reduces the incident light on the
photo-detector and the voltage output of the photo-detector is momentarily reduced. The
changes in the voltage register as electrical pulses that are converted by the instrument into
the number of particles present. The method is non-specific and measures particles regardless
of their origin. The particle sizes that were monitored were 10 µm, and 25 µm.
In this example, HIAC analysis was performed using samples that had been stored at
4°C. Specifically, samples of 11F1 formulations in Table 32a were subject to vacuum (also
called “degassing”) in order to remove air bubbles that could be detected as particles in the
particle-counting system. For the 11F1 samples, the method was to subject the samples to
vacuum at 75 torr for 1 to 2 hours. Particle counting was performed within 2 hours of
completing the degassing process.
Figure 30A and 30B show the results of the HIAC assays for the above-identified
formulations incubated in containers for 0 weeks, and four months. 10 µm and 25 µm
particles were counted. Figures 30A and 30B demonstrate that all of the formulations of
11F1 were stable as measured with HIAC. Particle counts for all formulations are below
USP limits for each particle size (10 µm and 25 µm). USP limits for 10 µm particles is 6000
per container and for 25 µm particles, 600 per container.
Example 33
11F1 Binding Specificity
Results from this assay demonstrate that 11F1 binds to PCSK9 and not to PCSK1,
PCSK2, PCSK7, or furin, demonstrating the specificity of 11F1 for PCSK9.
Biotinylated PCSK9, diluted in buffer A (25 mM Tris, 150 mM NaCl, 0.1% BSA,
0.05% tween, pH 7.5) was bound to neutravidin coated 96 well plates at a concentration of
0.2 µg/mL, for one hour incubation at room temperature. Separately, 0.4 µg/mL of 11F1
was incubated for one hour at room temperature with various concentrations (ranging from 0
to 20 µg/mL) of either PCSK1, PCSK2, PCSK7, PCSK9 or furin (R&D Systems,
Minneapolis, MN) (diluted in buffer A w/o tween). Furin inhibitor, at 4.5 µg/mL, was
included with all furin containing reactions. The PCSK9 coated streptavidin plate was
washed with buffer A and the antibody/proprotein convertase mixture was added to the plate
and incubated at room temperature for one hour. After washing, bound antibody was
detected by incubation with goat- α-human Fc-HRP (160 ng/mL, diluted in buffer A) (Jackson
Laboratories, Bar Harbor, ME) followed by TMB substrate. The reaction was stopped with 1
N HCl and the absorbance was read at a wavelength of 450 nm on a Spectramax Plus 384
spectrophotometer (Molecular Devices Inc., Sunnyvale, CA).
This assay relied on the ability of proprotein convertase in solution to compete for the
binding of 11F1 to plate-captured PCSK9. Pre-incubation of 11F1 and PCSK9 in solution
dose dependently and robustly reduced the amount of 11F1 binding to plate-captured PCSK9
detected as reduced OD450 (Figure 31). All results were expressed as the mean OD450 value
± standard deviation versus concentration of the proprotein convertase. Pre-incubation of
11F1 with PCSK1, PCSK2, PCSK7, or furin, in solution, did not significantly impact the
binding of 11F1 to plate-captured PCSK9. Therefore, at the protein concentrations studied,
11F1 binds only to PCSK9 and not to the other proprotein convertase family members tested.
Example 33
Efficacy of 11F1 Inhibition of LDLR:PCSK9 Binding
The example demonstrates that nanomolar concentrations of 11F1 can inhibit binding
of both D374Y and wild-type PCSK9 to the LDLR under the conditions of this assay.
Briefly, clear, 384 well plates were coated with 2 μg/mL of goat anti-LDL receptor
antibody (R&D Systems, Minneapolis, MN), diluted in PBS, by overnight incubation at 4°C.
Plates were washed thoroughly with buffer A (100 mM sodium cacodylate pH 7.5) and then
blocked with buffer B (1% non-fat dry milk [Bio-Rad Laboratories, Hercules, CA] in buffer
A) for 2 hours at room temperature. After washing, plates were incubated with 0.4 μg/mL of
LDL receptor (R&D Systems, Minneapolis, MN) diluted in buffer C (buffer B supplemented
with 10 mM CaCl2) for 1.5 hours at room temperature. Concurrent with this incubation, 20
ng/mL of biotinylated D374Y PCSK9 or 100 ng/mL of biotinylated WT PCSK9 was
incubated with various concentrations of anti-PCSK9 antibody 11F1 diluted in buffer A (final
concentrations ranging from 6.0 ng/mL to 200 ug/mL for the D374Y PCSK9 assay or 3.1
ng/mL to 25 ug/mL for the WT PCSK9 assay). The LDLR-coated plates were washed and the
biotinylated PCSK9/antibody mixture was added. The LDLR plate was incubated at room
temperature for 1 hour. Binding of the biotinylated PCSK9 to the LDLR was detected by
incubation with streptavidin-HRP (500 ng/mL in buffer C) followed by TMB substrate. The
reaction was stopped with 1N HCl and the absorbance was read at a wavelength of 450 nm
on a SpectraMax Plus 384 Spectrophotometer (Molecular Devices Inc., Sunnyvale, CA).
GraphPad Prism (v 4.01) software was used to plot log of antibody concentration versus
OD450 to determine IC50 values by nonlinear regression.
11F1 inhibited LDLR:PCSK9 binding. The IC50 values for 11F1 in the D374Y
PCSK9 assay ranged from 7.3 nM to 10.1 nM with an average (± SD) of 9.1 nM ± 1.5 nM
(n=3). The IC50 values for 11F1 in the wild-type PCSK9 assay ranged from 4.4 nM to 8.1
nM with an average (± SD) of 5.9 nM ±1.9 nM (n=3). It should be noted that these IC50
values are dependent on the amount of recombinant D374Y PCSK9 or WT PCSK9 used in
the binding assay. A representative dose response curve for both the D374Y and wild-type
assays are presented in Figure 32 and Figure 33, respectively.
Example 34
Efficacy of 11F1 in Blocking Cell LDL Uptake
11F1 blocks the interaction between PCSK9 and LDLR in vitro and can prevent the
PCSK9-mediated reduction of LDL uptake in HepG2 cells.
Briefly, human` HepG2 cells were seeded in black, clear bottom 96-well plates
(Fisher Scientific CO LLC, Santa Clara, CA) at a density of 5x104 cells per well in DMEM
(Mediatech Inc., Herndon, VA) supplemented with 10% FBS and 1% of antibiotic-
antimycotic solution (Mediatech Inc., Herndon, VA). Cells were incubated at 37°C (5%
CO2) overnight. To form the complex between D374Y PCSK9 and antibody or WT PCSK9
and antibody, serial dilutions (1:2) of 11F1, from 666.7 nM to 0.7 nM (for blocking D374Y
PCSK9) or from 3.3 µm to 3.3 nM (for blocking WT PCSK9), were prepared in formulation
buffer (25 mM HEPES, pH 7.5, 0.15 M NaCL). Either D374Y PCSK9 (2 µg/mL) or WT
PCSK9 (25 µg/mL) were diluted in uptake buffer (DMEM containing 1% FBS) and
incubated with the various concentrations of 11F1 or uptake buffer alone (negative control)
for 1 hour at room temperature with shaking. BODIPY-LDL (Invitrogen, Carlsbad, CA) was
diluted in uptake buffer to a concentration of 12 µg/mL. Following overnight incubation,
HepG2 cells were rinsed twice with DPBS (Mediatech Inc., Herndon, VA). Twenty-five
microliters of the D374Y PCSK9 or WT PCSK9 complex with 11F1 and 25 µL of diluted
BODIPY-LDL (Invitrogen, Carlsbad, CA) were added to the cells and incubated at 37°C (5%
CO2) for 3 hours. Cells were washed with DPBS 5 times and resuspended in 100 µL DPBS.
Fluorescent signals were detected using a Safire plate reader (Tecan Systems Inc., San Jose,
CA) at 480~520 nm (excitation) and 520~600 nm (emission) and expressed as relative
fluorescence unit (RFU).
GraphPad Prism (Version 4.02, GraphPad Software Inc., San Diego, CA) software
was used to plot log of antibody concentration versus RFU and to determine EC50 values by
nonlinear regression using the sigmoidal dose-response (variable slope) curve fitting
program.
This example shows that 11F1 blocked D374Y PCSK9 or WT PCSK9 -mediated
decrease of LDL uptake in HepG2 cells in a dose-dependent manner. Adding recombinant
purified D374Y PCSK9 (2 µg/mL) or WT PCSK9 (25 µg/mL) to HepG2 cells reduced the
uptake of BODIPY-LDL to ~50 to 60% and ~40% of the level measured in untreated cells,
respectively. The antibodies dose-dependently restored LDL uptake to the level observed in
untreated cells. The mean (± SD) EC50 value for the ability of 11F1 to block D374Y
PCSK9-mediated decrease of LDL uptake was 35.3 ± 9.1 nM (n = 6, Figure 34). The EC50
value for the ability of 11F1 to block WT PCSK9-mediated decrease in LDL uptake was
124.2 ± 28.5 nM (n = 3, Figure 35). It should be noted that these EC50 values are a function
of the amount of recombinant D374Y PCSK9 or WT PCSK9 used in the cell assay. The
EC50 value is lower against D374Y PCSK9 than WT PCSK9 since less D374Y PCSK9 was
used in the assay because its binding affinity to the LDLR is 5- to 30-fold greater than that of
WT PCSK9 (Cunningham et al, 2007; Fisher et al, 2007; Kwon et al, 2008).
The EC50 values reported here are representative for mean values derived from 3 to 6
separate measurements for 11F1.
Example 35
Efficacy of 11F1 and 8A3 in Blocking Human PCSK9 Expressed Via an Adeno-Associated
Virus in a mouse model
A single intravenous bolus administration of the anti-PCSK9 antibodies 11F1 or 8A3
leads to a significant decrease in serum non-HDL-C and TC in mice expressing human
PCSK9 by AAV. This example demonstrates the effectiveness of both anti-PCSK9
antibodies in blocking the function of human PCSK9 in vivo.
Briefly, 120 C57BL/6 mice expressing human PCSK9 were generated by infection
with an engineered adeno associated virus (AAV) coding for human PCSK9, resulting in
elevated levels of circulating low density lipoprotein cholesterol (LDL-C). Serum cholesterol
analysis was performed using the Cobas Integra 400 plus chemistry analyzer (Roche
Diagnostics, Indianapolis, IN). Animals were randomized into treatment groups with similar
levels of non-HDL-C (LDL-C and VLDL-C), HDL-C and TC. On treatment day 0 (T=0) a
subset of mice was euthanized and serum collected to establish that day’s baseline levels.
Remaining mice were then administered 11F1, 8A3 or anti-keyhole limpethemocyanin
(KLH) IgG2 control antibody at 30 mg/kg. via tail vein injection. At days 1 through 5
following injection, subsets of mice were euthanized and whole blood was collected from the
vena cava and allowed to coagulate for 30 minutes at room temperature. Following
centrifugation at 12,000 rpm with a bench top centrifuge for 10 minutes, serum was collected.
Serum cholesterol analysis was performed using the Cobas Integra 400 plus chemistry
analyzer.
Serum concentrations of PCSK9 were determined using a sandwich ELISA assay.
Clear 96 well plates were coated overnight with 2 µg/ml of monoclonal anti-PCSK9
antibody (31H4) diluted in 1X PBS. Plates were washed thoroughly with 1X PBS/.05%
tween and then blocked for 2 hours with 3% BSA/1XPBS. After washing, plates were
incubated for 2 hours with serum diluted in general assay diluents (Immunochemistry
Technologies, Bloomington, MN). Recombinant human PCSK9 (1 ng/ml to 500 ng/ml) was
assayed concurrently and used to generate a standard curve on each ELISA plate. A rabbit
polyclonal biotinylated anti-PCSK9 antibody (D8773, Amgen Inc, CA) was added at 1
ug/ml (in 1%BSA/PBS), followed by neutravidin-HRP at 200 ng/ml (in 1% BSA/PBS).
Bound PCSK9 was detected by incubation with TMB substrate. The reaction was stopped
with addition of 1N HCl and the absorbance measured at 450 nm on a Spectra Max Plus 384
Spectrophotometer (Molecular Devices Inc, Sunnyvale, CA). The standard curve (4-
parameter logistic fit) generated with recombinant human PCSK9 was used to determine the
corresponding concentration of PCSK9 in the serum samples.
Serum concentrations of antibody were determined using a sandwich ELISA assay.
Polyclonal goat anti-human Fc IgG and an HRP-labeled goat anti-human IgG Fc γ polyclonal
reagent (both from Jackson ImmunoResearch Laboratories Inc, West Grove, PA) were used
as the capture and the detection antibody, respectively. A 3,3’,5,5’tetramethylbenzidine
(TMB) substrate solution reacted with peroxide, and in the presence of horse radish
peroxidase (HRP), created a colorimetric signal that was proportional to the amount of the
respective anti-PCSK9 antibody bound by the capture reagent. The intensity of the color
(optical density, OD) was measured at 450 nm minus 650 nm using a microplate reader
(Spectra Max Plus 384). Data was analyzed using Watson version 7.0.0.01 (Thermo
Scientific, Waltham, MA) data reduction package with a Logistic (auto-estimate) regression
of separately prepared standard curves. The lower limit of quantification (LLOQ) for the
assay was ng/mL. 34.4.
Calculation of Pharmacokinetic Parameters in AAV Mice
Non-compartmental analysis (NCA) was performed on serum concentrations using
the pre-determined nominal time points for each subject using WinNonlin Enterprise, version
.1.1 (Pharsight, St. Louis, MO). Data points for estimating the terminal elimination rate
constants and half-lives were chosen by visual inspection of the concentration-time profiles.
NCA parameters reported include: apparent half-life (t1/2), area under the serum
concentration-time curve from time zero to the last measured concentration (AUC0-t), and
apparent serum clearance (CL0-t). AUC0-t was determined using the linear log-linear
trapezoidal method, and CL0-t was calculated by Dose/AUC0-t. For 11F1, 8A3, and 31H4
antibodies. Post-study dose solution analysis showed actual doses were within 20% of the 30
mg/kg target. However, for the IgG2 control, analysis showed actual dose was only 40% of
the intended target. Therefore, a corrected dose of 12 mg/kg was used for CL0-t calculation
for IgG2 control. Parameters were reported to three significant figures, except for half-life
which was reported to two significant figures.
Statistical Analysis
All cholesterol results were expressed as the mean ± standard error of the mean. All
pharmacokinetic data were expressed as the mean ± standard deviation. The p value of 0.05,
determined by 1-way ANOVA was used as a threshold to determine statistical significance
between the anti-KLH IgG2 control antibody injected animals and those dosed with anti-
PCSK9 antibody at the same time point.
Effect of Anti-PCSK9 Antibodies on Serum non-HDL-C, HDL-C, and TC
To establish a baseline, a subset of mice expressing human PCSK9 was euthanized
prior to injection of antibodies and blood was collected. Non-HDL-C, HDL-C and TC levels
in these animals were 33 ± 4, 117 ± 4 and 183 ± 9 mg/dL, respectively (mean ± SEM). Levels
of PCSK9 in naïve animals were determined to be 4921 ng/mL ± 2044 ng/mL.
Compared to mice injected with anti-KLH IgG2 control antibody (control animals),
injection of 11F1 produced significant lowering of non-HDL-C at days 1, 2, and 4 post-
injection (with a maximum of 59%), while TC was significantly lowered at day 4 only (by
22%) (Figure 36, Figure 37). No significant lowering of HDL-C was observed at any time
point (Figure 38).
Compared to control animals, injection of 8A3 produced significant lowering of non-
HDL-C at days 1, 2, and 4 post-injection (with a maximum of 65%), while TC was
significantly lowered at day 2 post-injection (with a maximum of 24%) (Figure 36, Figure
37). No significant lowering of HDL-C was observed at any time point (Figure 38).
Pharmacokinetics
At an intravenous dose of 30 mg/kg, 11F1 and 8A3 had very similar pharmacokinetic
behavior (Figure 39). For these two molecules, AUC0-t exposures, estimated CL0-t, and
apparent half-lives were equivalent (Table of Figure 40). The anti-KLH IgG2 control
antibody had an unexpectedly lower AUC0-t exposure than 11F1 and 8A3, but this is likely
due to the antibody being administered at a lower dose than intended (12 mg/kg as opposed to
mg/kg; dose solution analysis showed antibody concentration to be 40% of target. Anti-
KLH IgG2 control antibody CL0-t was similar to that of 11F1 and 8A3, when calculated
using the corrected dose, and the apparent half-life of the anti-KLH IgG2 control antibody
was estimated at >120 hours. These data suggested that affects of the PCSK9 ligand on
antibody disposition are less pronounced for 11F1 and 8A3 when compared to other
antibodies dosed in the AAV model because 11F1 and 8A3 CL0-t values are more similar to
anti-KLH IgG2 control antibody.
Summary.
Expression of human PCSK9 by AAV in mice (approximately 5 ug/mL) resulted in a
serum non-HDL-C level of approximately 33 mg/dL. Following a 30 mg/kg injection of
11F1, significant serum non-HDL-C lowering was observed at days 1, 2 and 4 post-injection
(with a maximum of 59% as compared to control animals). Significant lowering of TC was
seen at day 4 only. Injection of 8A3 resulted in a similar pattern of non-HDL-C lowering with
a maximum of 65% as compared to control animals. However, 8A3 administration resulted in
significant TC lowering at day 2 only, post-injection, with a maximum of 24%. No
significant lowering of HDL-C was observed in animals administered either 11F1 or 8A3.
Analysis of serum antibody levels of 11F1 and 8A3 demonstrated a similar profile to anti-
KLH IgG2 control antibody.
Example 36
Effect of a Single Subcutaneous Dose of 11F1, 21B12 and 8A3 on Serum Lipids in
Cynomolgus Monkeys
Single SC administration of 11F1, 8A3 or 21B12 to cynomolgus monkeys leads to the
significant lowering of serum LDL-C, and TC. This study demonstrated the ability of anti-
PCSK9 antibodies to lower serum cholesterol in non-human primates.
Briefly, naive male cynomolgus monkeys were acclimated to their environment for at
least 2 weeks prior to experimentation. Animals were randomized into treatment groups
based on a pre-screen of their serum TC, HDL-C, LDL-C, and triglyceride levels, and their
body weight. After 1 week, animals were fasted overnight, and bled from the peripheral
vasculature (cephalic or saphenous vein), for measurement of baseline serum lipid levels at a
time point designated T = 0. Animals were then injected SC with either anti-KLH IgG2
control antibody, 11F1, 21B12, or 8A3 (all in 10 mM NaOAc pH 5.2, 9% sucrose) at 0.5
mg/kg (all at 0.4 mL/kg body weight). Fasting blood samples were then collected from
animals at designated time points over a 45 day period.
Experimental Design
Group No Dose Level Conc. Volume
No. Males Route Treatment (mg/kg) (mg/mL) (mL/kg)
1 5 SC Anti-KLH 0.5 1.09 0.4
2 5 SC 21B12 0.5 1.19 0.4
3 5 SC 11F1 0.5 1.11 0.4
4 5 SC 8A3 0.5 1.25 0.4
At specified time points, blood was collected from animals under overnight fasting
conditions from the peripheral vasculature (cephalic or saphenous vein). Whole blood was
allowed to coagulate for 30 minutes at room temperature. Following centrifugation
at 3,000 rpm for 20 minutes, serum was collected. Direct serum cholesterol analysis was
performed using the Cobas Integra 400 analyzer (Roche Diagnostics Inc, Indianapolis, IN).
Apolipoprotein B serum levels were determined at specified time points (day 0, 3, 6, 15, 24
and 33) by Anilytics, MD, with the following methodology. A 17 µL aliquot of the sample
(no preparation) was used for analysis with a Hitachi 717 Analyzer using a 6 points standard
curve. If the initial value of the sample was higher than the standard curve linearity, then the
sample was diluted and repeated with the result multiplied by the appropriate dilution factor.
The reagents for the assay (APO-B Reagent Kit # 86071, Antibody Set # 86060, Control Set
# 86103) were obtained from DiaSorin (Stillwater, MN).
Antibody concentrations in serum were determined using an enzyme-linked
immunosorbent assay (ELISA) with an assay range of 34.4 to 3000 ng/mL (34.4 ng/mL being
the lower limit of quantitation [LLOQ]).
Non-compartmental analysis (NCA) was performed on the serum concentrations
using the pre-determined nominal time points for each subject using Watson LIMS, version
7.0.0.01 (Thermo Scientific, Waltham, MA). Data points for estimating the terminal
elimination rate constants and half-lives were chosen by visual inspection of the
concentration-time profile and best linear fit (typically from 360 h until the antibody
concentrations dropped below the lower limit of quantitation). NCA parameters reported
include: terminal half-life (t1/2,z), the maximum serum concentration (Cmax), area under the
serum concentration-time curve from time zero to infinity (AUC0-inf), and apparent serum
clearance (CL/F). AUC0-inf was calculated using the linear log-linear trapezoidal method.
All parameters were all reported to three significant figures, except for half-life which was
reported to two significant figures.
Statistical Analysis
A statistical model that considers baseline as a covariate and treatment group as a
fixed effect was fit to the log transformed response at each time point for LDL-C, HDL-C,
TC, and triglycerides. Tukey's multiple comparison correction was applied to adjust the pair
wise comparisons at each time point. The statistical significance was evaluated at alpha=0.05
using adjusted p-values.
Effect of 11F1, 21B12, and 8A3 on Serum LDL Cholesterol
Maximal LDL-C lowering for 11F1 was observed 9 days after injection, with a 57%
lowering of LDL-C as compared to anti-KLH IgG2 control antibody-treated monkeys
(control animals). LDL-C returned to levels similar to those observed in control animals by
day 27. Maximal LDL-C lowering for 21B12 was observed 3 days after injection, with a 64%
lowering of LDL-C as compared to control animals. LDL-C returned to levels similar to
control animals by day 6. Maximal LDL-C lowering for 8A3 was observed 4 days after
injection, with a 54% lowering of LDL-C as compared to control animals. LDL-C returned to
levels similar to those observed in control animals by day 27 (Figure 41).
Effect of 11F1, 21B12, and 8A3 on Serum Total Cholesterol
. Maximal TC lowering for 11F1 was observed 9 days after injection, with a 27%
lowering of TC as compared to anti-KLH IgG2 control antibody-treated monkeys (control
animals). TC returned to levels similar to those observed in control animals by day 27.
Maximal TC lowering for 21B12 was observed 3 days after injection, with a 20% lowering of
TC as compared to control animals. TC transiently returned to levels similar to those
observed in vehicle-treated monkeys by day 4, but were significantly lower between days 14
and 18, inclusively. Maximal TC lowering for 8A3 was observed 9 days after injection, with
a 22% lowering of TC as compared to control animals. TC returned to levels similar to those
observed in control animals by day 30 (Figure 42).
Effect of 11F1, 21B12, and 8A3 on Serum HDL Cholesterol and Triglycerides
On average and at each time point, HDL-C or triglyceride levels for animals treated
with 11F1 or 8A3 were not significantly different (based on an alpha = 0.05 significance
level) from those observed in anti-KLH IgG2 control antibody-treated monkeys. However,
21B12 did induce a statistically significant change in HDL-C at a single time point (day 18
following injection) (Figure 43 and Figure 45).
Effect of 11F1, 21B12, and 8A3 on Apolipoprotein B (ApoB)
Serum ApoB levels were measured at days 3, 6, 15, 24 and 33, post-injection. 11F1
and 8A3 were associated with ApoB lowering at days 3 to 24, as compared to anti-KLH IgG2
control antibody-treated monkeys (Figure 46). 21B12 was associated with statistically
significant lower ApoB levels at day 3 only.
Pharmacokinetic Profiles of 11F1, 21B12, and 8A3
A summary plot of the mean concentration-time profiles by treatment is shown in
Figure 48. The estimated mean pharmacokinetic parameters for animals receiving 11F1,
21B12, 8A3, and anti-KLH IgG2 control antibody are displayed in Table of Figure 47.
Antibody absorption in all groups was consistent and characteristic of subcutaneous
antibody administration. 21B12 pharmacokinetic behavior with regard to CL/F, Cmax, and
AUC0-inf was consistent with that observed in previous studies where 21B12 was
administered at the same dose. Pharmacokinetics of 11F1 and 8A3 differed significantly
from 21B12, where lower CL/F was observed (approximately 15% of 21B12 CL/F) and
longer half-lives were estimated (approximately 200 h compared to 40 h for 21B12).
Notably, pharmacokinetics of 11F1 and 8A3 were indistinguishable both from one another
and the anti-KLH IgG2 control antibody. These data suggest that disposition of 11F1 and
8A3 is impacted to a far lesser extent by association with the PCSK9 target than 21B12,
given that 11F1 and 8A3 have the same exposure profile as anti-KLH IgG2 control antibody
with no affinity for PCSK9.
Summary of Results
Over the course of the 45 day study, statistically significant lowering of TC and LDL-
C was observed in animals administered 11F1, 21B12, or 8A3 as compared to anti-KLH
IgG2 control antibody. 11F1 was associated with statistically significant LDL-C lowering
(vs. anti-KLH IgG2 control antibody) from day 2 to day 24 inclusively. 21B12 demonstrated
statistically significant LDL-C lowering (vs anti-KLH IgG2 control antibody) from day 1 to
day 4 inclusively. 8A3 demonstrated statistically significant LDL-C lowering (vs anti-KLH
IgG2 control antibody) from day 1 to day 24 inclusively. Changes in TC and ApoB mirrored
changes observed in LDL-C for all groups. 11F1 achieved a maximal lowering of LDL-C (vs
anti-KLH IgG2 control antibody at the same time point) 9 days following injection (-57%).
21B12 achieved a maximal lowering of LDL-C (vs anti-KLH IgG2 control antibody at the
same time point) 3 days following injection (-64%). 8A3 achieved a maximal lowering of
LDL-C (vs anti-KLH IgG2 control antibody at the same time point) 4 days following
injection (-54%). 21B12 lowered HDL-C at a single time point, 18 days after injection. No
statistically significant changes were observed in HDL-C levels following 11F1 or 8A3
administration. No statistically significant changes were observed in triglycerides levels
following 11F1, 21B12, or 8A3 administration.
Example 37
A Two Part Study to Assess the Safety, Tolerability and Efficacy of a Human Anti-
PCSK9 Antibody on LDL-C in Subjects with Homozygous Familial Hyperchoesterolemia
Study Design: This is a 2 part study. Part A is an open label, single arm, multicenter
pilot study. Part B is a double-blind, randomized, placebo-controlled, multicenter, study of
human antibody, 21B12, with expanded enrollment but otherwise identical design to Part A.
Both inclusion/exclusion criteria and the Schedule of Assessments will be the same for Parts
A and B.
Inclusion Criteria includes:
Males and females ≥ 12 to ≤ 65 years of age
Diagnosis of homozygous familial hypercholesterolemia
Stable lipid-lowering therapies for at least 4 weeks
LDL cholesterol >130 mg/dl (3.4 mmol/L)
Triglyceride < 400 mg/dL(4.5 mmol/L)
Bodyweight of > 40 kg or greater at screening.
Exclusion Criteria includes:
LDL or plasma apheresis within 8 weeks prior to randomization
New York Heart Failure Association (NYHA) class III or IV or last known left
ventricular ejection fraction < 30%
Myocardial infarction, unstable angina, percutaneous coronary intervention (PCI),
coronary artery bypass graft (CABG) or stroke within 3 months of randomization
Planned cardiac surgery or revascularization
Uncontrolled cardiac arrhythmia
Uncontrolled hypertension
Schedule of Assessments include, but are not limited to, collection of adverse event (AE)
and significant adverse event (SAE) data, vital signs, concomitant medication, laboratory
tests, etc.
Subjects who meet inclusion/exclusion criteria will be instructed to follow an NCEP
Adult Treatment Panel TLC (or comparable) diet and be required to maintain their current
lipid lowering therapy throughout the duration of the studies.
The 21B12 formulation will be presented as a sterile, clear, colorless frozen liquid.
Each sterile vial is filled with a 1-mL deliverable volume of 70 mg/mL 21B12 formulated
with 10 mM sodium acetate, 9% (w/v) sucrose, 0.004% (w/v) polysorbate 20, pH 5.2. Each
vial is for single use only. Placebo will be presented in identical containers as a clear,
colorless, sterile, protein-free frozen liquid and is formulated as 10 mM sodium acetate, 9%
(w/v) sucrose, 0.004% (w/v) polysorbate 20, pH 5.2.
In Part A, between 4-16 subjects will be enrolled and receive open label 21B12
formulation (420 mg Q4W). Study visits will occur every 4 weeks. These visits will entail
collection of adverse event (AE) and significant adverse event (SAE) data, vital signs,
concomitant medication, laboratory tests, etc. A fasting lipid panel will be collected at week
6 to assess the nadir LDL-C level in response to treatment with 21B12 formulation. The
21B12 formulation will be administered at day 1, week 4, and week 8. The end-of-study
(EOS) visit and the last estimation of lipids will occur at week 12.
Approximately 51 new subjects will be enrolled into Part B. Subjects enrolled will be
randomized to a 2:1 allocation into 2 treatment groups: 420 mg 21B12 Q4W SC or placebo
Q4W SC. Randomization will be stratified by baseline LDL-C levels. Study visits will occur
every 4 weeks, with two optional visits occurring at week 2 and week 10. Visits will entail
collection of AE and SAE data, vital signs, concomitant medication, laboratory tests, etc. A
fasting lipid panel will be collected at week 6 to assess the nadir LDL-C level in response to
21B12 treatment. 21B12 formulation will be administered at day 1, week 4, and week 8. The
end-of-study (EOS) visit and the last estimation of lipids will occur at week 12 for all
subjects.
Incorporation by Reference
All references cited herein, including patents, patent applications, papers, text books,
and the like, and the references cited therein, to the extent that they are not already, are
hereby incorporated herein by reference in their entirety. To the extent that any of the
definitions or terms provided in the references incorporated by reference differ from the
terms and discussion provided herein, the present terms and definitions control.
Equivalents
The foregoing written specification is considered to be sufficient to enable one skilled
in the art to practice the invention. The foregoing description and examples detail certain
preferred embodiments of the invention and describe the best mode contemplated by the
inventors. It will be appreciated, however, that no matter how detailed the foregoing may
appear in text, the invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any equivalents thereof.
In embodiments of the invention, there is provided a stable formulation comprising at
least one monoclonal antibody that specifically binds to PCSK9, wherein PCSK9 comprises
the amino acids of SEQ ID NO 1, the monoclonal antibody in an amount of about 40 mg/ml
to about 300 mg/ml, and a pharmaceutically acceptable buffer in an amount of about .05 mM
to about 40 mM, and a pharmaceutically acceptable surfactant in an amount that is about
.01% w/v to about 20% w/v, and at least one pharmaceutically acceptable stabilizer of about
0.5% w/v to about 10% w/v, wherein the stable formulation has a pH of between about 4.0 to
about 6.0.
In embodiments of the formulation, the monoclonal antibody comprises:
a) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 23 and a heavy chain variable
region that comprises and amino acid sequence that is at least 90%
identical to that of SEQ ID NO:49,
b) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO:465 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:463,
c) ,a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 461 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:459,
d) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 485 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:483; or
e) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO:582 and a heavy chain
variable region that comprises an amino acid sequence that is at least 90%
identical to that of SEQ ID NO:583.
In embodiments of the invention, there is provided a stable formulation, comprising
a. a monoclonal antibody in an amount of about 70 mg/ml to about 200
mg/ml, said monoclonal antibody comprising:
i) a light chain variable region that comprises the amino
acid sequence having at least 90% identity to the
sequence of SEQ ID NO: 577 and a heavy chain variable
region that comprises the amino acid sequence having at
least 90% identity to the sequence of SEQ ID NO: 576;
ii) a light chain variable region that comprises the amino
acid sequence of SEQ ID NO: 577 and a heavy chain
variable region that comprises the amino acid sequence of
SEQ ID NO:576;
iii) a light chain variable region that comprises the amino
acid sequence having at least 90% identity to the
sequence of SEQ ID NO: 588 and a heavy chain variable
region that comprises the amino acid sequence having at
least 90% identity to the sequence of SEQ ID NO: 599; or
iv) a light chain variable region that comprises the amino
acid sequence of SEQ ID NO: 588 and a heavy chain
variable region that comprises the amino acid sequence of
SEQ ID NO:589;
(b) about 10 mM sodium acetate;
(c) about 9.0% w/v sucrose;
(d) about 0.004% to about 0.01% w/v polysorbate 20 or polysorbate 80, and
(e) a pH of about 5.2.
In embodiments of the invention, there is provided a stable formulation comprising: a
monoclonal antibody in an amount of about 70 mg/ml to about 200 mg/ml, said monoclonal
antibody comprising:
i) a light chain variable region that comprises the amino
acid sequence having at least 90% identity to the
sequence of SEQ ID NO: 577 and a heavy chain variable
region that comprises the amino acid sequence having at
least 90% identity to the sequence of SEQ ID NO: 576;
ii) a light chain variable region that comprises the amino
acid sequence of SEQ ID NO: 577 and a heavy chain
variable region that comprises the amino acid sequence of
SEQ ID NO:576;
iii) a light chain variable region that comprises the amino
acid sequence having at least 90% identity to the
sequence of SEQ ID NO: 588 and a heavy chain variable
region that comprises the amino acid sequence having at
least 90% identity to the sequence of SEQ ID NO: 589; or
iv) a light chain variable region that comprises the amino
acid sequence of SEQ ID NO: 588 and a heavy chain
variable region that comprises the amino acid sequence of
SEQ ID NO:589,
(b) about 10 mM sodium acetate;
(c) between about 2.0% to 3.0% w/v proline;
(d) about 0.01% w/v polysorbate 20 or polysorbate 80, and
(e) a pH of about 5.0.
In embodiments of the invention, there is provided a method of lowering serum LDL
cholesterol in a patient comprising administering at least one anti-PCSK9 antibody to the
patient in need thereof at a dose of about 10 mg to about 3000 mg, thereby lowering said
serum LDL cholesterol level by at least about 15%.
In embodiments of the invention, there is provided a method of treating or preventing
a cholesterol related disorder in a patient having a serum LDL cholesterol level comprising
administering at least one anti-PCSK9 antibody to the patient in need thereof at a dose of
about 10 mg to about 3000 mg, thereby treating or preventing the cholesterol related disorder
in said patient.
In embodiments of the methods, the anti-PCSK9 antibody comprises,
(a) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 23 and a heavy chain variable
region that comprises and amino acid sequence that is at least 90% identical to
that of SEQ ID NO:49;
(b) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 12 and a heavy chain variable
region that comprises an amino acid sequence that is at least 90% identical to
that of SEQ ID NO:67;
(c) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 461 and a heavy chain variable
region that comprises an amino acid sequence that is at least 90% identical to
that of SEQ ID NO:459;
(d) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO:465 and a heavy chain variable
region that comprises an amino acid sequence that is at least 90% identical to
that of SEQ ID NO:463;
(e) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO: 485 and a heavy chain variable
region that comprises an amino acid sequence that is at least 90% identical to
that of SEQ ID NO:483; or
(f) a light chain variable region that comprises an amino acid sequence that is
at least 90% identical to that of SEQ ID NO:582 and a heavy chain variable
region that comprises an amino acid sequence that is at least 90% identical to
that of SEQ ID NO:583.
In embodiments of the methods, wherein the anti-PCSK9 antibody comprises,
(a) a light chain variable region that comprises an amino acid sequence, SEQ
ID NO: 23, and a heavy chain variable region that comprises and amino acid
sequence, SEQ ID NO:49;
(b) a light chain variable region that comprises an amino acid sequence, SEQ
ID NO: 12, and a heavy chain variable region that comprises an amino acid
sequence, SEQ ID NO:67;
(c) a light chain variable region that comprises amino acid sequence SEQ ID
NO: 461 and a heavy chain variable region that comprises amino acid
sequence SEQ ID NO:459;
(d) a light chain variable region that comprises the amino acid sequence of
SEQ ID NO:465 and a heavy chain variable region that comprises the amino
acid sequence of SEQ ID NO:463; or
(e) a light chain variable region that comprises the amino acid sequence of
SEQ ID NO: 485 and a heavy chain variable region that comprises the amino
acid sequence of SEQ ID NO:483; or
(f) a light chain variable region that comprises the amino acid sequence of
SEQ ID NO: 582 and a heavy chain variable region that comprises the amino
acid sequence of SEQ ID NO:583.
In embodiments of the methods, the anti-PCSK9 antibody is administered to a patient
at a dose selected from the group consisting of: a) about 45 mg to about 450 mg, b) about
140 mg to about 200 mg, c) about 140 mg to about 180 mg, d) about 140 mg to about 170
mg, e) about 140 mg, f) about 150 mg, g) about 420 mg, h) about 450 mg, i) about 600 mg,
j) about 700 mg, k) about 1400 mg, l) about 1200 mg, m) about 420 mg to about 3000 mg, n)
about 1000 mg to about 3000 mg, o) about 3000 mg.
In embodiments of the methods, the anti-PCSK9 antibody is administered to a patient
on a schedule selected from the group consisting of: (1) once a week, (2) once every two
weeks, (3) once a month, (4) once every other month, (5) once every three months (6)once
every six months and (7) once every twelve months.
In embodiments of the methods, the administering step comprises administering the at
least one anti-PCSK9 antibody parenterally.
In embodiments of the methods, the administering step comprises administering the at
least one anti-PCSK9 antibody intravenously.
In embodiments of the methods, the administering step comprises administering the at
least one anti-PCSK9 antibody subcutaneously.
In embodiments of the methods, the anti-PCSK9 antibody is administered to a patient
at a dose of about 105 mg to about 280 mg subcutaneously once every two weeks, and
wherein the serum LDL cholesterol level of the patient is lowered at least about 30 -50% for
about 7-14 days.
In embodiments of the methods, the anti-PCSK9 antibody is administered to a patient
at a dose of about 280 to about 450 mg subcutaneously once every month, and wherein the
serum LDL cholesterol level of the patient is lowered at least about 30 -50% for about 21 to
31 days.
In embodiments of the methods, the anti-PCSK9 antibody is administered to a patient
at a dose of about 150 mg subcutaneously once every other week wherein the serum LDL
cholesterol level of the patient is lowered at least about 30-50% for about 7-14 days.
In embodiments of the methods, the anti-PCSK9 antibody is administered to a patient
at a dose of about 150 mg subcutaneously once every four weeks wherein the serum LDL
cholesterol level of the patient is lowered at least about 30-50% for about 21-31 days.
In embodiments of the methods, the anti-PCSK9 antibody is administered to a patient
at a dose of about greater than 150 mg to about 200 mg subcutaneously once every four
weeks wherein the serum LDL cholesterol level of the patient is lowered at least about 30-
50% for about 21-31 days.
In embodiments of the methods, the at least one anti-PCSK9 antibody is administered
to the patient before, after or with at least one other cholesterol-lowering agent selected from
the group consisting of: statins, including, atorvastatin, cerivastatin, fluvastatin, lovastatin,
mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, Nicotinic acid, Fibric acid,
Bile acid sequestrants, Cholesterol absorption inhibitor, lipid modifying agents, PPAR
gamma agonists, PPAR alpha/gamma agonists, squalene synthase inhibitors, CETP
inhibitors, anti-hypertensives, anti-diabetic agents, including sulphonyl ureas, insulin, GLP-1
analogs, DDPIV inhibitors, ApoB modulators, MTP inhibitoris and /or arteriosclerosis
obliterans treatments, oncostatin M, estrogen, berbine and a therapeutic agent for an immune-
related disorder.
In embodiments of the methods, the at least one anti-PCSK9 antibody is administered
to the patient before, after or with at least one other cholesterol-lowering agent.
In embodiments of the methods, the at least one other cholesterol lowering agent is
selected from the group consisting of: statins, including, atorvastatin, cerivastatin, fluvastatin,
lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, Nicotinic acid,
Fibric acid, Bile acid sequestrants, Cholesterol absorption inhibitor, lipid modifying agents,
PPAR gamma agonists, PPAR alpha/gamma agonists, squalene synthase inhibitors, CETP
inhibitors, anti-hypertensives, anti-diabetic agents, including sulphonyl ureas, insulin, GLP-1
analogs, DDPIV inhibitors, ApoB modulators, MTP inhibitoris and /or arteriosclerosis
obliterans treatments, oncostatin M, estrogen, berbine and a therapeutic agent for an immune-
related disorder.
We
Claims (28)
1. Use of at least one monoclonal antibody that specifically binds to PCSK9, or a fragment of said monoclonal antibody that specifically binds to PCSK9, wherein PCSK9 comprises the amino acids of SEQ ID NO 1, in the manufacture of a medicament for lowering serum LDL cholesterol in a patient in need thereof and/or treating or preventing a cholesterol related disorder in a patient, wherein said medicament is capable of lowering said serum LDL cholesterol level by at least about 15%, wherein said medicament is to be administered to said patient at a dose of about 300 mg to about 480 mg of said monoclonal antibody or said fragment, wherein said medicament is to be administered every two to four weeks; and wherein said monoclonal antibody or said fragment comprises: (a) a light chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:23 and a heavy chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:49; or (b) a light chain complementarity determining region (CDR) of the CDRL1 sequence in SEQ ID NO:23, a CDRL2 of the CDRL2 sequence in SEQ ID NO:23, and a CDRL3 of the CDRL3 sequence in SEQ ID NO:23, and a heavy chain complementarity determining region (CDR) of the CDRH1 sequence in SEQ ID NO:49, a CDRH2 of the CDRH2 sequence in SEQ ID NO:49, and a CDRH3 of the CDRH3 sequence in SEQ ID NO:49.
2. The use according to claim 1, wherein said medicament is capable of lowering said serum LDL cholesterol level by an amount selected from the group consisting of a) at least about 30%, b) at least about 40%, c) at least about 50%, and d) at least about 60%.
3. The use according to any one of claims 1 to 2, wherein the cholesterol related disorder is familial hypercholesterolemia.
4. The use according to claim 3, wherein the familial hypercholesterolemia is heterozygous familial hypercholesterolemia or homozygous familial hypercholesterolemia.
5. The use according to any one of claims 1 to 4, wherein said monoclonal antibody or said fragment comprises a light chain variable region that comprises the amino acid sequence of SEQ ID NO:23 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:49.
6. The use according to any one of claims 1 to 5, wherein the said monoclonal antibody or said fragment comprises: (a) a light chain complementarity determining region (CDR) of the CDRL1 sequence in SEQ ID NO:23, a CDRL2 of the CDRL2 sequence in SEQ ID NO:23, and a CDRL3 of the CDRL3 sequence in SEQ ID NO:23, and a heavy chain complementarity determining region (CDR) of the CDRH1 sequence in SEQ ID NO:49, a CDRH2 of the CDRH2 sequence in SEQ ID NO:49, and a CDRH3 of the CDRH3 sequence in SEQ ID NO:49; or (b) a light chain complementarity determining region (CDR) CDRL1 comprising the amino acid sequence of SEQ ID NO: 158, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 162, a CDRL3 comprising the amino acid sequence of SEQ ID NO: 395; and a heavy chain complementarity determining region (CDR) CDRH1 comprising the amino acid sequence of SEQ ID NO: 368, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 175, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 180.
7. The use according to claim 6, wherein each CDR is defined in accordance with the Kabat definition, the Chothia definition, the combination of the Kabat definition and the Chothia definition, the AbM definition, or the contact definition of the CDR.
8. The use of any one of claims 1 to 7, wherein said monoclonal antibody comprises a human lambda light chain and a human IgG2 heavy chain.
9. The use according to any one of claims 1 to 8, wherein said medicament is to be administered to said patient parenterally.
10. The use according to claim 9, wherein said medicament is to be administered to said patient subcutaneously.
11. The use according to claim 9, wherein said medicament is to be administered to said patient intravenously.
12. The use according to any one of claims 1 to 11, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of 350 mg to 480 mg.
13. The use according to any one of claims 1 to 11, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 420 mg.
14. The use according to any one of claims 1 to 11, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 350 mg.
15. The use according to any one of claims 1 to 11, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 400 mg.
16. The use according to any one of claims 1 to 11, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 450 mg.
17. The use according to any one of claims 1 to 11, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 480 mg.
18. The use according to any one of claims 1 to 11, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 350 mg and wherein said medicament is capable of lowering said serum LDL cholesterol level by at least about 30-50% for about 24-28 days.
19. The use according to any one of claims 1 to 11, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 420 mg and wherein said medicament is capable of lowering said serum LDL cholesterol level by at least about 30-50% for about 24-28 days.
20. The use according to any one of claims 1 to 12, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 420 mg and wherein said medicament is capable of lowering said serum LDL cholesterol level by at least about 30-50% for about 7-10 days.
21. The use according to any one of claims 1 to 20, wherein said medicament is to be administered to said patient before, after or with at least one other cholesterol-lowering agent.
22. The use according to claim 21, wherein the at least one other cholesterol lowering agent is selected from the group consisting of: statins, including, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, Nicotinic acid, Fibric acid, Bile acid sequestrants, Cholesterol absorption inhibitor, lipid modifying agents, PPAR gamma agonists, PPAR alpha/gamma agonists, squalene synthase inhibitors, CETP inhibitors, anti-hypertensives, anti- diabetic agents, including sulphonyl ureas, insulin, GLP-1 analogs, DDPIV inhibitors, ApoB modulators, MTP inhibitors and /or arteriosclerosis obliterans treatments, oncostatin M, estrogen, berbine and a therapeutic agent for an immune-related disorder.
23. The use according to any one of claims 1 to 22, wherein the medicament is to be administered once every four weeks.
24. The use according to claim 1, wherein said medicament is to be administered to said patient at a dose of said monoclonal antibody or said fragment of about 420 mg subcutaneously once every four weeks and wherein said medicament is capable of lowering said serum LDL cholesterol level by at least about 30%.
25. The use according to claim 24, wherein said monoclonal antibody comprises: a light chain complementarity determining region (CDR) CDRL1 comprising the amino acid sequence of SEQ ID NO: 158, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 162, a CDRL3 comprising the amino acid sequence of SEQ ID NO: 395; and a heavy chain complementarity determining region (CDR) CDRH1 comprising the amino acid sequence of SEQ ID NO: 368, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 175, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 180.
26. The use according to any one of claims 1 to 25, wherein the monoclonal antibody or said fragment is in a stable formulation, wherein the stable formulation comprises: (a) about 10 mM to about 20 mM sodium acetate; (b) about 2.0% to about 3.0% w/v proline; (c) about 0.01% w/v polysorbate 20 or polysorbate 80, and and wherein the pH of the formulation is about 5.0.
27. The use according to claim 26, wherein stable formulation comprises about 100mg/ml to about 200mg/ml of the monoclonal antibody or said fragment thereof.
28. The use of any one of claims 1 to 27 substantially as herein described with reference to any one or more of the examples or figures but excluding comparative examples.
Applications Claiming Priority (11)
Application Number | Priority Date | Filing Date | Title |
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US201161484610P | 2011-05-10 | 2011-05-10 | |
US61/484,610 | 2011-05-10 | ||
US201161562303P | 2011-11-21 | 2011-11-21 | |
US61/562,303 | 2011-11-21 | ||
US201261595526P | 2012-02-06 | 2012-02-06 | |
US61/595,526 | 2012-02-06 | ||
US201261614417P | 2012-03-22 | 2012-03-22 | |
US61/614,417 | 2012-03-22 | ||
US201261642363P | 2012-05-03 | 2012-05-03 | |
US61/642,363 | 2012-05-03 | ||
NZ717550A NZ717550B2 (en) | 2011-05-10 | 2012-05-10 | Methods of treating or preventing cholesterol related disorders |
Publications (2)
Publication Number | Publication Date |
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NZ734570A NZ734570A (en) | 2021-11-26 |
NZ734570B2 true NZ734570B2 (en) | 2022-03-01 |
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