METHODS FOR TREATING HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA

PROCEDES DE TRAITEMENT D'UNE HYPERCHOLESTEROLEMIE FAMILIALE HOMOZYGOTE

English Abstract

The present invention relates to methods for treating homozygous familial hypercholesterolema using antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9).

French Abstract

La présente invention concerne des procédés de traitement d'une hypercholestérolémie familiale homozygote au moyen d'anticorps dirigés contre la proprotéine convertase subtilisine/kexine de type 9 (PCSK9).

Claims

What is claimed is:
1. Use of a pharmaceutical composition comprising a pharmaceutically
acceptable
carrier and an anti-PCSK9 antibody comprising 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, for lowering serum low-
density lipoprotein (LDL) cholesterol by at least 10% in a patient having
defective LDL
receptor activity comprising 2% to 25% LDL receptor function, wherein said
anti-
PCSK9 antibody is for use at a dose of 420 mg every two weeks.
2. Use of a pharmaceutical composition comprising a pharmaceutically
acceptable
carrier and an anti-PCSK9 antibody comprising 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, for lowering serum low-
density lipoprotein (LDL) cholesterol by at least 10% in a patient having
defective LDL
receptor activity comprising 2% to 25% LDL receptor function, wherein said
anti-
PCSK9 antibody is for use at a dose of 420 mg once a month.
3. The use of claim 1 or 2, wherein the serum LDL cholesterol level of said

patient is lowered by at least 15%, at least 20%, at least 30%, at least 40%,
or at least
50%.
4. The use of any one of claims 1 to 3, wherein the amino acid sequence of
CDRL1 is represented by SEQ ID NO: 158; the CDRL2 is represented by SEQ ID NO:
162; the
CDRL3 is represented by SEQ ID NO: 395, the CDRH1 is represented by SEQ ID
NO:308 or SEQ
ID NO:368; the CDRH2 is represented by SEQ ID NO:175; and the CDRH3 is
represented by SEQ
ID NO:180.
5. The use of any one of claims 1 to 4, wherein the anti-PCSK9 antibody
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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 of any one of claims 1 to 5, wherein the anti-PCSK9 antibody
further
comprises:
(a) the light chain constant sequence of SEQ ID NO: 156;
(b) the light chain constant sequence of SEQ ID NO: 157;
(c) the heavy chain constant sequence of SEQ ID NO: 154;
(d) the heavy chain constant sequence of SEQ ID NO: 155;
(e) the light chain constant sequence of SEQ ID NO: 156 and the heavy chain
constant
sequence of SEQ ID NO: 154;
(f) the light chain constant sequence of SEQ ID NO: 157 and the heavy chain
constant
sequence of SEQ ID NO: 154;
(g) the light chain constant sequence of SEQ ID NO: 156 and the heavy chain
constant
sequence of SEQ ID NO: 155; or
(h) the light chain constant sequence of SEQ ID NO: 157 and the heavy chain
constant
sequence of SEQ ID NO: 155.
7. The use of any one of claims 1 to 6, wherein the anti-PCSK9 antibody
further
comprises a glycine residue at the C-terminal end of the light chain variable
domain.
8. The use of any one of claims 1 to 4, wherein the anti-PCSK9 antibody
comprises a light chain that comprises the amino acid sequence of SEQ ID NO:
593 and
a heavy chain that comprises the amino acid sequence of SEQ ID NO: 592.
9. The use of any one of claims 1 to 4, wherein the anti-PCSK9 antibody
comprises a heavy chain variable region that comprises the amino acid sequence
of SEQ
ID NO: 590 and a light chain variable region that comprises the amino acid
sequence of
SEQ ID NO: 591.
10. The use of any one of claims 1 to 9, wherein the pharmaceutical
composition is
for use parenterally.
11. The use of any one of claims 1 to 9, wherein the pharmaceutical
composition is
for use intravenously.
190
Date Recu/Date Received 2021-10-13

12. The use of
any one of claims 1 to 9, wherein the pharmaceutical composition is
for use subcutaneously.
191
Date Recu/Date Received 2021-10-13

Description

METHODS FOR TREATING HOMOZYGOUS FAMILIAL
HYPERCHOLESTEROLEMIA
10
FIELD OF THE NVENTION
The present invention relates to methods for treatment of homozygous familial
hypercholesterolemia using antigen binding proteins, including antibodies,
against
proprotein convertase subtilisin/kexin type 9 (PCSK9).
BACKGROUND
Homozygous familial hypercholesterolemia is a rare, but serious clinical
disorder caused by substantial reduction in low density lipoprotein (LDL)
receptor
function. As a result, LDL cholesterol levels are severely elevated, leading
to
cardiovascular disease, and often death, in childhood (Goldstein JL, Hobbs HH,
Brown
MS, eds. Familial hypercholesterolemia. 8th Edition ed: McGraw-Hill; 2001).
Over
95% have a mutation in the LDL receptor, less than 4% in apolipoprotein B and
less
than 0.5% in proprotein convertase subtilisin/kexin 9 (PCSK9)(Abifadel M,
Varret M,
Rabes JP, et al. Mutations in PCSK9 cause autosomal dominant
hypercholesterolemia.
Nat Genet 2003;34:154-6; Rader DJ, Cohen J, Hobbs HH. Monogenic
hypercholesterolemia: new insights in pathogenesis and treatment. J Clin
Invest
2003;111:1795-803). While true genetic homozygous familial
hypercholesterolemia is
not uncommon, the majority of patients are compound heterozygotes (Usifo E,
Leigh
SE, Whittall RA, et al. Low-density lipoprotein receptor gene familial
hypercholesterolemia variant database: update and pathological assessment. Ann
Hurn
Genet 2012;76:387-401). The residual LDL receptor activity, either negative
(<2%
function) or defective (2% to 25% function), is associated with severity of
LDL
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cholesterol elevation and propensity for earlier cardiovascular disease
(Goldstein JL,
Hobbs HH, Brown MS, eds. Familial hypercholesterolemia. 8th Edition ed: McGraw-

Hill; 2001).
Response to conventional therapies, such as statins and ezetimibe, the most
commonly used drugs for homozygous familial hypercholesterolemia, is modest,
and
patients usually also require LDL apheresis when it is available. Reductions
in LDL
cholesterol with statins tend to correlate with LDL receptor function,
although receptor
negative patients have shown decreases (Raal FJ, Pappu AS, illingworth DR, et
al.
Inhibition of cholesterol synthesis by atorvastatin in homozygous familial
hypercholesterolaemia. Atherosclerosis 2000;150:421-8). The improvements in
LDL
cholesterol with statins appear to reduce cardiovascular disease morbidity and
mortality
(Raal FJ, Pilcher GJ, Panz VR, et al. Reduction in mortality in subjects with
homozygous familial hypercholesterolemia associated with advances in lipid-
lowering
therapy. Circulation 2011;124:2202-7). Recently two drugs, lomitapide and
.. mipomersen, which both reduce hepatic lipoprotein production, have been
approved
solely for the treatment of homozygous familial hypercholesterolemia. Even
with the
introduction of these two new drugs, there remains a need to identify new
methods for
treating patients diagnosed with homozygous familial hypercholesterolemia.
SUMMARY OF VARIOUS EMBODIMENTS
In some aspects, the invention provided comprises a method of lowering serum
LDL cholesterol in a patient diagnosed with homozygous familial
hypercholesterolemia
comprising administering at least one anti-PCSK9 antibody to the patient in
need
thereof at a dose of about 120 mg to about 3000 mg, thereby lowering said
scrum LDL
cholesterol level by at least about 10%, 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 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%, a least about 85%, or
at least
about 90% as compared to a predose level of serum LDL cholesterol in the
patient.
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In some embodiments of this aspect of the invention, the anti-PCSK9 antibody
is
administered to a patient diagnosed with homozygous familial
hypercholesterolemia at
a dose of about 140 mg to about 3000 mg, of about 140 mg to about 2800 mg, of
about
140 mg to about 2500 mg, of about 140 mg to about 2000 mg, of about 140 mg to
about 1800 mg, of about 140 mg to about 1400 mg, of about 120 mg to about 1200
mg,
of about 120 mg to about 1000 mg, of about 120 mg to about 700 mg, of about
140 mg
to about 700 mg, of about 140 mg to about 600 mg, of about 140 mg to about 450
mg,
of about 120 mg to about 450 mg, of about 120 mg to about 450 mg, of about 140
mg
to about 450 mg, of about 210 mg to about 450 mg, or of about 280 mg to about
450
mg, of about 210 mg to about 420 mg, of about 280 mg to about 420 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-PC SK9 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 ant-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) one or more heavy chain complementary determining regions
(CDRHs)
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selected from the group consisting of: (i) a CDRH1 from a CDRH1 in a sequence
selected from the group consisting of SEQ ID NO: 49, 67, 459, 463 and 483;
(ii) a
CDRH2 from a CDRH2 in a sequence selected from the group consisting of SEQ ID
NO: 49, 67, 459, 463 and 483; (iii) a CDRH3 from a CDRH3 in a sequence
selected
from the group consisting of SEQ ID NO: 49, 67, 459, 463 and 483; and (iv) a
CDRH
of (i), (ii), and (iii) that contains one or more amino acid substitutions,
deletions or
insertions of no more than 4 amino acids; B) one or more light chain
complementary
determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1
from
a CDRL1 in a sequence selected from the group consisting of SEQ ID NO: 23, 12,
461,
465, and 485; (ii) a CDRL2 from a CDRL2 in a sequence selected from the group
consisting of SEQ ID NO: 23, 12, 461, 465, and 485; (iii) a CDRL3 from a CDRL3
in a
sequence selected from the group consisting of SEQ ID NO: 23, 12, 461, 465,
and 485;
and (iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acid
substitutions,
deletions or insertions of no more than 4 amino acids; or C) one or more heavy
chain
CDRHs of A) and one or more light chain CDRLs of B). In some embodiments, the
isolated antigen binding protein comprises at least one CDRH of A) and at
least one
CDRL of B). In some embodiments, the isolated antigen binding protein
comprises at
least two CDRH of A) and at least two CDRL of B). In some embodiments, the
isolated antigen binding protein comprises at least three CDRH of A) and at
least three
CDRL of B).
In some embodiments, the isolated antigen binding protein comprises a light
chain complementarity 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 1D 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.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:465, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:465, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:465, and a heavy chain complementarity determining
region
(CDR) of the CDRH1 sequence in SEQ ID NO: 463, a CDRH2 of the CDRH2
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sequence in SEQ ID NO: 463, and a CDRH3 of the CDRH3 sequence in SEQ ID
NO:463.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:12, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:12, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:12, and a heavy chain complementarity determining region

(CDR) of the CDRH1 sequence in SEQ ID NO: 67, a CDRH2 of the CDRH2 sequence
in SEQ ID NO: 67, and a CDRH3 of the CDRH3 sequence in SEQ ID NO:67.
in some embodiments, the isolated antigen bindng protein comprises a light
chain complimentarity region (CDR) of the CDRL1 sequence in SEQ ID NO:461, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:461, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:461, and a heavy chain complementarity determining
region
(CDR) of the CDRH1 sequence in SEQ ID NO: 459, a CDRH2 of the CDRH2
sequence in SEQ ID NO: 459, and a CDRH3 of the CDRH3 sequence in SEQ ID
NO:459.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:485, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:485, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:485, and a heavy chain complementarity determining
region
(CDR) of the CDRH1 sequence in SEQ ID NO: 483, a CDRH2 of the CDRH2
sequence in SEQ ID NO: 483, and a CDRH3 of the CDRH3 sequence in SEQ ID
NO :483.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:582, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:582, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:582, and a heavy chain complementarity determining
region
(CDR) of the CDRH1 sequence in SEQ ID NO: 583, a CDRH2 of the CDRH2
sequence in SEQ ID NO: 583, and a CDRH3 of the CDRH3 sequence in 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
that is
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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 1D 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; 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; or a light chain
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variable region that comprises an amino acid sequence, SEQ ID NO:591, and a
heavy
chain variable region that comprises and amino acid sequence, SEQ ID NO:590.
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 a patient
diagnosed with homozygous familial hypercholesterolemia comprising
administering at
least one anti-PCSK9 antibody to the patient in need thereof at a dose of
about 120 mg
to about 3000 mg, thereby treating the homozygous familial
hypercholesterolemia in
said patient. In some embodiments of this aspect, the serum LDL cholesterol
level of
.. said patient diagnosed with homozygous familial hypercholesterolemia is
lowered by at
least about 10%, 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
comprises: A) one or more heavy chain complementary determining regions
(CDRHs)
selected from the group consisting of: (i) a CDRH1 from a CDRH1 in a sequence
selected from the group consisting of SEQ ID NO: 49, 67, 459, 463 and 483;
(ii) a
CDRH2 from a CDRH2 in a sequence selected from the group consisting of SEQ ID
NO: 49, 67, 459, 463 and 483; (iii) a CDRH3 from a CDRH3 in a sequence
selected
from the group consisting of SEQ ID NO: 49, 67, 459, 463 and 483; and (iv) a
CDRH
of (i), (ii), and (iii) that contains one or more amino acid substitutions,
deletions or
insertions of no more than 4 amino acids; B) one or more light chain
complementary
determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1
from
a CDRL1 in a sequence selected from the group consisting of SEQ ID NO: 23, 12,
461,
465, and 485; (ii) a CDRL2 from a CDRL2 in a sequence selected from the group
consisting of SEQ ID NO: 23, 12, 461, 465, and 485; (iii) a CDRL3 from a CDRL3
in a
sequence selected from the group consisting of SEQ ID NO: 23, 12, 461, 465,
and 485;
and (iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acid
substitutions,
deletions or insertions of no more than 4 amino acids; or C) one or more heavy
chain
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CDRHs of A) and one or more light chain CDRLs of B). In some embodiments, the
isolated antigen binding protein comprises at least one CDRH of A) and at
least one
CDRL of B). In some embodiments, the isolated antigen binding protein
comprises at
least two CDRH of A) and at least two CDRL of B). In some embodiments, the
isolated antigen binding protein comprises at least three CDRH of A) and at
least three
CDRL of B).
In some embodiments, the isolated antigen binding protein comprises a light
chain complementarity 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.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:465, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:465, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:465, and a heavy chain complementarity determining
region
(CDR) of the CDRH1 sequence in SEQ ID NO: 463, a CDRH2 of the CDRH2
sequence in SEQ ID NO: 463, and a CDRH3 of the CDRH3 sequence in SEQ ID
NO:463.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:12, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:12, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:12, and a heavy chain complementarity determining region

(CDR) of the CDRH1 sequence in SEQ ID NO: 67, a CDRH2 of the CDRH2 sequence
in SEQ ID NO: 67, and a CDRH3 of the CDRH3 sequence in SEQ ID NO:67.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complimentarity region (CDR) of the CDRL1 sequence in SEQ ID NO:461, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:461, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:461, and a heavy chain complementarity determining
region
(CDR) of the CDRH1 sequence in SEQ ID NO: 459, a CDRH2 of the CDRH2
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sequence in SEQ ID NO: 459, and a CDRH3 of the CDRH3 sequence in SEQ ID
NO:459.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:485, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:485, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:485, and a heavy chain complementarity determining
region
(CDR) of the CDRH1 sequence in SEQ ID NO: 483, a CDRH2 of the CDRH2
sequence in SEQ ID NO: 483, and a CDRH3 of the CDRH3 sequence in SEQ ID
NO:483.
In some embodiments, the isolated antigen bindng protein comprises a light
chain complementarity region (CDR) of the CDRL I sequence in SEQ ID 10:582, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:582, and a CDRL3 of the CDRL3
sequence in SEQ ID NO:582, and a heavy chain complementarity determining
region
(CDR) of the CDRH1 sequence in SEQ ID NO: 583, a CDRH2 of the CDRH2
sequence in SEQ ID NO: 583, and a CDRH3 of the CDRH3 sequence in 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
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
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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; 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; or a light chain

variable region that comprises an amino acid sequence, SEQ ID NO:591, and a
heavy
chain variable region that comprises and amino acid sequence, SEQ ID NO:590.
In
some embodiments of this aspect of the invention the anti-PCSK9 antibody is
selected
from the group consisting of 21B12, 31H4, 8A3, I IF1 and 8A1.
In particular embodiments the anti-PCSK9 antibody comprises a light chain
complementarity 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. 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

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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
comprises a light chain variable region that comprises the amino acid sequence
of SEQ
ID NO:591and a heavy chain variable region that comprises the amino acid
sequence of
SEQ ID NO:590. In some embodiments, the anti-PCSK9 antibody is 21B12. In a
particular embodiment wherein the anti-PCSK9 antibody comprises a light chain
complementarity 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 N0:23, 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 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 comprises a light chain
variable region that comprises the amino acid sequence of SEQ ID NO:591 and a
heavy
chain variable region that comprises the amino acid sequence of SEQ ID NO:590,
or
comprises the antibody is 21B12, the anti-PC SK9 antibody is administered to a
patient
at a dose of about 120 mg to about 450 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 120 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
140 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 120 mg to about 450 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
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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 350 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 420 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 a
light chain complementarity 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
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
12

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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
comprises a light chain variable region that comprises the amino acid sequence
of SEQ
ID NO:591and a heavy chain variable region that comprises the amino acid
sequence of
SEQ ID NO:590, or comprises the 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 3-10 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 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 15-50% 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 scrum 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
nag 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
13

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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 a
light chain complementarity 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
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 the
antibody is 21B12, the anti-PCSK9 antibody is administered to a patient at a
dose of
about 120 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 140 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 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
350 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
14

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administered to a patient at a dose of about 420 mg subcutaneously once every
other
week; is administered to a patient at a dose of about 280 mg to about 450 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 comprises a
light
chain variable region that comprises the amino acid sequence of SEQ ID NO:591
and a
heavy chain variable region that comprises the amino acid sequence of SEQ ID
NO :590, or comprises the 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-
10 days; is administered to a patient at a dose of about 700 mg intraveneously
every
week, wherein the scrum 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

CA 02916259 2015-12-19
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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
complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:465, a CDRL2
of the CDRL2 sequence in SEQ ID NO:465, and a CDRL3 of the CDRL3 sequence in
SEQ ID NO:465, and a heavy chain complementarity determining region (CDR) of
the
CDRH1 sequence in SEQ ID NO: 463, a CDRH2 of the CDRH2 sequence in SEQ ID
NO: 463, and a CDRH3 of the CDRI-13 sequence in SEQ ID NO:463. 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
16

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antibody is 11F1. In a particular embodiment, wherein the anti-PCSK9 antibody
comprises a light chain complementarity region (CDR) of the CDRL I sequence in
SEQ
ID NO:465, a CDRL2 of the CDRL2 sequence in SEQ ID NO:465, and a CDRL3 of
the CDRL3 sequence in SEQ ID NO:465, and a heavy chain complementarity
determining region (CDR) of the CDRH1 sequence in SEQ ID NO: 463, a CDRH2 of
the CDRH2 sequence in SEQ ID NO: 463, and a CDRH3 of the CDRH3 sequence in
SEQ ID NO:463, or 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 1D
NO:465 and a
heavy chain variable region that comprises the amino acid sequence of SEQ ID
NO:463, or comprises the antibody is 11F1, the anti-PCSK9 antibody is
administered
to a patient at a dose of about 150 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
17

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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 scrum
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
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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 a light chain complementarity region (CDR) of the CDRL1 sequence in
SEQ
ID NO:465, a CDRL2 of the CDRL2 sequence in SEQ ID NO:465, and a CDRL3 of
the CDRL3 sequence in SEQ ID NO:465, and a heavy chain complementarity
determining region (CDR) of the CDRH1 sequence in SEQ ID NO: 463, a CDRH2 of
the CDRH2 sequence in SEQ ID NO: 463, and a CDRH3 of the CDRH3 sequence in
SEQ ID NO:463. In some embodiments, the anti-PCSK9 antibody comprises, or
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 TD 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
comprises the antibody is 11F1, the anti-PCSK9 antibody is administered to a
patient at
a dose of about 150 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
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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 scrum 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
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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 a light chain complementarity region (CDR) of the CDRL1 sequence in
SEQ
ID NO:465, a CDRL2 of the CDRL2 sequence in SEQ ID NO:465, and a CDRL3 of
the CDRL3 sequence in SEQ ID NO:465, and a heavy chain complementarity
determining region (CDR) of the CDRI-11 sequence in SEQ ID NO: 463, a CDRH2 of
the CDRH2 sequence in SEQ ID NO: 463, and a CDRH3 of the CDRH3 sequence in
SEQ ID NO:463. In some embodiments, the anti-PCSK9 antibody comprises, or
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
the
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 scrum 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
21

patient is lowered 30-50% for about 10-14 days; is administered to a patient
at a dose
of about 1200 mg intravcneously 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-
NIACINTm),
laropiprant (CORDAPTIVETm), fibric acid (LOP1DTm (Gemfibrozil), TRICORTm
22
CA 2916259 2019-11-01

(fenofibrate)), Bile acid sequestrants, sucha as cholestyTamine (QUESTRANTNI),

colesvelam (WELCHOLTNI), COLESTIDTm (colestipol)), cholesterol absorption
inhibitor (ZETIATm (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 diagnosed with homozygous familial
hypercholesterolemia. The method comprises administering to a patient
diagnosed
with homozygous familial hypercholesterolemia a dose of about 120 mg to about
3000
mg of at least one anti-PCSK9 antibody described herein. In some embodiments,
the
dose is about 120 mg to about 450 rng of at least one anti-PCSK9 antibody
administered once weekly (QW). In some embodiments, the dose is about 140 mg
to
about 450 mg of at least one anti-PCSK9 antibody administered once weekly. In
some
embodiments, the dose is about 280 mg to about 450 mg of at least one anti-
PCSK9
antibody administered once weekly. In some embodiments, the dose is about120
mg to
about 450 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 450 mg of at
least
one anti-PCSK9 antibody administered once every 2 weeks (Q2W). In some
embodiments, the dose is about 280 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 400 mg to about 450 mg of at least one anti-PCSK9 antibody administered
once
every 2 weeks (Q2W). In some embodiments, the dose is about 420 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
23
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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-PC SK9 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 10% as compared to a predose
serum LDL
cholesterol level. In some embodiments, the serum LDL cholesterol level is
reduced by
at least about 15%. 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
scrum
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
24

CA 02916259 2015-12-19
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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 diagnosed with homozygous familial
hypercholesterolemia, 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 120 mg every week (QW); (2) at least an amount of about 140
mg
every week (QW); (3) at least an amount of about 120 mg every two weeks or
every
other week (Q2W); (4) at least an amount of about 140 mg every two weeks or
every
other week (Q2W); (5) at least an amount of about 150 mg every two weeks or
every
other week (Q2W) (6) at least an amount of about 280 mg every two weeks or
every
other week (Q2W); (7) at least an amount of about 350 mg every two weeks or
every
other week (Q2W); (8) at least an amount of about 420 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 10% as compared to a
predose serum
LDL cholesterol level. In some embodiments, the serum LDL cholesterol level is

reduced by at least about 10% as compared to a predose serum LDL cholesterol
level.
In some embodiments, the serum LDL cholesterol level is reduced by at least
about
15%. In some embodiments, the serum LDL cholesterol level is reduced by at
least

CA 02916259 2015-12-19
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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 the serum
PCSK9 level in a patient diagnosed with homozygous familial
hypercholesterolema,
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 120 mg
every week
(QW); (2) at least an amount of about 140 mg every week (QW); (3) at least an
amount
of about 120 mg every two weeks or every other week (Q2W); (4) at least an
amount of
about 140 mg every two weeks or every other week (Q2W); (5) at least an amount
of
about 150 mg every two weeks or every other week (Q2W) (6) at least an amount
of
about 280 mg every two weeks or every other week (Q2W); (7) at least an amount
of
about 350 mg every two weeks or every other week (Q2W); (8) at least an amount
of
about 420 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
26

CA 02916259 2015-12-19
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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 20% as compared to a predose serum PCSK9 level. In some embodiments, the

serum PCSK9 value is reduced by at least about 30%. In some embodiments, the
serum PCSK9 value is reduced by at least about 40%. In some embodiments, the
serum PCSK9 value is reduced by at least about 50%. 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 some aspects, the invention comprises a method of lowering the total
cholesterol level in a patient diagnosed with homozygous familial
hypercholesterolemia, 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 120 mg every week (QW); (2) at least an amount of about
140 mg
every week (QW); (3) at least an amount of about 120 mg every two weeks or
every
other week (Q2W); (4) at least an amount of about 140 mg every two weeks or
every
other week (Q2W); (5) at least an amount of about 150 mg every two weeks or
every
other week (Q2W) (6) at least an amount of about 280 mg every two weeks or
every
other week (Q2W); (7) at least an amount of about 350 mg every two weeks or
every
other week (Q2W); (8) at least an amount of about 420 mg every two weeks or
every
other week (Q2W); and (9) at least an amount of about 150 mg every four weeks
27

CA 02916259 2015-12-19
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(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 20% 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 diagnosed with homozygous familial
hypercholesterolemia, 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 120 mg every week (QW); (2) at least an amount of about 140
mg
every week (QW); (3) at least an amount of about 120 mg every two weeks or
every
other week (Q2W); (4) at least an amount of about 140 mg every two weeks or
every
other week (Q2W); (5) at least an amount of about 150 mg every two weeks or
every
other week (Q2W) (6) at least an amount of about 280 mg every two weeks or
every
other week (Q2W); (7) at least an amount of about 350 mg every two weeks or
every
other week (Q2W); (8) at least an amount of about 420 mg every two weeks or
every
28

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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 non-
HDL cholesterol level is reduced by at least about 30% as compared to a
predose non-
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 diagnosed with homozygous familial hypercholesterolemia, 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 120 mg every week
(QW); (2)
at least an amount of about 140 mg every week (QW); (3) at least an amount of
about
120 mg every two weeks or every other week (Q2W); (4) at least an amount of
about
140 mg every two weeks or every other week (Q2W); (5) at least an amount of
about
29

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150 mg every two weeks or every other week (Q2W) (6) at least an amount of
about
280 mg every two weeks or every other week (Q2W); (7) at least an amount of
about
350 mg every two weeks or every other week (Q2W); (8) at least an amount of
about
420 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 ApoB level is reduced by at least about 10% as
compared to
a predose ApoB level. In some embodiments, the ApoB level is reduced by at
least
about 15%. In some embodiments, the ApoB level is reduced by at least about
20%.
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 LS 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 diagnosed with homozygous familial
hypercholesterolemia,
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 120 mg
every week

CA 02916259 2015-12-19
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(QW); (2) at least an amount of about 140 mg every week (QW); (3) at least an
amount
of about 120 mg every two weeks or every other week (Q2W); (4) at least an
amount of
about 140 mg every two weeks or every other week (Q2W); (5) at least an amount
of
about 150 mg every two weeks or every other week (Q2W) (6) at least an amount
of
about 280 mg every two weeks or every other week (Q2W); (7) at least an amount
of
about 350 mg every two weeks or every other week (Q2W); (8) at least an amount
of
about 420 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 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 a patient
diagnosed
with homozyougous familial hypercholesterolemia, the method comprising
administering
to a patient diagnosed with homozygous familial hypercholesterolemia a dose of
about
120 mg to about 3000 mg of at least one anti-PCSK9 antibody described herein.
In
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some embodiments, the dose is about 120 mg to about 450 mg of at least one
anti-
PCSK9 antibody administered once weekly (QW). In some embodiments, the dose is

about 140 mg to about 450 mg of at least one anti-PCSK9 antibody administered
once
weekly. In some embodiments, the dose is about 120 mg to about 450 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 420 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-PC SK9 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 400 mg to about 450 mg of at least one anti-PC SK9 antibody
administered
once every two weeks (Q2W). In some embodiments, the dose is about 420 mg of
at
least one anti-PCSK9 antibody administered once every two weeks (Q2W). In some

embodiments, the dose is about 120 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 420 mg of at least one anti-PC SK9 antibody
administered
once every four weeks (Q4W). In some embodiments, the dose is about 400 mg to
about 450 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-PC SK9 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
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about 10% as compared to a predose serum LDL cholesterol level. In some
embodiments, the serum LDL cholesterol level is reduced by at least about 15%.
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.
Other embodiments of this invention will be readily apparent from the
disclosure provided herewith.
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BRIEF DESCRIPTION OF THE FIGURES
FIG. lA depicts an amino acid sequence of the mature form of the PCSK9 with
the pro-domain underlined.
FIGs. 1B1-1B4 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. FIG. 2C continues the sequence started in FIG. 2A.
FIG. 2D
continues the sequence started on FIG. 2B.
FIGs. 3A-3D are sequence comparison tables of various heavy chains of various
antigen binding proteins. FIG. 3C continues the sequence started in FIG. 3A.
FIG. 3D
continues the sequence started on FIG. 3B.
FIGs. 3E-3JJ depict the amino acid and nucleic acid sequences for the variable

domains of some embodiments of the antigen binding proteins.
FIG. 3KK 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.
FIG. 3LLL is a set of ABP sequences identifying various differences between
the human ABP sequences and the ABP sequences that were raised in E. co/i..
(U.S.P.
8,030,457).
FIG. 4A is a binding curve of an antigen binding protein to human PCSK9.
FIG. 4B is a binding curve of an antigen binding protein to human PCSK9.
FIG. 4C is a binding curve of an antigen binding protein to cynomolgus PCSK9.
FIG. 4D is a binding curve of an antigen binding protein to cynomolgus
PCSK9.
FIG. 4E is a binding curve of an antigen binding protein to mouse PCSK9.
FIG. 4F is a binding curve of an antigen binding protein to mouse PCSK9.
FIG. 5A depicts the results of an SDS PAGE experiment involving PCSK9 and
various antigen binding proteins demonstrating the relative purity and
concentration of
the proteins.
34

FIG. 5B and 5C depict graphs from Biacore solution equilibrium assays for
21B12.
FIG. 5D depicts the graph of the kinetics from a Biacore capture assay.
FIG. 6A is an inhibition curve of antigen binding protein 31H4 IgG2 to PCSK9
in an in vitro PCSK9:LDLR binding assay
FIG. 6B is an inhibition curve of antigen binding protein 3IH4 IgG4 to PCSK9
in an in vitro PCSK9:LDLR binding assay.
FIG. 6C is an inhibition curve of antigen binding protein 21B12 IgG2 to PCSK9
in an in vitro PCSK9:LDLR binding assay.
FIG, 6D is an inhibition curve of antigen binding protein 21B12 IgG4 to
PCSK9 in an in vitro PCSK9:LDLR binding assay.
FIG. 7A 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
FIG. 78 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
FIG. 7C is an inhibition curve of antigen binding protein 211312 IgG2 in the
cell
LDL uptake assay showing the effect of the ABP to reduce the LDL uptake
blocking
effects of PCSK9
FIG. 7D 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
FIG. 8A is a graph depicting the serum cholesterol lowering ability in mice of
ABP 31H4 (shown by A), changes relative to the IgG control treated mice (shown
by
M1) (* p< 0.01).
FIG. 8B is a graph depicting the serum cholesterol lowering ability in mice of
ABP 31114, changes relative to time = zero hours (# p, 0.05).
FIG. 8C is a graph depicting the effect of ABP 31H4 on HDL cholesterol levels
in C57131/6 mice (* p< 0.01).
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FIG. 9 depicts a western blot analysis of the ability of ABP 31H4 to enhance
the
amount of liver LDLR protein present after various time points.
FIG. 10A is a graph depicting the ability of an antigen binding protein 31H4
to
lower total serum cholesterol in wild type mice, relative.
FIG. 10B is a graph depicting the ability of an antigen binding protein 31H4
to
lower HDL in wild type mice.
FIG. 10C is a graph depicting the serum cholesterol lowering ability of
various
antigen binding proteins 31H4 and 16F12.
FIG. 11A depicts an injection protocol for testing the duration and ability of
antigen binding proteins to lower serum cholesterol.
FIG. 11B is a graph depicting the results of the protocol in FIG. 11A.
FIG. 12A depicts LDLR levels in response to the combination of a statin and
ABP 21B12 in HepG2 cells.
FIG. 12B depicts LDLR levels in response to the combination of a statin and
ABP 31H4 in HepG2 cells.
FIG. 12C 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.
FIG. 12D depicts LDLR levels in response to the combination of a statin and
ABP 21B12 in HepG2 cells over expressing PCSK9.
FIG. 12E depicts LDLR levels in response to the combination of a statin and
ABP 31H4 in HepG2 cells over expressing PCSK9.
FIG. 12F 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.
FIG. 13A depicts the various light chain amino acid sequences of various ABPs
to PCSK9. The dots 0 indicate no amino acid.
FIG. 13B depicts a light chain cladogram for various ABPs to PCSK9.
FIG. 13C depicts the various heavy chain amino acid sequences of various
ABPs to PCSK9. The dots 0 indicate no amino acid.
FIG. 13D depicts a heavy chain dendrogram for various ABPs to PCSK9.
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FIG. 13E depicts a comparison of light and heavy CDRs and designation of
groups from which to derive consensus.
FIG. 13F depicts the consensus sequences for Groups 1 and 2.
FIG. 13G depicts the consensus sequences for Groups 3 and 4.
FIG. 13H depicts the consensus sequences for Groups 1 and 2. The dots (.)
indicated identical residues.
FIG. 131 depicts the consensus sequences for Group 2. The dots (.) indicated
identical residues.
FIG. 131 depicts the consensus sequences for Groups 3 and 4. The dots (.)
indicated identical residues.
FIG. 14 is a graph illustrating the binding specificity of 11F1 in a
competition
assay with PCSKP, PCSK2, PCSK1 PCSK7 and Furin with 0D450 plotted on the
vertical axis and concentration of PCSK9 ug/ml) plotted on the horizontal
axis.
FIG. 15 is a graph showing the dose response curve for inhibition of
LDLR:D374Y PCSK9 binding by 11F1 in a competition assay with 0D450 plotted on
the vertical axis and Log [1 1F1] (pM) plotted on the horizontal axis.
FIG. 16is a graph depicting the dose response curve for the inhibition of
LDLR:
WT PCSK9 binding by 11Flin a competition assay with 0D450 plotted on the
vertical
axis and Log [11f1] (pM) plotted on the horizontal axis.
FIG. 17 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 (x104) plotted on the vertical axis and Log [11F]]
(nM)
plotted on the horizontal axis.
FIG. 18 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 (x104) on the vertical axis and Log [11F1]
(nM)
plotted on the horizontal axis.
FIG. 19 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.
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FIG. 20 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.
FIG. 21 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/m1)
on the vertical axis and time following injection (days) plotted on the
horizontal axis.
FIG. 22 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.
FIG. 23 is a table summarizing PK parameters for IgG2, 11F1 and 8A3 in mice
expressing human PCSK9 by AAV.
FIG. 24 is a graph depicting the effect of a single subcutaneous
administration
of an ant-KLH antibody (control), 21B12, 8A3 and 11F1 on serum LDL
concentration
(LDL-C) in cynomolgus monkeys with LDL-C (mg/di) plotted on the vertical axis
and
time following administration in days on the horizontal axis.
FIG. 25 is a graph depicting the effect of a single subcutaneous
administration
of an ant-KLH antibody (control), 21B12, 8A3 and 11F1 on Serum Total
Cholesterol in
cynomolgus monkeys with Total Cholesterol concentration (mg/di) plotted on the

vertical axis and time following administration in days on the horizontal
axis.
FIG. 26 is a graph depicting the effect of a single subcutaneous
administration
of an ant-KLH antibody (control), 21B12, 8A3 and 11F1 on Scrum HDL Cholesterol

in cynomolgus monkeys with HDL-C (mg/di) plotted on the vertical axis and time
following administration in days on the horizontal axis.
FIG. 27 is a graph depicting the effect of a single subcutaneous
administration
of an ant-ICLH antibody (control), 21B12, 8A3 and 11F1 on Serum Triglycerides
in
cynomolgus monkeys with Serum Triglyceride concentration (mg/di) plotted on
the
vertical axis and time following administration in days on the horizontal
axis.
FIG. 28 is a graph depicting the effect of a single subcutaneous
administration
of an ant-KLH antibody (control), 21B12, 8A3 and 11F1 on Apolipoprotein B
(ApoB)
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in cynomolgus monkeys with APOB concentration (mg/di) plotted on the vertical
axis
and time following administration in days on the horizontal axis.
FIG. 29 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.
FIG. 30 is a table summarizing PK parameters for the anti--KLH antibody
(control), 21B12, 8A3 and 11F1 in cynomolgus monkeys.
FIG. 31A 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.
FIG. 31B 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
Provided herein are methods of treating a patient diagnosed with homozygous
familial hypercholesterolemia are also included, said method comprises
administering
at least one antigen binding protein, including antibodies, against proprotein
convertase
subtilisin/kexin type 9 (PCSK9) to the patient. Moreover, methods of lowering
serum
LDL cholesterol in a patient diagnosed with homozygous familial
hypercholesterolemia
using antigen binding proteins, including antibodies, against proprotein
convertase
subtilisin/kexin type 9 (PCSK9) are provided herein.
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
39

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. 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 kcxin type 9" or "PCSK9" refers to
a
polypeptide as set forth in SEQ ID NO: I 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
interspccies 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,
FICHOLA3, 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"
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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 Vase 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
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regulated by nutritional status via insulin and sterol regulatory element-
binding protein
lc" 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 "homozygous familial hypercholesterolemia" or "HoFE" as used
herein, refers a cholesterol-related disorder that is determined by genetic
confirmation
or clinical diagnosis (such as history of an untreated LDL-cholesterol
concentration
greater than 13 rnmolfL plus either xanthoma before 10 years of age or
evidence of
heterozygous familial hypercholesterolaemia in both parents).
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
internucleofide 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. Oligonucicotides 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
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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
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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 tetin "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
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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

CA 02916259 2015-12-19
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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 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 .1
Applied
Math. 481073.
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
46

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 etal., 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)). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino
acids,
unnatural amino acids such as
47
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a-, a-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,
y-carboxyglutamate, E-N,N,N-trimethyllysine, a-N-acetyllysine, 0-
phosphoserine, N-
acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, cr-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: norlcucinc, Met, Ala, Val, Leu,11e;
2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
3) acidic: Asp, Glu;
4) basic: His, Lys, Arg;
5) 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
48

CA 02916259 2015-12-19
WO 2014/209384 PCT/US2013/048714
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); setine (-0.8); tryptophan (-
0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartatc (-
3.5); &sparagine (-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.
Mot. 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 immunogcnicity 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
49

CA 02916259 2015-12-18
WO 2014/209384 PCT/US2013/048714
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
'Ot1gj,n4LRO.iatkniMt::*tiitOta':OI$tihSlitHt:jfihkMilgttae1*.aStiklltlliiki.iC
i
MEINOMMEMEMMERPRIONOIMPARENNIMii
Ala Val, Leu, Ile Val
Arg Lys, Gin, Asn Lys
ASTI Gin Gin
Asp Glu Glu
Cys Ser, Ala Ser
Gin Asn Asn
Glu Asp Asp
Gly Pro, Ala Ala
His Asn, Gin, 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, Gin, Asn
Met Leu, Phe, Ile Leu
Leu, Val, Ile, Ala,
Phe Leu
Tyr
Pro Ala Gly
Ser Thr, Ala, Cys Thr

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WO 2014/209384 PCT/US2013/048714
teetigi#4040:011c8NE :]K#11100y.*400-4-orogi eigeff110.! 40.0q$4010
Thr Ser Ser
Trp Tyr, Phe Tyr
Tyr Trp, Phe, Thr, Ser Phe
Ile, Met, Len, Phe,
Val L eu
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
51

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.
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, .1., Adv. Drug Res., 15:29 (1986); Veber & Freidinger, TINS, p.392
(1985);
and Evans et al., J, Med. Chem., 30:1229 (1987. Such compounds are often
developed
with the aid of computerized molecular modeling. Peptide mimeties 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: --CH2 NH--, --CH2 S--
, --CH2 -
CH2 --, --CH=CH-(cis and trans), --COCH2 --
CH(011)CH2 --, and --CH2 SO--, by
methods well 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)); 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
52
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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 arc 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
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antibody (e.g., 25A7 versus 21B12). 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). Unless otherwise stated, the ABP
name is
understood to be a generic designation denoting an antibody.
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. Moreover, examples of
antibodies are
provided in e.g., U.S.P. 8,030,457, U.S.P. 8,168,762. Further examples of
antibodies
are provided in e.g., U.S.P. 8,188,233, U.S.P. 8,188,234, U.S.P. 8,080,243,
U.S.P.
8,062,640, WO 2008/06332, WO 2009/055783, WO 2011/053759, WO 2012/054438,
WO 2012/088313, W02012/109530, and WO 2013/039958.
An "Fe" region comprises two heavy chain fragments comprising the C111 and
CH2 domains of an antibody. The two heavy chain fragments are held together by
two
or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
A "Fab fragment" comprises one light chain and the CH1 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 VII domain and the CH 1 domain and also the region between
the CHI
and CH2 domains, such that an interchain disulfide bond can be formed between
the
two heavy chains of two Fab' fragments to form an F(alf)2 molecule.
A "F(ab')2 fragment" contains two light chains and two heavy chains containing

a portion of the constant region between the CH 1 and CH2 domains, such that
an
interchain disulfide bond is formed between the two heavy chains. A F(ab)2
fragment
54

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,
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 VH regions are covalently joined
with a
peptide linker to create a bivalent domain antibody. The two VH 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
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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 (I(d) is <lir M. The ABP specifically binds antigen
with
"high affinity" when the Kd is <5 X le M, and with "very high affinity" when
the Kd is
<5x 10-1 M. In one embodiment, the ABP has a Kd of <10-9 M. In one
embodiment,
the off-rate is <1 x 10-5. In other embodiments, the ABPs will bind to human
PCSK9
with a Kd of between about IV M and 10-13 M, and in yet another embodiment the

ABPs will bind with a Kd <5 x 10-10. 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-3.1.11, 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);
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WO 2014/209384 PCT/US2013/048714
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,
minibodics,
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 cornposed 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-
57

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, 1gG, IsA,
and IgE,
respectively. IgG has several subclasses, including, but not limited to, IgGI,
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,
IgAl 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)).
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 FRI, 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 etal., 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
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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 etal., 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); "AbM1N, 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 Li, L2, and L3 and are numbered sequentially in the direction
from the
amino terminus to the carboxy teiminus.
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The term "light chain" includes a full-length iiit chain and fragments thereof

having sufficient variable region sequence to confer binding specificity. A
full-length
light chain includes a variable region domain, VL, and a constant region
domain, CL.
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, VH, and three constant
region
domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the
polypeptide, and the CH domains are at the carboxyl-terminus, with the CH3
being
closest to the carboxy-terminus of the polypeptide. Heavy chains can be of any
isotype,
including igG (including IgG 1, IgG2, IgG3 and IgG4 subtypes), IgA (including
IgAl
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 etal., 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 arc the basic units of
which
antibody polypeptides are composed. In humans, the IgA and 1gD isotypcs
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 Cul, C112 and C113. The antibodies that are provided
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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 IC50 or EC50 value. In some embodiments, 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
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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 (ETA), 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.
Immunot
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 1-125 label (see,
e.g., Morel et
al., 1988, Malec. Irnmunol. 25:7-15); solid phase direct biotin-avidin ETA
(see, e.g.,
Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA
(Moldenhauer et
al., 1990, Scand. J. Innnunol. 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
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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 arc
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.
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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: radioisotopes or radionuclides (e.g., 3H, 14C,
15N, 35s, 90y,
99Tc, 111In, 1251, 1310, fluorescent labels (e.g., FITC, rhodamine, lanthanide
phosphors),
enzymatic labels (e.g., horseradish peroxidasc, f3-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
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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)).
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 NO: 1 in FIG.
IA (depicting the "pro" domain of the protein as underlined) and SEQ ID NO:3
in FIG.
1B (depicting the signal sequence in bold and the pro domain underlined).
An
exemplary human PCSK9 coding sequence is presented as SEQ ID NO: 2 (FIG. 1B).
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.,
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Structure, 15:1-8, 2007). 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 used in the methods provided herein. In some embodiments, the antigen
binding
proteins 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 that can be used in the methods 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
useful
in the methods provided herein 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 cilia,
interactions
between PCSK9 and LDLR and other physiological effects mediated by PCSK9. In
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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 disclosed antigen binding proteins that are useful in the methods provided

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.
In some embodiments, the antigen binding proteins that are useful in the
methods provided herein 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
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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')2, or a scFv). The various structures are further described and
defined
herein.
Certain of the antigen binding proteins that are useful in the methods
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 FIG. lA (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 piotein, with or
without a
signal sequence.
ln 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,
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the ABP has a Kd of less (binding more tightly) than le, 10-8, le, 10-10, 10 -
ii, 10-12,
10-13M. In some embodiments, the ABP has an IC50 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, lOnM 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, FIG. 31(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, FIG. 3KK.
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, FIG. 3KK.
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, FIG. 3KK.
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: FRI, 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-3JJJ and 3LLL. In some embodiments, each
of
these variable regions can be attached to the above heavy and light chain
constant

CA 02916259 2015-12-18
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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
SEQ ID NO
!11)kijt164oiiBiESMMUiibtiBiiiiie
30A4 s 5/74'
3C4 7/85
23B5 9/71
2564 10/72
31114 12/67
27B2 13/87
25A7 15/58
27115 16/52
26115 17/51
31D1 18/53
20D10 19/48
27E7 20/54
30B9 21/55
19119 22/56
26E10 23/49
21B12 23/49
21B12v1 591/590
17C2 24/57
2364 26/50
13111 28/91
9C9 30/64
9H6 31/62
31A4 32/89
1Al2 33/65
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16F12 35/79
22E2 36/80
27A6 37/76
28E112 38/77
28D6 39/78
31G11 40/83
13B5 42/69
31B12 44/81
3136 46/60
5H5 421/419
24F7 425/423
22B11 429/427
30F1 433/431
24B9.1 437/435
24139.2 441/439
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
Again, each of 8A1 485/483 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
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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 3KKK and 13A-13J). 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 FIG. 2A-2D, 3A-3D, and other embodiments in 3CCC-3JJJ and 3LLL
and 13A-13J) 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 3LLL and 13A-J). 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, 30, 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 FIG. 2A-
3D and
FIG. 3CCC-3JJJ and 3LLL 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 FIG. 2C has the same FR1, CDR1, and FR2 as 19H9 in FIG.

2A as the only difference is noted in FIG. 2C. 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.
73

Indeed, in sonic embodiments, only one or neither of the specific heavy or
light chain
nucleic acids need be present.
In some embodiments, the antibodies useful in the methods described herein
include the antibodies provided in U.S.P. 8,030,457 or U.S.P. 8,168,762.
Further
examples of antibodies that are useful in the methods described herein are
provided in
e.g., U.S.P. 8,188,233, U.S.P. 8,188,234, U.S.P. 8,080,243, U.S.P. 8,062,640,
WO
2008/06332, WO 2009/055783, WO 2011/053759, WO 2012/054438, WO
2012/088313, W02012/109530, and WO 2013/039958.
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, 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). 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
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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 at. 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. hi
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, 1000-10,000 fold or more to the mutant than the wild type
(as
measured via a Ku). 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 1050). 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

CA 02916259 2015-12-19
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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 FIG. lA and/or FIG. 1B. 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 FIG. IA and/or FIG. 1B. In some embodiments,
the ABP binds to and prevents variants of PCSK9 that arc 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 FIG. 1 A and/or FIG. 1B 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 FIG. 1B 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., Antibodies1
As described herein, an antigen binding protein to PCSK9 can comprise a
humanized antibody and/or part thereof. An in 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
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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
5,869,619.
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 etal., 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 humanind 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
Inimunol 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
77

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, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101, and in
Jones et
at, 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).
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 complementarily 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
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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.
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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 FIGs 2A-3JJ and 3LL-JJJ and 3LLL 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) and FIGs 2A-3JJ and 3LL-JJJ and 3LLL. 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
FIG.
2A-3D (and 13A-13J, other embodiments in FIGs. 31A and 31B) 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, 131, 13J, and/or 31A and 31B and variations are allowed in the
positions identified as variable in the figures. The CDRs shown in FIGs. 13A,
13C,
13F, 13G, 31A and 31B 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 31A and 31B). The CDRs in FIGs. 13H, 131, and 13J were obtained by
the
Kabat method alone. Unless specified otherwise, the defined consensus
sequences,

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CDRs, and FRs in FIGs. 13H-13J will define and control the noted CDRs and FRs
for
the referenced ABPs in FIG. 13.
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, 60, 419, 423, 427, 431, 435, 439, 443, 447, 451,
455, 459,
463, 467, 471, 475, 479, and 483. 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, 60, 419, 423, 427,
431, 435,
439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, and 483. 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, 60, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459,
463, 467,
471, 475, 479, and 483.
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, 60,
419, 423, 427,
431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, and 483. 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: 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
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embodiments, 1, 2, 3, or 4 FR (each being at least 90 A, 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, 46, 421, 425, 429, 433, 437, 441, 445, 49, 453, 457,
461, 465,
469, 473, 477, 481, and 485. 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, 41, and 46. In certain
embodiments,
an antigen binding protein comprises a light chain cornprising 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, 46,
421, 425, 429, 433, 437, 441, 445, 49, 453, 457, 461, 465, 469, 473, 477, 481,
and 485.
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, 46, 421,
425, 429, 433,
437, 441, 445, 49, 453, 457, 461, 465, 469, 473, 477, 481, and 485 . 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, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 421, 425,
429, 433,
437, 441, 445, 49, 453, 457, 461, 465, 469, 473, 477, 481, and 485. 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.
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In light of the present disclosure, a skilled artisan will be able to
deteimine
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.
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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
84

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).
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
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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, 296,
418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446,
448, 450,
452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480,
482, and 484
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, 296, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442,
444, 446,
448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476,
478, 480,
482, and 484.
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, 151, 296, 418, 420, 422, 424, 426, 428, 430, 432,
434, 436,
438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466,
468, 470,
472, 474, 476, 478, 480, 482, and 484) 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,
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CA 02916259 2015-12-19
WO 2014/209384 PCT/US2013/048714
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. 0., 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
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CA 02916259 2015-12-19
WO 2014/209384 PCT/US2013/048714
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 fowl of PCSK9 shown in FIG. 1A,
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. WO
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
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WO 2014/209384 PCT/US2013/048714
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. 5,885,793. In certain embodiments, phage arc
"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
Proc Natl Acad Sci (USA), 87: 8095-8099 (1990).
89

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).
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., Jimmunol. 160: 2051-2058 (1998); Carballido etal., 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.

lmmunol., 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.,
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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 phagc 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. See also Mendez et al., Nature
Genetics,
15:146-156 (1997).
Through the use of such technology, fully human monoclonal antibodies to a
variety of antigens have bccn 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
91
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such cell lines, including nucleotide and amino acid sequence analyses of the
heavy and
light chains of such antibodies.
The production of the XenoMousee 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,
tiled April 27, 1995, 08/464,584, tiled 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 5,939,598 and Japanese Patent
Nos. 3
068 180 B2, 3 068 506 12, and 3 068 507 B2. See also European Patent No., EP 0
463
151 BI, 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.
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 VII genes, one or more Du genes, one or more in genes, a mu
constant
region, and 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. 5,545,807 to Surani et al. and U.S. Patent Nos. 5,545,806,
5,625,825,
5,625,126, 5,633,425, 5,661,016, 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. 5,643,763 to Choi & Dunn, and
92
CA 2916259 2019-11-01

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. 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. 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 etal., (1996).
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.
Additionally, KM1'm 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
Genpharrn mice (Ishida etal., 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 IgGI. The antibodies possessed high affinities, typically
possessing
a Kd of from about 10-6 through about 10-13 M or below, when measured by
various
techniques.
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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. 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 transfeetion, 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 arc 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 or other cell lines. Cell lines or 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 or
the following hybridomas: 21B12, 31H4, 16F12, any of 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 (KO 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 imtnunoglobulin
molecule of at least one of the 1g61, IgG2, Ig63, IgG4, 1g 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 IgGI, IgG2, IgG3, IgG4, lgE, IgA, 1gD, or IgM isotype. In certain
embodiments,
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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 IgG 1, 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 IgGI, igG3, IgE, IgA, 1gD or 1gM 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: 21612, 3164 and I6F12. In addition,
certain
other modifications to the heavy and light chains of the antibodies from at
least one of
the hybridoma lines: 21612, 31H4 and 16F12 (and corresponding modificatiosn 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 (such as, for example, SEQ ID NOS:592-593). 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 insertions or substitutions (whether conservative or non-
conservative) can be determined by those skilled in the art at the time such
insertions or
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. 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
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hamster ovary (CHO) cells, HcLa 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 (lnvitrogen, 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 (PharMingenTm, San Diego, CA). In certain embodiments,
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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-SepharoseTM 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
etal., 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 NaC1, 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,
pll 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
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SDS polyacrylamide gel (See, e.g., Sambrook et al., supra). In certain
embodiments,
such a gel can be soaked in 0.4 M KC1 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 polypcptides 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
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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.
100

In certain instances, the electrophoretic migration of a polypeptide can vary,

sometimes significantly, with different conditions of SDS/PAGE. See, e.g.,
Capaldi et
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 useful in the methods provided herein
bind are described. 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 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.)
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 (sec, e.g., Nanevicz, T., etal., 1995õ1 Biol. Chem., 270:37, 21619-
21625 and
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Zupnick, A., et al., 2006, 1 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), EC50, 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
900/0 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
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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, 10, or 244) of the following mutations:
R207E,
D208R, R185E, R439E, E513R, V53 8R, E539R, Ti 32R, S351R, A390R, A413R,
E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E,
H521R, and Q554R as compared to a wild-type 1 CSK9 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: lor 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.
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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, 1132R,
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,
S3511., 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 TD 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
cpitopc, 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
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embodiments can be compared to an allelic sequence, rather than purely the
wild-type
sequence shown in FIG. lA
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. 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 3114.
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.
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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
arc 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.
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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
Provided herein are pharmaceutical formulations containing antigen binding
proteins to PCSK9 that are useful in the described methods. 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
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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 2-8 C 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 ("H1AC") and/or
visual
inspection.
In certain embodiments, the pharmaceutical formulation comprises any of the
antigen binding proteins to PCSK9 comprising: one or more heavy chain
complementary determining regions (CDRHs) and one or more light chain
complementary determining regions (CDRLs) depicted in Table 2 and FIGs. 2
and/or 3
and FIGS. 31A and 31B. In
certain other embodiments, the pharmaceutical
formulation comprises an antigen binding protein to PCSK9 comprising: a light
chain
variable region that comprises an amino acid sequence that is at least 90%
identical the
antigen binding proteins to PCSK9 depicted in Table 2 and FIGs. 2 and/or 3 and
FIGs
31A and 31B, and a heavy chain variable region that comprises and amino acid
sequence that is at least 90% identical to that of any of the antigen binding
proteins to
PCSK9 depicted in Table 2 and FIGs. 2 and/or 3 and FIGs 31A and 31B. In still
other
embodiments, the pharmaceutical foimulation comprises any of the antigen
binding
108

proteins to PCSK9 depicted in Table 2 and FIGs. 2 and/or 3 and FIGs 31A and
31B. In
certain other 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, 311-14, 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.
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,e. 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
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phosphate ("Na0AC"), Tris-HC1, 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, sucroseõ 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 polypcptides; salt-forming counter
ions
(such as sodium); preservatives (such as benzalkonium chloride, benzoic acid,
salicylic
acid, thirnerosal, 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, trornethamine, 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, 18th 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
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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/m1 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
Na0AC. 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 folutulation 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,
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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 duIcitol) 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 10% sorbital. In certain
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embodiments, the pharmaceutical foititulation 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 30 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 c13-1 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 aspect, 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 2-8 C 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 sarnple. 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
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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 Subvisiblc Particle Detection by Light Obscuration ("HIAC"). An
electronic,
liquid-borne particle-counting system (HIAC/Royco 9703 or equivalent)
containing a
light-obscuration sensor (H1AC/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
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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 urn. In the case of HIAC, a stable pharmaceutical formulation
should
exhibit no more than 6000 101.tm 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, 101.1m particles per container (or unit) as compared to a
control
sample. In still other embodiments, a stable pharmaceutical formulation should
exhibit no more than 600 251.1m 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 25ptm 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/nil of antigen binding protein
to
PCSK9; (ii) about 0.05 nriM to about 40 mM of a buffer such as sodium acetate
("Na0AC") 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
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formulation has a pH in the range of about 4.0 to 6Ø 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 250 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 250 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 250
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, rnethionine, 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/m1 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 foimulations of this invention comprise (i) at
least about
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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
founulation 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 20 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
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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 homozygous familial
hypercholesterolemia. In
certain embodiments, 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, parentcrally
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
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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 thrmulated 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 stcarate, 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
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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(-)-3-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
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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 appropiate 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 comprising: one or more heavy
chain complementary determining regions (CDRHs) and one or more light chain
complementary determining regions (CDRLs) depicted in Table 2 and FIGs. 2
and/or 3
and FIGS. 31A and 31B can be administered to a patient diagnosed with
homozygous
familial hypercholesterolemia according to the methods of the present
invention. In
certain other embodiments, antigen binding protein to PCSK9 comprising: a
light
chain variable region that comprises an amino acid sequence that is at least
90%
identical the antigen binding proteins to PCSK9 depicted in Table 2 and FIGs.
2 and/or
3 and FIGs 31A and 31B, and a heavy chain variable region that comprises and
amino
acid sequence that is at least 90% identical to that of any of the antigen
binding proteins
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to PCSK9 depicted in Table 2 and FIGs. 2 and/or 3 and FIGs 31A and 31B can be
administered to a patient diagnosed with homozygous familial
hypercholesterolemia
according to the methods of the present invention. In still other embodiments,
any of
the antigen binding proteins to PCSK9 depicted in Table 2 and FIGs. 2 and/or 3
and/or
13 and/or FIG.3 lA and 31B can be administered to a patient diagnosed with
homozygous familial hypercholesterolemia according to the methods of the
present
invention. In certain other embodiments, 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 can be administered to a patient
diagnosed with homozygous familial hypercholesterolemia. In some embodiments
any
one of 21B12, 26H5, 31H4, 8A3, 11F1 or 8A1 can be administered to a patient
diagnosed with homozygous familial hypercholesterolemia.
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 jig/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 jig/kg up to about 100 mg/kg; or 1 jig/kg up to
about 100
mg/kg; or 5 jig/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 120 mg to about 3000 mg, of about 140 mg
to
about 2800 mg, of about 140 mg to about 2500 mg, of about 140 mg to about 2000
mg,
of about 140 mg to about 1800 mg, of about 140 mg to about 1400 mg, of about
120
mg to about 1200 mg, of about 120 mg to about 1000 mg, of about 120 mg to
about
700 mg, of about 140 mg to about 700 mg, of about 140 mg to about 600 mg, of
about
140 mg to about 450 mg, of about 120 mg to about 450 mg, of about 120 mg to
about
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450 mg, of about 140 mg to about 450 mg, of about 210 mg to about 450 mg, or
of
about 280 mg to about 450 mg, of about 210 mg to about 420 mg, of about 280 mg
to
about 420 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 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
subcutancious 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 an 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.
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In certain embodiments, a dose of at least about 120 mg; or up to about 140
mg;
or up to about 210 mg; or up to about 280 mg; or up to about 350 mg, or up to
about
420 mg, or up to about 450 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 120 mg, or up to about 140
mg; or up to about 150 mg, or up to about 210 mg, or up to about 280 mg; or up
to
about 350 mg, or up to about 420 mg; or up to about 450 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 about 600 mg; or up to about 700
mg; or
up to about 1000 mg; or up to about 2000 mg; or up to about 3000 mg every four
weeks
of a an antigen binding protein to PCSK9 is administered once every four
weeks, (or
once a month)(Q4W), to a patient in need thereof.
In certain other embodiments, a dose of at least about 400 mg; or up to about
420 mg; or up to about 450 mg; or up to about 600 mg; or up to about 700 mg;
or up to
about 1000 mg; or up to about 2000 mg; or up to about 3000 mg every other
month of a
an antigen binding protein to PCSK9 is administered once every 8 weeks, (or
once
every other month), to a patient in need thereof.
In some embodiments, the serum LDL cholesterol level is reduced by at least
about 10%, as compared to a predose serum LDL cholesterol level. In some
embodiments, the serum LDL cholesterol level is reduced by at least about
15%.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
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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 10%, 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 10 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 15%, 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 10 days, at least about 14 days, at least about 21 days,
at least about
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 20%, 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
20 days, at least about 10 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 25%, 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
25 days, at least about 10 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 30%, 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 10 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.
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In some embodiments, the serum LDL cholesterol level is reduced by at least
about 35%, 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 10 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 10 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 10 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 10 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 10 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 10 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.
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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 10 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 10 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 10 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 10 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 10 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 10 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.
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Certain Therapeutic Applications
As will be appreciated by one of skill in the art, the methods provided herein

are for treatment of patients diagnosed with homozygous familial
hypercholesterolemia
using antigen binding proteins, including antibodies, against proprotein
convertase
subtilisin/kexin type 9 (PCSK9).
In one aspect, an antigen binding protein to PCSK9 is used to modulate serum
LDL cholesterol levels in a patient diagnosed with homozygous familial
hypercholesterolema. In some embodiments, the antigen binding protein to PCSK9
is
used to decrease the amount of scrum 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 10% as compared to a predose level. In certain
embodiments,
the serum LDL cholesterol level is reduced by at least about 15%. In certain
embodiments, the serum LDL cholesterol level is reduced by at least about 20%.
In
certain embodiments, the serum LDL cholesterol level is reduced by at least
about
.. 25%. 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 scrum 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 diagnosed with homozygous familial
hypercholesterolema.
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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 20% as compared to a predose
level.
.. In some embodiments, the serum PCSK9 value is reduced by at least about
25%. In
some embodiments, the serum PCSK9 value is reduced by at least about 30%. In
some
embodiments, the serum PCSK9 value is reduced by at least about 35%. In some
embodiments, the serum PCSK9 value is reduced by at least about 40%. In some
embodiments, the serum PCSK9 value is reduced by at least about 45%. In some
embodiments, the serum PCSK9 value is reduced by at least about 50%. In some
embodiments, the serum PCSK9 value is reduced by at least about 55%. 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 diagnosed with homozygous familial
hypercholesterolemia. 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 hielh level or
even a
normal level. In some embodiments, the total cholesterol level is reduced by
at least
about 20% as compared to a predose 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
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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 diagnosed with homozygous familial
hypercholesterolemia. 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-H DL 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 diagnosed with homozygous familial
hypercholesterolemia. 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 10% as compared to a predose
level. In
some embodiments, the ApoB level is reduced by at least about 15%. In some
embodiments, the ApoB level is reduced by at least about 20%. 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
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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 motein to PCSK9 is used to modulate the
Lp(a) levels in a patient diagnosed with homozygous familial
hypercholesterolemia. 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 10% as compared to a predose
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%.
Combination Therapies
In certain embodiments, methods are provided of treating
homozygous familial hypercholesterolemia, 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,
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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) (NIACORTM, NIASPANTM (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 (ZETIAT" (ezetimibe)), Combining nicotinic
acid
with statin (ADVICORTM (LOVASTATINTm and NIASPAN), Combining a statin with
an absorption inhibitor (VYTORINT" (ZOCORTM 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,
DDPI V inhibitors, e.g., metaforrnin), 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 eytokines 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.
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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. It is noted that the ABP names are used
generically
herein (e.g., "21B12" can be used as outlined in Example 7, as in Table 2, or
as in
Figures 2 and/or 3 and/or 13A, 13C, 13F-13J), unless explicitly denoted
otherwise.
EXAMPLE 1
Immunization and Titering
Generation of Anti-PCSK9 Antibodies and Hybridomas
Antibodies to the mature form of PCSK9 (depicted as the sequence in FIG. 1A,
with the pro-domain underlined), were raised in XenoMouseg 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 IgG2X, 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 XenoMouseg
strain XMG4-KL, which produce fully human IgG4õ and lgG4k 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 jig 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 TiterMaxg
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 jig of antigen in the adjuvant alum gel. A
final injection
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of 5 p.g of antigen per mouse is delivered in Phospho buffered saline and
delivered into
2 sites 50% IF into the abdomen and 50% SQ at the base of tail. The
immunization
programs are summarized in Table 1.1, shown below.
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TABLE 1.1
mouse strain XMG2/k1 XMG4/k1
# of animals 10 10
immunogen PCSK9-V5/His PCSK9-V5/His
1st boost IP injection IP injection
bug each bug 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/m1 (50u1/well) and
incubated at 4 C in 1XPBS/0.05% azide overnight. They were washed using
TiterTek
3-cycle wash with RO water. Plates were blocked using 250u1 of 1XPBS/1%milk
and
135

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 (0, 2ug/m1 in
I XPBS/1%milk/lOmM Ca2+ (assay diluent) 50u1/well and incubated for I hr 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
50u1/well and incubated for I hr at RT. One then washed using TiterTek 3-cycle
wash
with RO water. The secondary antibody was goat anti Human IgG Fe HRP @, 400
ng/ml in assay diluent at 50u1/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 (NeogenTM, Lexington, Kentucky) was
used
(50u1/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 (CorningTM Life Sciences) were employed. The plates
were
coated with neutravadin at 8 pg/m1 in 1XPBS/0.05%Azide, (50 l/well). The
plates were
incubated at 4 C 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 1,11 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 tig/m1 in I XPBS/1%milk/lOmM Ca2+ (40 ul/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 pi/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/l0mM Ca21- (50 .ii/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 p.1/well) was added to the plate and was
quenched
with IN hydrochloric acid (50111/w-ell) after 30 minutes at room temperature.
OD's were
read immediately at 450 nm using a Titertek plate reader.
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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/m1 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 Fe at a concentration of 400 ng/m1 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 [tg/m1 in
1XPBS/0.05%Azide,
(50 11well). The plates were incubated at 4 C 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 I 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 pg/m1 in 1XPBS/1%milk/1OmM Ca2I (40
1/we11).
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 Fe HRP at

400 ng/ml in 1XPBS/1%milk/l0mM Ca2 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 IN hydrochloric acid (50 I/well) after 30 minutes at room
temperature. OD's were read immediately at 450 rim using a Titertek plate
reader.
Positive control was LDLR, rabbit anti-PCSK9 titrated 1:3 in duplicate from 3
g/m1 in assay diluent. LDLR detect with goat anti-LDLR (R&D Systems, Cat
#AF2148) and rabbit anti-goat IgG Fe HRP at a concentration of 400 ng/ml;
rabbit poly
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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 p.g/m1
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 XM64-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 1.2) were selected for harvest, and splenocytes and lymphocytes were
isolated
from the spleens and lymph nodes respectively, as described herein.
TABLE 1.2
Summary of ELISA Results
Titer Titer
Animal b-hu PCSK9 b-hu PCSK9 @
ID (V5His) @ 2ug/m1 2ug/m1
P175807 >72900 @ OD 2.2 68359
P175808 >72900 @ OD 2.3 >72900 @ OD 2.5
P175818 >72900 @ OD 3.2 >72900 @ OD 3.0
P175819 >72900 @ OD 3.4 >72900 @ OD 3.2
Group 1 - P175820 >72900 @ OD 2.4 >72900 @ OD 2.5
IgG2k/I P175821 >72900 @00 3.4 >72900 @ OD 3.0
P175830 >72900 @ OD 2.6 >72900 @ OD 2.5
P175831 >72900 @ OD 3.1 >72900 @ OD 3.1
P175832 >72900 @ OD 3.8 >72900 @ OD 3.6
P175833 >72900 @ OD 2.6 >72900 @ OD 2.3
P174501 19369 17109
P174503 31616 23548
P174508 48472 30996
P174509 23380 21628
Group 2 - P174510 15120 9673
IgG4k/I P175773 19407 15973
P175774 54580 44424
P175775 60713 55667
P175776 30871 22899
P175777 16068 12532
Naïve
G2 < 100 @ OD 0.54 < 100 @ OD 0.48
Naïve <100 @ OD 1.57 <100 @ OD 1.32
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Titer Titer
Animal b-hu PCSK9 b-hu PCSK9 @
ID (V5His) @ 2ugiml 2ugiml
G4
EX.A.MPLE 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
resu.spend 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/DMS0 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, penistrep, MEM non-essential amino acids (all from Invitrogen),
was then
added over 2 minutes with gentle agitation. Finally preheated IDMEM (8 ml per
106
B-cells) was added over 3 minutes.
The fused cells were spun down 400 x g 6 mm 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-Azaserin.e Hypoxanthine and OPT
(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
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resuspended in 200 ml selection media and cultured for 3-4 days in T175 flask
pi ior to
96 well plating. Thus, hybridomas that produced antigen binding proteins to
PCSK9
were produced.
EXAMPLE 3
Selection of PCSK9 Antibodies
The present exarrtple 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.
BlAcore (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/m1 in
1XPBS/0.05%Azide, at a volume of 40 p.1/well. The plates were incubated at 4 C
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
I
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/l0mM Ca2+ 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 I of supernatant was transferred
into 40 p,1
of 1XPBS/1%milk/10mM Ca2+ and incubated 1.5 hours at room temperature. Again
the plates were washed using the Titertek plate washer operated using a 3-
cycle wash.
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40 l/well of Goat anti-Human IgG Fc POD at a concentration of 100 nglml
(1:4000) in
IXPBS/1 Amilk/lOmM Ca2+ 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 (Coming Life Sciences) were
employed.
The plates were coated with neutravadin at 3 g/m1 in 1XPBS/0.05%Azide at a
volume
of 40 l/well. The plates were incubated at 4 C 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 .1 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/m1 in 1XPBS/1%milk/10mM Ca2I
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 1 of
supernatant
was transferred into 40 I of 1XPBS/1%milc/ 10mM Ca2 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 1.11/well of Goat anti-Human IgG Fc POD at a

concentration of 100 ng/ml (1:4000) in 1XPBS/1%milk/l0mM Ca2' was added to the

plate, and the plate was incubated 1 hour at mom temperature. The plates were
washed
.. once again, using the Titertek plate washer operated using a 3-cycle wash.
Finally, 40
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l/well of One-step TMB (Neogen, Lexington, Kentucky) was added to the plate
and
was quenched with 40 1/well of IN 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 (Coming Life Sciences) were employed. The
plates were coated with neutravadin at 3 g/m1 in 1XPBS/0.05%Azide at a volume
of
40 l/well. The plates were incubated at 4 C 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 I 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/m1 in 1XPBS/1%milk/lOmM Ca2+
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 I of supernatant was transferred to the plates and
incubated I
hour at room temperature. Again the plates were washed using a 3-cycle wash.
40
l/well of Goat anti-Human IgG Fe POD at a concentration of 100 ng/ml (1:4000)
in
1XPBS/1%milk/10mM Ca2+ 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 .1/well of 1N hydrochloric acid after 30
minutes at
room temperature. OD's were read immediately at 450 nm using a Titcrtek plate
reader. 579 antibodies were observed to cross-react with mouse PCSK9. These
antibodies were then used in the subsequent screenings.
142

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: CostarTM 3702 medium binding 384 well plates (Corning Life Sciences)
were
employed in the screen. The plates were coated with neutravadin at 4 1.1.g/m1
in
1XPBS/0.05% Azide at a volume of 40 ul/well. The plates were incubated at 4 C
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
1
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 ug/m1 in 1 XPBS/1%milk/lOmMCa2+ 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/Ca2 (10 ml plus 40 ml) and incubated for I 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 lug/m1 in I XPBS/1%milldl OmMCa2+ 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 Pc HRP at a concentration of 100 ng/ml (1:4000) in
IXPBS/1%milk/l0mM Ca2+ was added to the plate and the plate was incubated 1
hour
at room temperature. 40 H.1/well of Goat anti-rabbit IgG Fc HRP at a
concentration of
100 ng/ml (1:4000) in IXPBS/1%milk/10mM Ca' was added to the plate and the
plate
was incubated I hour at room temperature. The plates were then washed using a
Titertek plate washer. A 3-cycle wash was performed. Finally, 40 pl/well of
One-step
TMB (Neogen, Lexington, Kentucky) was added to the plate and was quenched with
40
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.t1/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 jig/m1 in 1XPBS/0.05%Azide at a volume
of
40 IA/well The plates were incubated at 4 C 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 pl 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 #21.48LD/CF), and was added at 0.4 jig/m.1 in 1. XPBS/1%milk/1.0mM
at a volume of 40 gl/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 plhArell 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 pi/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 1/well of One-step TMB (Neogen, Lexington, Kentucky) was added to the
plate
and was quenched with 40 Owe11 of 1N hydrochloric acid after 30 minutes at
room
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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
Mocker subset assay as was done in the large scale receptor ligand blocking
screen.
This repeat screen confiiiited 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 pg/ml in 1XPBS/0.05% Azide at a volume of 40 1.11/well.
The
plates were incubated at 4 C 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 IA 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,
#21.48LD) was added at 5 vg/m1 in 1.X.PBS/1. %milk/10mM Ca2+ at a volume of 40
til/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.
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Contemporaneously, biotinylated human wild-type PCSK9 was pre-incubated with
hybridoma exhaust supernatant in Nunc polypropylene plates. 22 p.1 of
hybridoma sup
was transferred into 33u1 of b-PCSK9 at a concentration of 583 ng/ml in
1XPBS/1%milk/lOmMCa2+, 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 p1/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 pi/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 p.1/well of One-step TMB (Neogen, Lexington,
Kentucky) was added to the plate and was quenched with 40 p.1/well of IN
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
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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 50m1 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 1x106 cells per ml from the 31H4 hybridoma
line
was placed into the small chamber of an Integra flask in Integra media (see
Table 4.1
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 (0.22mn). 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 Scpharose column. Non-specific bindings were removed by an
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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 7.8-8Ø Antibody was eluted with a

NaC1 gradient of 10 mM-500 mM in 25 column volumes.
TABLE 4.1
Composition of Media
INTEGRA MEDIA
HSFM
10% Ultra Low IgG serum
2mmo1/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
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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
7.8-8Ø The antibody was eluted with a Nan gradient of 10 mM-500 mM in 25
column volumes.
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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 5). 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 lx106 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 lam).
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 inIVI Citrate, pH 3.0). Aggregated
antibody
pioteins 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 21612 proteins
are Q-
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Sepharose HP at pH 7.8-8Ø The antibody was eluted with a NaC1 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 NaCI 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 (didcoxy) 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 and 3LL-
BBB.
The cDNA sequences for the lambda light chain variable regions of 3114,
21B12, and 16F12 were determined and are disclosed as SEQ ID NOs: 153, 95, and

105 respectively.
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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 FIG.
3KK.
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 arc disclosed as SEQ ID
NOs:
12, 23, and 35 respectively, the lambda light chain constant region (SEQ 1D
NO: 156),
the heavy chain variable regions of 3114, 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 and FIG. 3CCC-3JJJ.
Based on the sequence data, the gerniline 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 FIG.
3CCC-3JJJ and each is represented by a unique SEQ ID NO. FIGs. 2A-3D and FIG.
3CCC-3JJJ also depict the determined amino acid sequences for additional
antibodies
that were characterized.
EXAMPLE 8
Production of Antibodies in E. coli
Some of the antibodies were also raised in E. coli and possess some minor
amino acid differences from antibodies produced as in, for example, Examples 4-
6.
The first residue in the variable region was a glutamic acid instead of a
glutamine for
the heavy and light chains of 21B12 and for the light chain for 31H4. in
addition to the
differences in the sequence of variable region, there were also some
differences in the
constant region of the antibodies described by the coordinates (due to the
fact that the
antibody was raised in E. coli). FIG. 3LLL highlights (via underlining
shading, or
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bold) the differences between the constant regions of the 21B12, 31H4, and
31A4 Fabs
(raised in E. coli) when compared to SEQ ID NOs: 156, and 155. For 21B12 31H4,

and 31A4, the light chain constant sequence is similar to human lambda (SEQ ID
NO:
156). The underlined glycine residue is an insertion between where the 21B12
and
31H4 variable sequences stop and the lambda sequence starts.
For both 21B12 and 31H4, the heavy chain constant is similar to human IgG4
(SEQ ID NO: 155). The highlighted differences in FIG. 3LLL are shown in Table
8.1:
Table 8.1
Crystal SEQ ID NO: 155
20
In regard to 31A4, while it also has the same distinctions noted above, there
are
three additional differences. As shown in FIG. 3LLL, there are two additional
amino
acids at the start, which comes from incomplete processing of the signal
peptide in E.
coli expression. In addition, there is one additional substitution in the 31A4
heavy
chain constant region when compared to SEQ ID NO: 155, which is the adjustment
of a
L (in SEQ ID NO: 155) to a H. Finally, 31A4 does have a glutamine as the
initial
amino acid of the Fab, rather than the the adjustment to glutamic acid noted
above for
21B12 and 31H4.
For all three antibodies, the end of the heavy chain (boxed in dark grey)
differs
as well, but the amino acids are not ordered in the structure so they do not
appear in the
cooridnates. As will be appreciated by one of skill in the art, his-tags are
not a required
part of the ABP and should not be considered as part of the ABP's sequence,
unless
explicitly called out by reference to a specific SEQ ID NO that includes a
histidinc tag
and a statement that the ABP sequence "includes the Histidine tag."
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EXAMPLE 9
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 KiinExA affinity analysis was performed. The
samples
and buffers employed in these studies are presented in Table 9.1 below.
TABLE 9.1
[sample] [sample]
sample mg/m1 Buffer pm
hPCSK9 1.26 PBS 16.6
mPCSK9-8xHIS 1.44 PBS 18.9
cPCSK9-V5-6xHIS 0.22 PBS 2.9
20mM Na0AC, pH
16F12, anti-PCSK9 huIgG4 4.6 5.2, 50mM NaCI 31.9
10mM NAOAC, pH
21B12, anti-PCSK9 huIgG4 3.84 5.2, 9% Sucrose 27.0
10mM NAOAC, pH
31H4, anti-PCSK9 huIgG4 3.3 5.2, 9% Sucrose 22.9
BIA core 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
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immobilized antibody surface (at a flow rate of 100 pl/min for 1.5 minutes).
Fresh
HBS-P buffer (pH 7.4, 0.01 M Hepes, 0.15 M NaC1, 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
NaC1, 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 (KD) 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 ka (association rate constant),
ki
(dissociation rate constant), and KD (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 KD values determined by the BIAcore affinity analysis are
presented
in Table 9.2, shown below.
TABLE 9.2
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
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Table 9.3 depicts the koo and koff rates.
TABLE 9.3
s-1) Kat(ki) KD
31H4.1, pH 7.4 2.45 e+5 5.348 e-5 210 pM
31H4.1,pH6 5.536e+6 6.936e-5 12.5 pM
21B12.1, pH 7.4 3.4918c14 6.634e-6 190 pM
21B12.1, pH 6 2.291 e+6 1.676e-5 7.3 pM
16F12.1, pH 7.4 1.064e+5 4,983e-5 470 pM
16F12.1, pH 6 2.392 e+6 7.007 e-5 29 pM
KinExA Affinity Measurements
A KinFxAc (Sapidyne Instruments, Inc., Boise, ID) affinity analysis of 16F12
and 31H4 was performed according to the manufacturer's instructions. Briefly,
Reacti-
GelTM (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 (KD) 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
157

antibodies tested using the KinExA e system, antibody 31H4 showed higher
affinity to
both human and cyno PCSK9 with 3 and 2 pM Kt), respectively. 16F12 showed
slightly
weaker affinity at 15pM Kp to human PCSK9 and 16 pM KD to cyno PCSK9.
The results of the KinExA affinity analysis are summarized in Table 9.4,
shown below.
TABLE 9.4
hPCSK9 cPCSK mPCSK
Sample KD (pM) 95% CI KD (pM) 95% CI KD (pM) 95% Cl
31H4.1 3 1-5 2 1-3 500 400-620'
In addition, a SDS PAGE was run to check the quality and quantity of the
samples and is shown in FIG. 5A. cPCSK9 showed around 50% less on the gel and
also from the active binding concentration calculated from KinExA assay.
Therefore,
the Kt) of the mAbs to cPCSK9 was adjusted as 50% of the active cPCSK9 in the
present.
A BlAcoreTM solution equilibrium binding assay was used to measure the Kd
values for ABP 211312. 211312.1 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/m1 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, KD was calculated
from
three sets of curves (0.3, 1 and 3nM fixed PCSK9 concentration) using a one-
site
homogeneous binding model in KinExA ProTM software. Although cPCSK9 has lower
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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
Kt). The results are displayed in Table 9.5 below and in FIGs. 5B-5D. FIG. 5B
depicts
the results from the solution equilibrium assay at three different hPCSK9
concentrations for hPCSK9. FIG. 5C depicts a similar set of results for
mPCSK9. FIG.
5D depicts the results from the above biacore capture assay.
TABLE 9.5
hPCSK9 cPCSK mPCSK
Sample Ka (pM) 95% Cl KD (pM) 95% CI KD (pM) 95% Cl
21612.1 15 9--23 11 7-16 17000 -
EXAMPLE 10
EFFICACY OF 31H4 AND 2IB12 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 (BiosourceTM) at 500 ng/ml in buffer C
followed by
TMB substrate (KPL). The signal was quenched with IN HC1 and the absorbance
read
at 450 nm.
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The results of this binding study are shown in FIGs. 6A-6D. Summarily, IC50
values were determined for each antibody and found to be 199 pM for 31H4 IgG2
(FIG. 6A), 156 pM for 31H4 IgG4 (FIG. 6B), 170 pM for 21B12 IgG2 (FIG. 6C),
and
169 pM for 21B12 IgG4 (FIG. 6D).
The antibodies also blocked the binding of wild-type PCSK9 to the LDLR in
this assay.
EXAMPLE 11
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 5x105 cells per well in DMEM
medium
(Mediatech, Inc) supplemented with 10% FBS and incubated at 37 C (5% CO2)
overnight. To form the PCSK9 and antibody complex, 2 pg/m1 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 PCSK9lantibody mixture was
transferred
to the cells, followed by LDL-BODIPY (Invitrogen) diluted in uptake buffer at
a final
concentration of 6 pg/ml. After incubation for 3 hours at 37 C (5% CO2), cells
were
washed thoroughly with PBS and the cell fluorescence signal was detected by
SafireTM
(TECAN) at 480-520nm (excitation) and 520-600nm (emission).
The results of the cellular uptake assay are shown in FIGs. 7A-7D. Summarily,
1050 values were determined for each antibody and found to be 16.7 nM for 31H4
1gG2
(FIG. 7A), 13.3 nM for 31H4 IgG4 (FIG. 7B), 13.3 nM for 21B12 lgG2 (FIG. 7C),
and
18 nM for 21B12 IgG4 (FIG. 7D). 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 12
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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
12.1.
TABLE 12.1
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
5 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 TmTC 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
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the mice also showed decreased HDL levels (FIG. 8C and FIG. 8D). For FIG. 8A
and
FIG. 8C, the percentage change is in relation to the control IgG at the same
time point
(*P<0.01, # P<0.05). For FIG. 8B 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,
#P0.05).
In respect to the lowered HDL levels, it is noted that one of skill in the art
will
appreciate that the decrease in IIDL in mice is not indicative that an HDL
decrease will
occur in humans and merely further reflects that the scrum 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 13
Effect of Antibody 311-14 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 11
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 BioRadTM protein assay reagents (BioRad
laboratories). 1001g of protein was denatured at 70 C for 10 minutes and
separated on
4-12% Bis-
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Tris SDS gradient gel (Invitrogen). Proteins were transferred to a 0.45
PVDF
membrane (hivitrogen) and blocked in washing buffer (50mM Tris PH7.5, 150mM
NaCL, 2mM CaC12 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-B 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
3114, as depicted in FIG. 9.
EXAMPLE 14
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 (C571L/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/mi 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 14.1. Animals were
sacrificed and livers were extracted and prepared as in Example 11.
TABLE 14.1
Group Treatment Time point after dosing Number Dose
1 IgG 72 hr 6 10mg/kg
2 31H4 72 hr 6 10mg/kg
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Group Treatment Time point after dosing Number Dose
3 31114 72 hr 6 lmg/kg
4 IgG 144 hr 6 10mg/kg
31H4 144 hr 6 10mg/kg
6 31H4 144 hr 6 lmg/kg
7 IgG 192 hr 6 10mg/kg
8 31114 192 hr 6 10mg/kg
9 31114 1.92 hr 6 1.m.g/kg
IgG 240 hr 6 10mg/kg
11 31H4 240hr 6 10mg/kg
12 31H4 240hr 6 lmg/kg
13 IgG 312 hr 6 10mg/kg
14 31H4 312 hr 6 10mg/kg
31H4 312 hr 6 1.mg/kg
16 Naive n/a 6 nia
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
5 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.
FIG. 10A depicts the results of this experiment. FIG. 10C depicts the results
of
repeating the above procedure with the 10mg/kg dose of 3114, and with another
antibody, 16F12, also at 10mg/kg. Dosing groups and time of sacrifice are
shown in
10 Table 14.2.
TABLE 14.2
Group Treatment Time point after dosing I Number Dose
1 IgG 24 hr 6 10mg/kg
2 16F12 24 hr 6 10mg/kg
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Group Treatment Time point after dosing Number Dose
3 31114 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 311714 144 hr 6 10mg/kg
IgG 192 hr 6 10mg/kg
11 1= 6F12 192 hr 6 10mg/kg
12 31H4 192 hr 6 10mg/kg
13 IgG2 240 hr 6 10mg/kg
14 1.012 240hr 6 10mg/kg
31H4 240hr 6 10mg/kg
16 IgG2 312 hr 6 10mg/kg
17 1= 6F12 312 hr 6 10mg/kg
18 31H4 312 hr 6 10mg/kg
19 N= aive n/a 6 10mg/kg
As shown in FIG. 10C 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
5 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 FIG. 10A and FIG. 10B,
the
percentage change is in relation to the control IgG at the same time point
(*P<0.01).
For FIG. 10C, the percentage change is in relation to the control IgG at the
same time
point (*P<0.05).
EXAMPLE 15
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Effect of Antibody 31H4 on HDL Levels in a 13 Day Study
The HDL levels for the animals in Example 14 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. FIG. 10B depicts the results of the experiment. There
was a
decrease in HDL levels of 34% on day 3. FIG. 10B 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 16
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 14
and 15 were repeated with the dosing schedule depicted in FIG. 11A. The
results are
displayed in FIG. 11B. As can be seen in the graph in FIG. 11B, 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 FIG. 11, 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
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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 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
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 19
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
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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
chemilumineseent
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.
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EXAMPLE 20
Consensus Sequences
Consensus sequences were determined using standard phylogenic analyses of
the CDRs corresponding to the VH and VL of anti-PCSK9 ABPs. The consensus
sequences were determined by keeping the CDRs contiguous within the same
sequence
corresponding to a VH or VL. Briefly, amino acid sequences corresponding to
the entire
variable domains of either VII or VL 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 VH or VL. VH or VL
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 2Ø 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.
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31A and 31B. In FIG. 13E, 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.
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Example 21
I IF) Binding Specificity
Results from this assay demonstrate that 11F1 binds to PCSK9 and not to PCSK I
,
PCSK2, PCSK7, or furin, demonstrating the specificity of 11F1 for PCSK9.
Biotinylated PCSK9, diluted in buffer A (25 mM Tris, 150 mM NaC1, 0.1% BSA,
0.05% tweenTM, pH 7.5) was bound to neutravidin coated 96 well plates at a
concentration of 0.2 g/mL, for one hour incubation at room temperature.
Separately,
0.4 pz/mL of 11F1 was incubated for one hour at room temperature with various
concentrations (ranging from 0 to 20 g/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 g/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-a-
human
Fc-FIFtP (160 ng/mL, diluted in buffer A) (Jackson Laboratories, Bar Harbor,
ME)
followed by TMB substrate. The reaction was stopped with 1 N HCI and the
absorbance was read at a wavelength of 450 nm on a SpectramaxTM 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 11FI and PCSK9 in
solution dose dependently and robustly reduced the amount of 11F1 binding to
plate-
captured PCSK9 detected as reduced 0D450 (FIG. 14). All results were expressed
as
the mean 0D450 value standard deviation versus concentration of the
proprotein
convertase. Pre-incubation of 1 I Fl 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, I1F1 binds only to PCSK9 and not to the
other
proprotein convertase family members tested.
Example 22
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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 ttg/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 p.g/mL of LDL receptor (R&D Systems,
Minneapolis, MN) diluted in buffer C (buffer B supplemented with 10 mM CaC12)
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 HC1 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 0D450 to determine 1050 values by
nonlinear regression.
11F1 inhibited LDLR:PCSK9 binding. The 1050 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 riM (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 FIG. 15 and FIG. 16, respectively.
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EXAMPLE 23
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 p.m 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 j.tg/mL) or WT PCSK9 (25 lag/mL) were diluted in uptake
buffer (DMEM containing 1% FBS) and incubated with the various concentrations
of
11F 1 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 Ag/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 uL 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 uL DPBS.
Fluorescent signals were detected using a Safirc plate reader (Tecan Systems
Inc., San
Jose, CA) at 480-520 nm (excitation) and 520-600 tun (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
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recombinant purified D374Y PCSK9 (2 ug/mL) or WT PCSK9 (25 iug/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, FIG. 17). 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, FIG. 18). 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 at, 2007; Fisher et at, 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 24
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 11F 1
or
8A3 leads to a significant decrease in serum non-HDL-C and IC 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 C5 7B116 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 (1=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 nag/kg
via
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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 p.g/m1 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 (i
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 Fe IgG and an HRP-labeled goat anti-human
IgG
Fcy 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
mn minus 650 nm using a microplate reader (Spectra Max Plus 384). Data was
analyzed using Watson version 7Ø0.01 (Thermo Scientific, Waltham, MA) data
reduction package with a Logistic (auto-estimate) regression of separately
prepared
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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 5.1.1 (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 (AUCO-t), and apparent serum clearance (CLO-t). AUCO-t
was
determined using the linear log-linear trapezoidal method, and CLO-t was
calculated by
Dose/AUCO-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 CLO-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,
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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%) (FIG. 19, FIG. 20). No significant lowering of HDL-C was
observed at any time point (FIG. 21).
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%) (FIG.
19, FIG.
20). No significant lowering of HDL-C was observed at any time point (FIG.
21).
Pharmacokinetics
At an intravenous dose of 30 mg/kg, 11F1 and 8A3 had very similar
pharmacokinetic behavior (FIG. 22). For these two molecules, AUCO-t exposures,

estimated CLO-t, and apparent half-lives were equivalent (Table of FIG. 23).
The anti-
KLH IgG2 control antibody had an unexpectedly lower AUCO-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 30 mg/kg; dose solution analysis showed
antibody
concentration to be 40% of target. Anti-KLH IgG2 control antibody CLO-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 11F 1 and 8A3 when compared to other antibodies dosed in the
AAV
model because 11F1 and 8A3 CLO-t values are more similar to anti-KLH lgG2
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).
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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 25
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 andPCSK9 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 Na0Ac 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
a45 day period.
Experimental Design
Group No Dose Level Conc. Volume
No. Males Route Treatment (mg/kg) (mg/mL) (mUkg)
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
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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 AL 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 [LLOQ1).
Non-compartmental analysis (NCA) was performed on the serum
concentrations using the pre-determined nominal time points for each subject
using
Watson' LIMS, version 7Ø0.01 (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 scrum
concentration (Cmax), area under the serum concentration-time curve from time
zero to
infinity (AUCO-inf), and apparent serum clearance (CL/F). AUCO-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.
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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 1111, 21B12, and 8A3 on Serum LDL Cholesterol
Maximal LDL-C lowering for IIFI 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 21Bl2 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 (FIG. 24).
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
(FIG. 25).
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Effect of 11F1, 211312, 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) (FIG. 26 and FIG. 27).
Effect of 11F1, 21B12, and 8A3 on Apolipoprotein B (ApoB)
Scrum 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 (FIG. 28). 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 29. The estimated mean pharmacokinetic parameters for animals
receiving
11F1, 21B12, 8A3, and anti-KLH IgG2 control antibody are displayed in Table of

Figure 30.
Antibody absorption in all groups was consistent and characteristic of
subcutaneous antibody administration. 21B12 pharmacokinetie behavior with
regard to
CL/F, Cmax, and AUCO-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 211312). 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.
181

Sum mary 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-ICLH
IgG2 control antibody) from day 1 to day 4 inclusively. 8A3 demonstrated
statistically
significant LDL-C lowering (vs anti-ICLH 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 26
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 antibody, 21B12, (heavy chain, SEQ ID NO :592 and light
chain,
SEQ ID NO:593) with expanded enrollment but otherwise identical design to Part
A.
Both inclusion/exclusion criteria and the Schedule of Assessments is the same
for Parts
A and B.
Inclusion Criteria includes:
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= 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/d1 (3.4 mmollL)
= 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 (N YHA) class ill 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 are instructed to follow an
NCEP Adult Treatment Panel TLC (or comparable) diet and are required to
maintain
their current lipid lowering therapy throughout the duration of the studies.
The 21B12 formulation is 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 is 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, eight subjects with genetically confirmed homozygous familial
hypercholesterolemia on stable lipid-lowering drug therapy for greater than
(or equal
183

CA 02916259 2015-12-19
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PCT/US2013/048714
to) 4 weeks are enrolled and received open label 21B12 formulation. Table 26.1
shows
the genotypes of the patients in the study.
Table 26.1: Patent Genotypes
Patient Mutation Allele 1 Mutation Allele 2
Overall LDL-r
(Estimated LDL-r (Estimated LDL-r Function
Function) Function)
Patient 1 Asp266Glu (15%-30%) Asp266Glu (15%-30%) Receptor defective
Patient 2 1187-10 G>At Asp266Glu Receptor
defective
(Not determined) (15%-30%)
Patient 3 Asp224Asn Cys296Tyr Negative
(<2%) (Not determined)
Patient 4 Deletion Exon 4-18 Cys197Gly Negative
(Not determined) .. (Not determined)
Patient 5 Asp221Gly Asp227Glu Receptor
defective
(<2%) (5%-15%)
Patient 6*# Asp227Glu Asp227Glu Receptor
defective
(5%-15%) (5%-15%)
Patient 7*# Asp227Glu Asp227Glu Receptor
defective
(5%-15%) (5%-15%)
Patient 8 Asp175Asn Asp227Glu Receptor
defective
(Not determined) .. (5%45%)
*True homozygous patient.
tMutation at splice acceptor site 10 nucleotides upstream of the first
nucleotide of exon 9, 1187.
I Pat icnts share the same gcnoty, pc.
LDL-r. Low density lipoprotein receptor.
184

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WO 2014/209384 PCT/US2013/048714
The 21B12 formulation (420 mg) is administered subcutaneously every 4 weeks
for 12 weeks, followed by an additional 12 weeks of treatment at 4 week
intervals, and
then 12 weeks with AMG 145 420 mg administered every 2 weeks. Study visits
occurr
at least every 4 weeks. During these visits adverse event (AE) and significant
adverse
event (SAE) data, vital signs, concomitant medication, laboratory tests, etc.
are
collected.
Changes and percentage changes in lipid related parameters are shown in Table
38.2. At week 12 of every 4-weeks treatment, the mean LDL cholesterol by
ultracentrifugation decreased from baseline by 16.5% (70.6 mg/dL; 1.8 mmol/L),
with
a range from +5.2% to -43.6%. Four (50%) patients have a reduction of >15%,
with 3
of the 4 (38%) achieving LDL cholesterol reductions >30%. Patients with
negative
LDL receptor activity have no LDL
cholesterol reduction.
After 12 weeks of every 2-week treatment, mean LDL cholesterol decrease
from baseline was 13.9% (60.8 mg/dL; 1.6 mmol/L). No LDL cholesterol reduction
is
observed in LDL receptor negative patients, but a greater reduction occurrs in
patients
with receptor defective function (Table 26.3). Three patients (38%) have an
LDL
cholesterol reduction >30%. Receptor defective patients have mean reductions
of
22.9% and 23.6% over the 12 week treatment period, respectively, for every 4-
and 2-
week dosing (Table 26.3).
The changes from baseline at week 12 in apolipoprotein B and related
lipoproteins with every 4- and 2-week dosing are shown in Table 26.2. The mean

change in Lp(a) is -11.7% and -18.6% with every 4- and 2-week dosing,
respectively;
this does not appear to be related to LDL receptor activity. Triglycerides
decrease by
5.7% and increase by 5.9% with every 4- and 2-week dosing, respectively. HDL-
cholesterol and apolipoprotein Al are essentially unchanged with either every
4- or 2-
week dosing (Table 26.2). Treatment with the 21B12 formulation 420 mg every 4
weeks reduces free PCSK9 by 22.7% and 87.6% at week 12 for every 4- and 2-week
dosing, respectively (Table 26.2).
185

CA 02916259 2015-12-19
WO 2014/209384 PCT/U82013/048714
Table 26.2: Efficacy Outcomes (Overall).
21B12 formulation (N = 8)
Week 12 Q4W Week 12 Q2W
..
Baselin
Change Percentage Percentage
e Value from change from Change change from
Parameter* Value . fro
baseline,..... ........õ 7.,.:.,,,,,..,,,,,,,,*,.../o,K7.)
baseline baseline (%) Value m baseline (%)
...
, , ,,' . H ! '.= 4...7,7k, 1
i,,,),%.5,,*,1'1::::::::.:::,,,:*,,, ,:.*:::::::::,0,,,*H::H:::'*',.:,
*<1::'IM:::::,'*.M,:,:M::::: :',,',..,',:,:,1:i:i:i
::::::::,,,,,,,,,,,i.Nt: ':: :,µ,,:.:,:,i.i,.i.:,:,:,:::::,,,,,,,,,i:i,i,i::::
*i .:.:..:.:.: :,:::::K, = = ..: = ,,,::::Ki,,,,??,
'Iiii51"HIHI'Wii01111 l'''''i,ERMlillin
'']...i.;i:'..iEiNH:r.,....:::1. nHH,],,i,..iiNn ,,= ':.,..,...:in
....õ.: , . H:::::==..= . i,:::::,,,, ,,:õ,,...,,,,, ... , , .
.,,,,:,...,,, , .... .... . :::
lifi6i40.0i..011PIEIIHMEIT:RH !µ',:]!*":7Mli:::;.,:..1
i:M:4;',..,.,i...:ii.',*''''''''''T,=:... ' ..jõ...õ:... L2õ:.,..,õ_
:.....,,,-. .... " :............ . .... :::,: i
ri, '':::::' ''' ':.:.::.:::::.:-' . ;:;:n . ''..:71' .
' :I.: . ... ..= =:`,.....= =:::::.::.::::' : '.'.':::::.::: .
*:: ' ``',.:.='':. . H :...:.:'.. :.E E:.... ::'''':.:.=
HI,:::::::;;','''
(l1-11rac:Fl`- .g:::::. i.::, :=:.:::..!.-..::.. .::,-
.,:.:õ.:dõ,:::::,õ..,,,,,,::.::.5.,....,,,,:.,,M.: ,::::: !:-..-...
d...;:;.,.-:;... .:=:::,..... :::::. dd:ii',,i, .,,HHW. :
:'',":' :: :::.... : d",!,i'',...'.,,i,
4,149P1i90',=';4i.P,i',:'.d::di:'4,,d'iiiii
.:,,=.:.,=.413.9.4.9.6)iiii':i
1 ii.)11,..,..,,i,,....,0./.,..,,i,..1LH:..:..,...:?......,,:: llilli
li,..1.1ill;1181hril;i1p11:!11100...(:64):õ.5' 380.9 60.8
iliii..46P39..7):.,.. :,.:.... . ::::......
M,040:4SE)ql! 'k,1';',1`..,,,j1..% .:.':::.(0'4"i',i':::- . (32.)
..-:::: -::: '.. . ::::,':..: . .õõ,:::õ:.:: ----.. ======== - -
..:.---.. '''''''''''''' 14ir:
.km,isi'-: :=if .:::::-:-.-:-.. = -:::::::::: .
:=:=:==== ' ... .. ..,,,,,... ' ,:,:,::=...]--,,-, .:,:*H,:
].:=,== =,,,,,='.=:';',,õ.m ,q:.,..i433::,..10.,õ*õ.,...:A
::::::,::,::,,,,-, = :-:-:::-:::-:::-:. =,:,:=:=,- ...,........
.."--=-= , :,...,,,:-::-. ,:.:,,....:,.= ,=-:,= L:,=:4,,,=-=].:;.:-
.=,,,..,,.,4,,,,.,4 196 to :;:,:',',',:,,7.41, f.===.,.":!WM
,:;?;',%,;::,,.',.0::,;:niiM;hU"';':,
.: :. ..' .4._ m
õ,,,,,.i::,,='),,';',,,=,:i.: H2: 8 tr, ::::.. '',,,41,44,',.!;.,Y,
.4ii:,.....;,:m ,',::n ?,::,,,,%niiii:44.6miiu
11 U..1=-=':' ..:: . 2.44,17,..H
11,,,%p?9,::!',".?..4=:':iH.'=`,':1': !.=....,.:,.'.:3Ri.=,,,"'::::':',iiHil
'''..;,:==',%=;"'ii M',0,14,0 ,.,,,,,gii!:1,,,r:..,iim
= UHk:13:,,,,E.,,,,..mimimiiiiin A],..,...,:,,b
f9gcl663,g'h''' ''''''''''''''''''.5 3: '''''':':':':':''''""'',E:i0M0 4
.;Niii:ii;HCM',d*H:*'',''''''''': .,,, ( 32.8 _0.9 (2.9) 1
-1. (7.3)
34.5 0.8 (3.0) 4.7 (7 u)
HIDL 33.8
cholesterol, (3-7)
(3.2) (3.4)
mg/dL
Apolipoprotei
-12.5 (6.7)
n 13, mg/dL (21689.7.1) (22218.3.8) (-1440..63) -14.9 (5,0)
(22345.7.3) (-1393..08)
,
Apolipoprotei 99.3
n Al , mgid L. (6.0) (949.8.3) 0.0 (4.8) 1.3 (5.0) ,
1(063Ø8) 4.5 (4.2) 5.2 (4.1)
Triglycerides, 5,9
(9.1)
-5.7 (5.6) ....., _incl.. .
1 -1.6
(14.4) (14.8)
mg/dL 1 10.8 (11070.8.8) -(170Ø0)
(22.8)
. _ .
-18.6 (4.3)
' -11.7 (3.8) (14628.4.0) -(277.8.3)
Lipoprotein
(a), nmol/L ' 246.5 (14710.2.6) -(284.2.6)
(61.5,2
76.0)1
Free PCSK9, 598.6 3 -22.7 (13.1) (7173..2 -
(54235.4) -87.6 (2.8)
ng/mL (42.8) (4743 -(18511-7) 0) .6
Values are mean (SE) unless otherwise stated.
* To convert values for cholesterol to millitmolesp oerleitr iteir,e,
multiplymrultiplyb y 0
y o0.002.591.. T
Too convertconvert vvaallu ueess
for Apolipoprotein Al or Apolipoprotein e o grams for triglycerides to
millimoles per liter, multiply by 0.0113. To convert values for free PCSK9 to
nanomoles per liter, divide by 72.
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CA 02916259 2015-12-19
WO 2014/209384 PCT/US2013/048714
t Median (interquartile range).
Q4W: every 4 weeks; Q2W: every 2 weeks; SE: standard error; LDL: low-density
lipoprotein;
HDL: high-density lipoprotein; PCSK9: proprotein convertase subtilisin/kexin
type 9.
Table 26.3: Efficacy Outcomes Based on Mutation Status
Percentage Change from Baseline, %, Mean (SE)
Week 12 Q4W Week 12 Q2W
Mutation UC LDL Apolipoprotein Lipoprotein UC LDL
Apolipoprotein Lipoprotein
Status B (a) B (a)
Total -16.5 -14.9 (5.0) -11.7 (3.8) -13.9
-12.5 (6.7) -18.6 (4.3)
(N=8) (6.7) (9.6)
Defective -22.9 - 18.3 (6.1) -10.0 (4.7) -23.6
-17.9 (7.3) -18.7 (5.7)
LDL (7.2) (7.6)
receptor
(N=6)
Negative 2.6 (2.6) -4.5 (2.6) -16.8 (5.7) 15.3
3.4 (9.9) -18.5 (3.7)
LDL receptor (24.6)
(N=2)
Average of Week 4, 8, and 12 Q4W Average
of Week 4, 8, and 12 Q2W
UC LDL Apolipoprotein Lipoprotein UC LDL Apolipoprotein
Lipoprotein
(a) B (a)
Total -13.3 -13.1 (5.1) -11.7 (3.8) -16.9
-16.0 (6.9) -20.7 (3.9)
(N=8) (6.2) (9.2)
Defective -19.3 - 18.0 (5.3) -10.0 (4.7) -26.3
-22.1 (7.7) -20.0 (5.0)
LDL (6.3) (8.3)
receptor
(N=6)
Negative 4.4 (7.3) 1.4 (4.0) -16.8 (5.7) 11.0
2.1 (5.6) -22.7 (7.9)
LDL receptor (16.7)
(N=2)
Q4W: every 4 weeks; Q2W: every 2 weeks; SE: standard error; UC LDL:
Ultracentrifugation
low-density lipoprotein
Approximately 51 new subjects are enrolled into Part B. Subjects enrolled are
randomized to a 2:1 allocation into 2 treatment groups: 420 mg 21B12 Q4W SC or

placebo Q4W SC. Randomization is stratified by baseline LDL-C levels. Study
visits
occur every 4 weeks, with two optional visits occurring at week 2 and week 10.
Visits
entail collection of AE and SAE data, vital signs, concomitant medication,
laboratory
187

tests, etc. A fasting lipid panel is collected at week 6 to assess the nadir
LDL-C level in
response to 21812 treatment. 21B12 formulation is administered at day 1, week
4, and
week 8. The end-of-study (EOS) visit and the last estimation of lipids occurs
at week
12 for all subjects.
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.
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