AU2020201012A1 - Methods for treating homozygous familial hypercholesterolemia - Google Patents

Methods for treating homozygous familial hypercholesterolemia Download PDF

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AU2020201012A1
AU2020201012A1 AU2020201012A AU2020201012A AU2020201012A1 AU 2020201012 A1 AU2020201012 A1 AU 2020201012A1 AU 2020201012 A AU2020201012 A AU 2020201012A AU 2020201012 A AU2020201012 A AU 2020201012A AU 2020201012 A1 AU2020201012 A1 AU 2020201012A1
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pcsk9
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Robert Andrew Donald Scott
Evan A. Stein
Scott Wasserman
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Amgen Inc
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Abstract

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

Description

METHODS FOR TREATING HOMOZYGOUS FAMILIAL
HYPERCHOLESTEROLEMIA
REFERENCE TO THE SEQUENCE LISTING
The present application is a divisional application of Australian Application
No. 2013396206, which is incorporated in its entirety herein by reference.
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled A-1837-WO-PCT-SequenceList062713.txt, created June 27, 2013 which is 322 KB in size. The information in the electronic format of the 0 Sequence Listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
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).
,5 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 20 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;lll:179525 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. Lowdensity lipoprotein receptor gene familial hypercholesterolemia variant database: update and pathological assessment. Ann Hum Genet 2012;76:387-401). The residual LDL receptor activity, either negative (<2% function) or defective (2% to 25% function), is associated with 30 severity of LDL
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2020201012 13 Feb 2020 cholesterol elevation and propensity for earlier cardiovascular disease (Goldstein JL, Hobbs HH, Brown MS, eds. Familial hypercholesterolemia. 8th Edition ed: McGrawHill; 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 serum LDL 25 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 30 least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% as compared to a predose level of serum LDL cholesterol in the patient.
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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 5 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 10 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 15 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 20 1400 mg, of about 1800 mg, of about 2000 mg, of about 2500 mg, of about 2800 mg, or about 3000 mg.
In some embodiments of this aspect of the invention the anti-PCSK9 antibody is administered to a patient on a schedule selected from the group consisting of: (1) once a week, (2) once every two weeks, (3) once a month, (4) once every other month, (5) 25 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)
WO 2014/209384
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2020201012 13 Feb 2020 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 5 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, 10 465, 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 15 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 20 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 25 (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 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 (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 25 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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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 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 25 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 30 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
WO 2014/209384
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2020201012 13 Feb 2020 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 2IB 12, 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 10 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 15 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 20 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 25 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; 30 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 5 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 10 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 15 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 25 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 30 (CDR) of the CDRH1 sequence in SEQ ID NO: 459, a CDRH2 of the CDRH2
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2020201012 13 Feb 2020 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 5 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 15 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 20 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 25 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 30 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
WO 2014/209384
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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 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 2IB 12, 31H4, 8A3, 11F1 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 25 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 30 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
WO 2014/209384
PCT/US2013/048714
2020201012 13 Feb 2020 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 5 comprises a light chain variable region that comprises the amino acid sequence of SEQ
ID NO:59land 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 10 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 an amino acid sequence that is at least 90% identical to that of SEQ ID NO:23 and a heavy chain 15 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 20 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 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 25 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 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 280 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 20 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 25 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 30 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
WO 2014/209384
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2020201012 13 Feb 2020 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:59land 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 10 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 15 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 20 patient is lowered 15-50% for about 7-14 days; is administered to a patient at a dose of about 1200 mg intraveneously every other week, wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 21-31 days; is administered to a patient at a dose of about greater than 1200 mg to about 3000 mg intraveneously every other week, wherein the serum LDL cholesterol level of the patient is lowered 15-50% for 25 about 7-14 days; is administered to a patient at a dose of about 420 mg to about 3000 mg intraveneously four weeks, wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 21-31 days, is administered to a patient at a dose of about 700 mg intraveneously every four weeks, wherein the serum LDL cholesterol level of the patient is lowered 15-50% for about 21-31 days; is administered to a patient at a 30 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
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2020201012 13 Feb 2020 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 5 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 10 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 15 antibody is 2IB 12, 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 20 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 25 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 30 350 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
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2020201012 13 Feb 2020 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 5 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 10 420 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% 15 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 20 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 710 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 25 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 30 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
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2020201012 13 Feb 2020 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 ΙΟΙ 4 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% 5 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 2410 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 15 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 2428 days.
In particular embodiments of the invention, the anti-PCSK9 antibody is 8A3, 11F1 and 8AL In some embodiments the anti-PCSK9 antibody comprises a light chain 20 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 25 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 antiPCSK9 antibody comprises a light chain variable region that comprises the amino acid 30 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
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2020201012 13 Feb 2020 antibody is 11F1. In a particular embodiment, 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, 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 10 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 15-50% for about 3-10 15 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 20 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 25 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 30 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
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2020201012 13 Feb 2020 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 5 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 10 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 4556 days; is administered to a patient at a dose of about 170 mg to about 180 mg 15 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 20 LDL cholesterol level of the patient is lowered 15-50% for about 45-56 days; at a dose of about 600 mg subcutaneously once every 8 weeks wherein the serum LDL cholesterol level of the patient is lowered at least about 15-50% for about 45-56 days; at a dose of about 700 mg subcutaneously once every 8 weeks wherein the serum LDL cholesterol level of the patient is lowered at least about 15-50% for about 45-56 days; 25 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 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 comprises the antibody is 1 IF 1, 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 20 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 25 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 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 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 5056 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 20 150 mg to about 170 mg subcutaneously every 8 weeks wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 50-56 days; is administered to a patient at a dose of about 450 mg subcutaneously every 8 weeks wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 50-56 days; at a dose of about 600 mg subcutaneously once every 8 weeks wherein the serum LDL 25 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; 30 at a dose of about 700 mg subcutaneously once every 12 weeks wherein the serum LDL cholesterol level of the patient is lowered at least about 30-50% for about 80-84
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2020201012 13 Feb 2020 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 5 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 10 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 15 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 1 IF 1, 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 20 intraveneously every week, wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 7-10 days; is administered to a patient at a dose of about 700 mg intraveneously every week, wherein the serum LDL cholesterol level of the patient is lowered 30-50% for about 7-10 days; is administered to a patient at a dose of about 1200 mg intraveneously every week, wherein the serum LDL cholesterol level of the 25 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% 30 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
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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 160 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 25 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, 30 rosuvastatin, simvastatin, nicotinic acid (niacin), slow relese niacin (SLO-NIACIN), laropiprant (CORDAPTIVE), fibric acid (LOPID (Gemfibrozil), TRICOR
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2020201012 13 Feb 2020 (fenofibrate)), Bile acid sequestrants, sucha as cholestyramine (QUESTRAN), colesvelam (WELCHOL), COLESTID (colestipol)), cholesterol absorption inhibitor (ZETIA (ezetimibe)), lipid modifying agents, PPAR gamma agonsits, PPAR alpha/gamma agonists, squalene synthase inhibitors, CETP inhibitors, anti5 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 10 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 mg of at least one anti-PCSK9 antibody 15 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 aboutl20 mg to about 450 mg of at least one anti-PCSK9 antibody administered once every 2 weeks 20 (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 25 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 30 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
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2020201012 13 Feb 2020 embodiments, the dose is about 420 mg to about 3000 mg of at least one anti-PCSK9 antibody administered once every week (QW). In some embodiments, the dose is about 1000 mg to about 3000 mg of at least one anti-PCSK9 antibody administered once every week (QW). In some embodiments, the dose is about 2000 mg to about 5 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 antiPCSK9 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 10 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 antiPCSK9 antibody administered once every month (Q4W). In some embodiments, the 15 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 20 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 25 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%. 30 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
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2020201012 13 Feb 2020 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 antiPCSK9 antibody is administered on a schedule selected from the group consisting of: 10 (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 20 180 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) 25 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
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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 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 30 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
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PCT/US2013/048714 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
2020201012 13 Feb 2020 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 serum PCSK9 value is serum PCSK9 value is reduced by at least about 40%. reduced by at least about 50%. reduced by at least about 60%.
In some embodiments, the
In some embodiments, the
In some embodiments, the serum PCSK9 serum PCSK9 serum
PCSK9 serum PCSK9 serum PCSK9 value is reduced by at least about 65%.
value is reduced by at least about 70%.
value is reduced by at least about 75%.
value is reduced by at least about 80%.
value is reduced by at least about 85%.
In some embodiments, the
In some embodiments, the
In some embodiments, the
In some embodiments, the
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 20 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 antiPCSK9 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 25 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
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PCT/US2013/048714 (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)
2020201012 13 Feb 2020 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
15 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 antiPCSK9 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
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2020201012 13 Feb 2020 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 5 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 10 amount of about 3000 mg every four weeks (Q4W). In some embodiments, the nonHDL cholesterol level is reduced by at least about 30% as compared to a predose nonHDL 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 15 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 nonHDL 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 20 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 25 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 30 120 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
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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 10 (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 15 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 20 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, 25 the ApoB level is reduced by at least about 70%. In some embodiments, the ApoB level is reduced by at least about 75%.
In some aspects, the invention comprises a method of lowering Lipoprotein A (“Lp(a)”) levels in a patient diagnosed with homozygous familial hypercholesterolemia, the method comprising administering to a patient in need thereof, a dose of at least one 30 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
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2020201012 13 Feb 2020 (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 amountof about 280 mg every two weeks or every other week (Q2W); (7) at least an amountof about 350 mg every two weeks or every other week (Q2W); (8) at least an amountof 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 10 (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 15 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 20 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 25 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 30 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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2020201012 13 Feb 2020 some embodiments, the dose is about 120 mg to about 450 mg of at least one antiPCSK9 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 5 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-PCSK9 antibody administered once every two weeks (Q2W). In some embodiments, the dose is about 140 mg to 10 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 15 is about 400 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 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 20 is about 150 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 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 25 embodiments, the dose is about 280 mg to about 420 mg of at least one anti-PCSK9 antibody administered once every four weeks (Q4W). In some embodiments, the dose is about 350 mg to about 420 mg of at least one anti-PCSK9 antibody administered once every four weeks. In some embodiments, the dose is about 1000 mg every four weeks (Q4W). In some embodiments, the dose is about about 2000 mg every four 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 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, 20 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. 1A depicts an amino acid sequence of the mature form of the PCSK9 with the pro-domain underlined.
FIGs. IB1-IB4 depict amino acid and nucleic acid sequences of PCSK9 with the 5 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 10 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 20 the human ABP sequences and the ABP sequences that were raised in E. coli.. (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 30 various antigen binding proteins demonstrating the relative purity and concentration of the proteins.
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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 31H4 IgG4 to PCSK9 in an in vitro PCSK9:LDLR binding assay.
FIG. 6C is an inhibition curve of antigen binding protein 2 IB 12 IgG2 to PCSK9 in an in vitro PCSK9:LDLR binding assay.
FIG. 6D is an inhibition curve of antigen binding protein 2IB 12 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. 7B 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 2IB 12 IgG2 in the cell LDL uptake assay showing the effect of the ABP to reduce the LDL uptake blocking 20 effects of PCSK9
FIG. 7D is an inhibition curve of antigen binding protein 2 IB 12 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 25 ABP 31H4, changes relative to the IgG control treated mice (* p< 0.01).
FIG. 8B is a graph depicting the serum cholesterol lowering ability in mice of ABP 31H4, 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 C57B1/6 mice (* p< 0.01).
FIG. 8D is a graph depicting the effect of ABP 31H4 on HDL cholesterol levels in C57B1/6 mice (# p< 0.05).
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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 10 antigen binding proteins to lower serum cholesterol.
FIG. 1 IB is a graph depicting the results of the protocol in FIG. 11 A.
FIG. 12A depicts LDLR levels in response to the combination of a statin and ABP 2IB 12 in HepG2 cells.
FIG. 12B depicts LDLR levels in response to the combination of a statin and 15 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 20 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 25 antibody) in HepG2 cells over expressing PCSK9.
FIG. 13A depicts the various light chain amino acid sequences of various ABPs to PCSK9. The dots (.) 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 30 ABPs to PCSK9. The dots (.) 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. 13J depicts the consensus sequences for Groups 3 and 4. The dots (.) 10 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 OD450 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 15 LDLR:D374Y PCSK9 binding by 11F1 in a competition assay with OD450 plotted on the vertical axis and Log [1 IF 1 ] (pM) plotted on the horizontal axis.
FIG. 16is a graph depicting the dose response curve for the inhibition of LDLR: WT PCSK9 binding by 1 IFlin a competition assay with OD450 plotted on the vertical axis and Log [11 fl] (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 (xlO4) plotted on the vertical axis and Log [HF1] (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 (xlO4) on the vertical axis and Log [HF1] (nM) plotted on the horizontal axis.
FIG. 19 is a bar graph depicting the effect of 1 IF 1 and 8A3 on serum non-HDL cholesterol in mice expressing human PCSK9 by AAV with non-HDL-C serum 30 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/ml) 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 10 (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 15 of an ant-KLH antibody (control), 2IB 12, 8A3 and 11F1 on serum LDL concentration (LDL-C) in cynomolgus monkeys with LDL-C (mg/dl) plotted on the vertical axis and time following administration in days on the horizontal axis.
FIG. 25 is a graph depicting the effect of a single subcutaneous administration of an ant-KLH antibody (control), 2 IB 12, 8A3 and 11F1 on Serum Total Cholesterol in 20 cynomolgus monkeys with Total Cholesterol concentration (mg/dl) 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), 2IB 12, 8A3 and 11F1 on Serum HDL Cholesterol in cynomolgus monkeys with HDL-C (mg/dl) plotted on the vertical axis and time 25 following administration in days on the horizontal axis.
FIG. 27 is a graph depicting the effect of a single subcutaneous administration of an ant-KLH antibody (control), 2IB 12, 8A3 and 11F1 on Serum Triglycerides in cynomolgus monkeys with Serum Triglyceride concentration (mg/dl) plotted on the vertical axis and time following administration in days on the horizontal axis.
FIG. 28 is a graph depicting the effect of a single subcutaneous administration of an ant-KLH antibody (control), 2IB 12, 8A3 and 11F1 on Apolipoprotein B (ApoB)
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2020201012 13 Feb 2020 in cynomolgus monkeys with APOB concentration (mg/dl) 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), 2IB 12, 8A3 and 11F1 in cynomolgus monkeys with antibody 5 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, 10 8A3 and 1 IF 1, as well as a consensus sequence derived from the the comparison. CDR sequences are underlined.
FIG. 3 IB depicts a comparison of heavy chain amino acid sequences of 8A1, 8A3 and 1 IF 1, 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 20 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 25 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
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2020201012 13 Feb 2020 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 10 not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have 15 the following meanings:
The term “proprotein convertase subtilisin kexin type 9” or “PCSK9” refers to a polypeptide as set forth in SEQ ID NO: 1 and/or 3 or fragments thereof, as well as related polypeptides, which include, but are not limited to, allelic variants, splice variants, derivative variants, substitution variants, deletion variants, and/or insertion 20 variants including the addition of an N-terminal methionine, fusion polypeptides, and interspecies homologs. In certain embodiments, a PCSK9 polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues. “PCSK9” has also been referred to as FH3, NARC1, 25 HCHOLA3, proprotein convertase subtilisin/kexin type 9, and neural apoptosis regulated convertase 1. The PCSK9 gene encodes a proprotein convertase protein that belongs to the proteinase K subfamily of the secretory subtilase family. The term “PCSK9” denotes both the proprotein and the product generated following autocatalysis of the proprotein. When only the autocatalyzed product is being referred 30 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 5 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 IB).
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 15 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 20 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 25 Molecular Genetics 14: 1161-1169, 2005 and Ouguerram K et al, “Apolipoprotein
Bl00 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 30 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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2020201012 13 Feb 2020 regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c” J. Biol, Chem. 281(10):6211-18, 2006).
The term “hypercholesterolemia,” as used herein, refers to a condition in which cholesterol levels are elevated above a desired level. In some embodiments, this 5 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 “HoFH” as used herein, refers a cholesterol-related disorder that is determined by genetic confirmation 10 or clinical diagnosis (such as history of an untreated LDL-cholesterol concentration greater than 13 mmol/L 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 15 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 intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate 20 and phosphoroamidate.
The term “oligonucleotide” means a polynucleotide comprising 200 or fewer nucleotides. In some embodiments, oligonucleotides are 10 to 60 bases in length. In other embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides can be single stranded or double stranded, e.g., 25 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, 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 “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 20 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 25 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 30 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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2020201012 13 Feb 2020 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 10 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 15 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 20 et al., 2001, Molecular Cloning: A Laboratory Manual, supra', Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.
The term “transformation” refers to a change in a cell's genetic characteristics, 25 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, 30 or can be maintained transiently as an episomal element without being replicated, or
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2020201012 13 Feb 2020 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 5 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 naturally10 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 15 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 20 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 25 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 30 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
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2020201012 13 Feb 2020 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 5 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 10 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 15 and is calculated based on the size of the smallest of the molecules being compared.
For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, 20 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., 25 eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.
In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program 30 package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm
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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, 5 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 10 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 15 for polypeptides or nucleotide sequences using the GAP program are the following:
• Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453 • Comparison matrix: BLOSum 62 from Henikoff et al., 1992, supra • Gap Penalty: 12 (but with no penalty for end gaps) • Gap Length Penalty: 4 · Threshold of Similarity: 0
Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method 25 (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. 30 (1991)), which is incorporated herein by reference for any purpose. Stereoisomers (e.g.,
D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as
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2020201012 13 Feb 2020 α-, α-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, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N5 acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-Νmethylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA and which are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”
Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.
Naturally occurring residues can be divided into classes based on common side chain properties:
1) hydrophobic: norleucine, Met, Ala, Vai, Leu, He;
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.
Lor 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
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2020201012 13 Feb 2020 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 5 considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (10 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar 15 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 20 made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological 25 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 (30 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
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2020201012 13 Feb 2020 certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One can also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as epitopic core regions.
Exemplary amino acid substitutions are set forth in Table 1.
TABLE 1
Amino Acid Substitutions
Original Residues Exemplary Substitutions Preferred Substitutions
Ala Val, Leu, He Val
Arg Lys, Gln, Asn Lys
Asn Gln Gln
Asp Glu Glu
Cys Ser, Ala Ser
Gln Asn Asn
Glu Asp Asp
Gly Pro, Ala Ala
His Asn, Gln, Lys, Arg Arg
He Leu, Val, Met, Ala, Phe, Norleucine Leu
Leu Norleucine, lie, Val, Met, Ala, Phe He
Lys Arg, 1,4 Diamino-butyric Acid, Gln, Asn Arg
Met Leu, Phe, lie Leu
Phe Leu, Val, lie, Ala, Tyr Leu
Pro Ala Gly
Ser Thr, Ala, Cys Thr
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Original Residues Exemplary Substitutions Preferred Substitutions
Thr Ser Ser
Trp Tyr, Phe Tyr
Tyr Trp, Phe, Thr, Ser Phe
Vai lie, Met, Leu, Phe, Ala, Norleucine Leu
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 5 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 10 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 15 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
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2020201012 13 Feb 2020 used to improve the therapeutic capacity for an antigen binding protein. In certain embodiments, PEG is used to improve the therapeutic capacity for a humanized antibody. Certain such methods are discussed, for example, in U.S. Patent No. 6,133,426, which is hereby incorporated by reference for any purpose.
Peptide analogs are commonly used in the pharmaceutical industry as nonpeptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics.” Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber & Freidinger, TINS, p.392 (1985); and Evans et al., J. Med. Chem., 30:1229 (1987), which are incorporated herein by 10 reference for any purpose. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce a similar therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological 15 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(OH)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 20 in certain embodiments to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation can be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein by reference for any purpose); for example, by adding internal cysteine residues capable of forming intramolecular 25 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
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2020201012 13 Feb 2020 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 5 immunoglobulin chain (heavy or light chain) antigen binding protein, as used herein, is a species of antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to the target 10 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, 15 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 20 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’)2, 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 25 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 30 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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2020201012 13 Feb 2020 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 10 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 15 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 “Fc” region comprises two heavy chain fragments comprising the ChI and Ch2 domains of an antibody. The two heavy chain fragments are held together by two 20 or more disulfide bonds and by hydrophobic interactions of the Ch3 domains.
A “Fab fragment” comprises one light chain and the ChI 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 25 that contains the VH domain and the ChI 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(ab’)2 molecule.
A “F(ab’)2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the ChI and Ch2 domains, such that an 30 interchain disulfide bond is formed between the two heavy chains. A F(ab’)2 fragment
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2020201012 13 Feb 2020 thus is composed of two Fab’ fragments that are held together by a disulfide bond between the two heavy chains.
The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
“Single-chain antibodies” are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and United States Patent Nos. 4,946,778 and No. 5,260,203, the disclosures of which are 10 incorporated by reference.
A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more Vh regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two Vh regions of a bivalent 15 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 20 “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 25 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. 30 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites
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2020201012 13 Feb 2020 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 (IQ) is <10’7 M. The ABP specifically binds antigen with 5 “high affinity” when the IQ is <5 x 10’9 M, and with “very high affinity” when the IQ is <5x IO’10 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 Kj of between about 10’9 M and 10’13 M, and in yet another embodiment the ABPs will bind with a Kd <5 x IO’10. As will be appreciated by one of skill in the art, in 10 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 15 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 20 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 25 regions are shown in FIGs. 2A-3D, 3CCC-3JJJ. In some embodiments, the sequences for CDRs for the light chain of antibody 3B6 are as follows: CDR1 TLSSGYSSYEVD (SEQ ID NO: 279); CDR2 VDTGGIVGSKGE (SEQ ID NO: 280); CDR3 GADHGSGTNFVVV (SEQ ID NO: 281), and the FRs are as follows: FR1 QPVLTQPLFASASLGASVTLTC (SEQ ID NO: 282); FR2 30 WYQQRPGKGPRFVMR (SEQ ID NO: 283);
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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 5 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 10 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. 15 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 20 term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody 25 mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.
Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, 30 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 amino57
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2020201012 13 Feb 2020 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 carboxyterminal portion of each chain typically defines a constant region that can be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and 10 IgA2. Within full-length light and heavy chains, typically, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each 15 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 20 specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Eesk, J. Mol. Biol., 25 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).
In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light 30 chain. In certain embodiments, an antibody binding region binds to an antigen in the
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2020201012 13 Feb 2020 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 5 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 10 definition, the Chothia definition, the AbM definition and the contact definition.
The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions.
See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford
Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).
By convention, the CDR regions in the heavy chain are typically referred to as Hl, 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 30 referred to as LI, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.
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The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, Cl. The variable region domain of the light chain is at the amino-terminus of the 5 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 fulllength heavy chain includes a variable region domain, VH, and three constant region domains, ChI, Ch2, and Ch3. The Vh domain is at the amino-terminus of the 10 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 IgGl, 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 15 having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).
Some species of mammals also produce antibodies having only a single heavy 20 chain.
Each individual immunoglobulin chain is typically composed of several “immunoglobulin domains,” each consisting of roughly 90 to 110 amino acids and having a characteristic folding pattern. These domains are the basic units of which antibody polypeptides are composed. In humans, the IgA and IgD isotypes contain 25 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 30 region domains known as ChI, Ch2 and Ch3. The antibodies that are provided can
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2020201012 13 Feb 2020 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 amino5 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 20 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%, 8590%, 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 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 25 137:3614-3619) 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, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et 30 al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled
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2020201012 13 Feb 2020 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 5 competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided 10 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 20 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 25 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 30 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 5 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 10 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 20 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, inIn, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline 25 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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2020201012 13 Feb 2020 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 5 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. 10 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 15 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, 20 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, 25 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 30 disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.
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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 5 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 10 more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures 15 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 25 exemplary human PCSK9 amino acid sequence is presented as SEQ ID NO: 1 in FIG. 1A (depicting the “pro” domain of the protein as underlined) and SEQ ID NOG in FIG. IB (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. IB). As described herein, PCSK9 proteins can also include fragments of the full length 30 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), the entireties of both of which are herein incorporated by reference. PCSK9 includes a signal sequence, a N-terminal prodomain, a subtilisin-like catalytic domain and a C-terminal domain.
Antigen binding proteins (ABPs) that bind PCSK9, including human PCSK9, 5 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 10 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 15 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” 20 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 25 the subject). In turn, this results in a reduction in the amount of serum cholesterol present in a subject.
In some embodiments, the antigen binding proteins provided herein are capable of inhibiting PCSK9-mediated activity (including binding). In some embodiments, antigen binding proteins binding to these epitopes inhibit, inter alia, interactions 30 between PCSK9 and LDLR and other physiological effects mediated by PCSK9. In
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2020201012 13 Feb 2020 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 5 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 10 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, 15 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 20 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 25 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 30 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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2020201012 13 Feb 2020 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 5 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, 10 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 20 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. 1A (SEQ ID NO: 1). In some embodiments, the antigen binding protein specifically binds to at least a fragment of the PCSK9 protein and/or a full length PCSK9 protein, with or without a signal sequence.
In embodiments where the antigen binding protein is used for therapeutic applications, an antigen binding protein can inhibit, interfere with or modulate one or more biological activities of PCSK9. In one embodiment, an antigen binding protein binds specifically to human PCSK9 and/or substantially inhibits binding of human PCSK9 to LDLR by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuring binding in an in vitro competitive binding assay). Some of the antigen binding proteins that are provided herein are antibodies. In some embodiments,
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2020201012 13 Feb 2020 the ABP has a Ka of less (binding more tightly) than ΙΟ’7, ΙΟ’8, ΙΟ’9, ΙΟ’10, ΙΟ’11, 10’12, 10’13 M. 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, lOOnM to 10 nM, lOnM to 1 nM, lOOOpM to 500pM, 500 pM to 200 pM, less than 200 5 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. 3KK.
One example of an IgG4 heavy chain constant domain of an anti-PCSK9 10 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 15 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 20 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 Nterminal to C-terminal, naturally-occurring light and heavy chain variable regions both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised for assigning numbers to 25 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 30 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
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2020201012 13 Feb 2020 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 5 of the antibodies that are provided and their corresponding amino acid sequences are summarized in TABLE 2.
TABLE 2: Exemplary Heavy and Light Chain Variable Regions
Antibody Light/Heavy SEQ ID NO
30A4 5/74
3C4 7/85
23B5 9/71
25G4 10/72
31H4 12/67
27B2 13/87
25A7 15/58
27H5 16/52
26H5 17/51
31D1 18/53
20D10 19/48
27E7 20/54
30B9 21/55
19H9 22/56
26E10 23/49
21B12 23/49
21B12vl 591/590
17C2 24/57
23G1 26/50
13H1 28/91
9C9 30/64
9H6 31/62
31A4 32/89
1A12 33/65
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16F12 35/79
22E2 36/80
27A6 37/76
28B12 38/77
28D6 39/78
31G11 40/83
13B5 42/69
31B12 44/81
3B6 46/60
5H5 421/419
24F7 425/423
22B11 429/427
30F1 433/431
24B9.1 437/435
24B9.2 441/439
20A5.1 445/443
20A5.2 449/447
20E5.1 453/451
20E5.2 457/455
8A3 461/459
11F1 465/463
12H11 469/467
11H4 473/471
11H8 477/475
11G1 481/479
8A1 485/483
Again, heavy chains 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 listed in the exemplary variable
Table 2 can be
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2020201012 13 Feb 2020 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 (CDRE1, CDRE2, CDRE3) and CDR1-3 from the heavy (CDRH1, CDRH2, and 5 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 3EFF 10 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 3EEE 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, 15 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, 20 455,459,463,467,471,475,479, and 483. In some of the entries in FIG. 2A-3D and
FIG. 3CCC-3JJJ and 3EEE variations of the sequences or alternative boundaries of the CDRs and FRs are identified. These alternatives are identified with a “vl” 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 25 light or heavy chain is the same as shown for the base ABP in the other panels. Thus, for example, 19H9vl 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 30 one such sequence need actually be used in the creation of an antibody or ABP.
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Indeed, in some 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 5 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 10 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 15 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, 20 100-200, 31-152, 153-692, 200-300, 300-400, 452-683, 400-500, 500-600, 31-692, 31449, 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 25 obstruct an area on the catalytic domain that interacts with the pro domain. In some embodiments, the ABP binds to the catalytic domain at a location or surface that the pro-domain interacts with as outlined in Piper et al. (Structure 15:1-8 (2007), the entirety of which is hereby incorporated by reference, including the structural representations therein). In some embodiments, the ABP binds to the catalytic domain 30 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 149152 of the pro-domain in Piper et al. In some embodiments, the ABPs bind to the 5 groove (as outlined in Piper et al.) on the V domain. In some embodiments, the ABPs bind to the histidine-rich patch proximal to the groove on the V domain. In some embodiments, such antibodies (that bind to the V domain) are not neutralizing. In some embodiments, antibodies that bind to the V domain are neutralizing. In some embodiments, the neutralizing ABPs prevent the binding of PCSK9 to LDLR. In some 10 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 Kd). In some embodiments, the antibody selectively inhibits variant 20 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 IC50). In some embodiments, the antibody binds 25 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 30 recycling receptors” Nature 388: 629-630, 1997). In some embodiments, the ABP can
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2020201012 13 Feb 2020 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 5 percent identity to the form of PCSK9 depicted in FIG. 1A and/or FIG. IB. 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. 1A and/or FIG. IB. In some embodiments, the ABP binds to and prevents variants of PCSK9 that are at least 50%, 50-60, 60-70, 10 70-80, 80-90, 90-95, 95-99, or greater percent identity to the form of PCSK9 depicted in FIG. 1A and/or FIG. IB 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. IB from interacting with LDLR. In some embodiments, the variant of PCSK9 15 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 20 antibodies described in Table 2. In some embodiments, the antigen binding proteins bind to a specific conformational state of PCSK9 so as to prevent PCSK9 from interacting with LDLR.
Humanized Antigen Binding Proteins (e.g., Antibodies)
As described herein, an antigen binding protein to PCSK9 can comprise a humanized antibody and/or part thereof. An important practical application of such a strategy is the “humanization” of the mouse humoral immune system.
In certain embodiments, a humanized antibody is substantially nonimmunogenic in humans. In certain embodiments, a humanized antibody has 30 substantially the same affinity for a target as an antibody from another species from
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2020201012 13 Feb 2020 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 5 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, 10 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 15 (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 antiidiotypic and anti-allotypic responses following repeated administration of the antibodies. Certain other methods for reducing immunogenicity are described, e.g., in 20 Gilliland et al., J. Immunol., 62(6): 3663-71 (1999).
In certain instances, humanizing antibodies results in a loss of antigen binding capacity. In certain embodiments, humanized antibodies are “back mutated.” In certain such embodiments, the humanized antibody is mutated to include one or more of the amino acid residues found in the donor antibody. See, e.g., Saldanha et al., Mol 25 Immunol 36:709-19 (1999).
In certain embodiments the complementarity determining regions (CDRs) of the light and heavy chain variable regions of an antibody to PCSK9 can be grafted to framework regions (FRs) from the same, or another, species. In certain embodiments, the CDRs of the light and heavy chain variable regions of an antibody to PCSK9 can be 30 grafted to consensus human FRs. To create consensus human FRs, in certain embodiments, FRs from several human heavy chain or light chain amino acid
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2020201012 13 Feb 2020 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 5 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 10 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 al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988);
Verhoeyen et al., Science, 239:1534-1536 (1988), Winter, FEBS Letts., 430:92-94 (1998), which are hereby incorporated by reference for any purpose.
Human Antigen Binding Proteins (e.g.. Antibodies)
As described herein, an antigen binding protein that binds to PCSK9 can comprise a human (i.e., fully human) antibody and/or part thereof. In certain embodiments, nucleotide sequences encoding, and amino acid sequences comprising, 20 heavy and light chain immunoglobulin molecules, particularly sequences corresponding to the variable regions are provided. In certain embodiments, sequences corresponding to complementarity determining regions (CDR's), specifically from CDR1 through CDR3, are provided. According to certain embodiments, a hybridoma cell line expressing such an immunoglobulin molecule is provided. According to certain 25 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 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 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, 25 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 30 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 5 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%, 8590%, 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 10 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 3IB) can be used in order to identify sections of the antibodies that can be modified by observing those variations 15 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, 20 13F, 13G, 13H, 131, 13J, and/or 31A and 3IB and variations are allowed in the positions identified as variable in the figures. The CDRs shown in FIGs. 13A, 13C, 13F, 13G, 31A and 3 IB 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, 25 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, 30 13G, and 31A and 3IB). 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 5 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 10 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 15 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 25 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, 30 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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2020201012 13 Feb 2020 embodiments, 1, 2, 3, or 4 FR (each being at least 90%, 90-95%, and/or 95-99% identical to the above sequences) is present.
In certain embodiments, an antigen binding protein comprises a light chain comprising a variable region comprising an amino acid sequence at least 90% identical 5 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 10 sequence at least 95% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In certain embodiments, an antigen binding protein comprises a light chain comprising a variable region comprising an amino acid sequence at least 99% identical to an amino acid sequence 15 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 20 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 determine suitable variants of the ABPs as set forth herein using well-known techniques. In certain embodiments, one skilled in the art can identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be 5 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 15 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, 20 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 25 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 30 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 5 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 10 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 15 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); 20 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 25 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 30 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
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2020201012 13 Feb 2020 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 Nlinked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants 5 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 10 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) 15 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 20 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 artrecognized polypeptide secondary and tertiary structures are described in Proteins, 25 Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company,
New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354:105 (1991), which are each incorporated herein by reference.
In some embodiments, the variants are variants of the nucleic acid sequences of 30 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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2020201012 13 Feb 2020 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, 5 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, 25 112, 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, 30 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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2020201012 13 Feb 2020 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 10 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 15 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 20 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 25 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 30 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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2020201012 13 Feb 2020 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 5 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. Lor 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 15 PCSK9, the catalytic domain alone, the mature form of PCSK9 shown in PIG. 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 20 human antibodies (see, for example, PCT Published Application No. W0 93/12227).
In certain embodiments, certain strategies can be employed to manipulate inherent properties of an antibody, such as the affinity of an antibody for its target. Such strategies include, but are not limited to, the use of site-specific or random mutagenesis of the polynucleotide molecule encoding an antibody to generate an 25 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 30 framework regions have been shown to contribute to the target binding properties of
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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 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 are “screened” to identify those antibody fragments having affinity for target. Thus, certain such 25 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 30 rearranged human V genes from peripheral blood lymphocytes. See, e.g., Mullinax et al., Proc Natl Acad Sci (USA), 87: 8095-8099 (1990).
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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 5 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 10 Genetics, 15:146-156 (1997), which are hereby incorporated by reference for any purpose.
Generally, fully human monoclonal ABPs (e.g., antibodies) specific for PCSK9 can be produced as follows. Transgenic mice containing human immunoglobulin genes are immunized with the antigen of interest, e.g. PCSK9, lymphatic cells (such as B15 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 20 provided.
In certain embodiments, fully human antibodies are produced by exposing human splenocytes (B or T cells) to an antigen in vitro, and then reconstituting the exposed cells in an immunocompromised mouse, e.g. SCID or nod/SCID. See, e.g., Brams et al., J.Immunol. 160: 2051-2058 (1998); Carballido et al., Nat. Med., 6: 10325 106 (2000). In certain such approaches, engraftment of human fetal tissue into SCID mice (SCID-hu) results in long-term hematopoiesis and human T-cell development. See, e.g., McCune et al., Science, 241:1532-1639 (1988); Ifversen et al., Sem. Immunol., 8:243-248 (1996). In certain instances, humoral immune response in such chimeric mice is dependent on co-development of human T-cells in the animals. See, 30 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); 5 Murphy et al., Blood, 86:1946-1953 (1995).
Thus, in certain embodiments, fully human antibodies can be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells. In other embodiments, antibodies can be produced using the phage display techniques described herein.
The antibodies described herein were prepared through the utilization of the
XenoMouse® technology, as described herein. Such mice, then, are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. Technologies utilized for achieving the same are disclosed in the patents, applications, and references 15 disclosed in the background section herein. In particular, however, a preferred embodiment of transgenic production of mice and antibodies therefrom is disclosed in U.S. Patent Application Serial No. 08/759,620, filed December 3, 1996 and International Patent Application Nos. WO 98/24893, published June 11, 1998 and WO 00/76310, published December 21, 2000, the disclosures of which are hereby incorporated by reference. See also Mendez et al., Nature Genetics, 15:146-156 (1997), the disclosure of which is hereby incorporated by reference.
Through the use of such technology, fully human monoclonal antibodies to a variety of antigens have been produced. Essentially, XenoMouse® lines of mice are immunized with an antigen of interest (e.g. PCSK9), lymphatic cells (such as B-cells) 25 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 30 PCSK9. Further, provided herein are characterization of the antibodies produced by
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2020201012 13 Feb 2020 such cell lines, including nucleotide and amino acid sequence analyses of the heavy and light chains of such antibodies.
The production of the XenoMouse® strains of mice is further discussed and delineated in U.S. Patent Application Serial Nos. 07/466,008, filed January 12, 1990, 5 07/610,515, filed November 8, 1990, 07/919,297, filed July 24, 1992, 07/922,649, filed
July 30, 1992, 08/031,801, filed March 15, 1993, 08/112,848, filed August 27, 1993, 08/234,145, filed April 28, 1994, 08/376,279, filed January 20, 1995, 08/430, 938, filed April 27, 1995, 08/464,584, filed June 5, 1995, 08/464,582, filed June 5, 1995, 08/463,191, filed June 5, 1995, 08/462,837, filed June 5, 1995, 08/486,853, filed June 10 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 B2, and 3 068 507 B2. See also European Patent No., EP 0 463 15 151 Bl, grant published June 12, 1996, International Patent Application No., WO
94/02602, published February 3, 1994, International Patent Application No., WO 96/34096, published October 31, 1996, WO 98/24893, published June 11, 1998, WO 00/76310, published December 21, 2000. The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference in their 20 entirety.
In an alternative approach, others, including GenPharm International, Inc., have utilized a “minilocus” approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more Vh genes, one or more DH genes, one or more Jh genes, a mu constant 25 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 30 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
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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, 5 08/155,301, filed November 18, 1993, 08/161,739, filed December 3, 1993,
08/165,699, filed December 10, 1993, 08/209,741, filed March 9, 1994, the disclosures of which are hereby incorporated by reference. See also European Patent No. 0 546 073 Bl, 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, 10 WO 97/13852, and WO 98/24884 and U.S. Patent No. 5,981,175, the disclosures of which are hereby incorporated by reference in their entirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et al., (1996), the disclosures of which are hereby incorporated by reference in their entirety.
Kirin has also demonstrated the generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced. See European Patent Application Nos. 773 288 and 843 961, the disclosures of which are hereby incorporated by reference. Additionally, KM™ mice, which are the result of cross-breeding of Kirin’s Tc mice with Medarex’s minilocus 20 (Humab) mice have been generated. These mice possess the human IgH transchromosome of the Kirin mice and the kappa chain transgene of the Genpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).
Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display (CAT, Morphosys, Dyax, Biosite/Medarex, 25 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 IgGl. The antibodies possessed high affinities, typically possessing 30 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 5 packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference). The transformation procedure used depends upon the host to be transformed. Methods for introducing 10 heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
Mammalian cell lines available as hosts for expression are well known in the art 15 and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Cell lines of particular preference are selected through 20 determining which cell lines have high expression levels and produce antibodies with constitutive PCSK9 binding properties.
In certain embodiments, antibodies and/or ABP are produced by at least one of the following hybridomas: 2IB 12, 31H4, 16F12, any of the other hybridomas listed in Table 2 or disclosed in the examples. In certain embodiments, antigen binding proteins 25 bind to PCSK9 with a dissociation constant (Kd) of less than approximately 1 nM, e.g., lOOOpM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, and/or 1 pM to 0.1 pM or less.
In certain embodiments, antigen binding proteins comprise an immunoglobulin molecule of at least one of the IgGl, IgG2, IgG3, IgG4, Ig E, IgA, IgD, and IgM isotype. In certain embodiments, antigen binding proteins comprise a human kappa 30 light chain and/or a human heavy chain. In certain embodiments, the heavy chain is of the IgGl, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments,
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2020201012 13 Feb 2020 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 IgGl, 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 IgGl, IgG3, IgE, IgA, IgD or IgM heavy chain. In other embodiments, 10 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 IgGl, IgG3, IgE, IgA, IgD or IgM heavy chain. In certain embodiments, antigen binding proteins comprise 15 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 iso type. In certain embodiments, antigen binding proteins have been cloned for expression in mammalian cells.
In certain embodiments, conservative modifications to the heavy and light 20 chains of antibodies from at least one of the hybridoma lines: 21B12, 31H4 and 16F12 (and corresponding modifications to the encoding nucleotides) will produce antibodies to PCSK9 having functional and chemical characteristics similar to those of the antibodies from the hybridoma lines: 21B12, 31H4 and 16F12. In addition, certain other modifications to the heavy and light chains of the antibodies from at least one of 25 the hybridoma lines: 21B12, 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 30 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
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2020201012 13 Feb 2020 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 5 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 non10 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 15 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 20 virus (or vector) or by transfection procedures known in the art, as exemplified by U.S.
Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference for any purpose). In certain embodiments, the transformation procedure used can depend upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are well known 25 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 30 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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2020201012 13 Feb 2020 hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. In certain embodiments, cell lines can be selected through determining which cell lines have high expression levels and produce antibodies with 5 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 10 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., 15 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 20 Expression System (Invitrogen, San Diego, CA), following the manufacturer's instructions. In certain embodiments, such a system relies on the pre-pro-alpha sequence to direct secretion. In certain embodiments, transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol.
In certain embodiments, a secreted polypeptide comprising one or more ABP 25 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 30 or more ABP components or the ABP itself is cloned into a baculovirus expression vector, such as pVL1393 (PharMingen, San Diego, CA). In certain embodiments, such
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2020201012 13 Feb 2020 a vector can be used according to the manufacturer's directions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free media and to produce recombinant polypeptide. In certain embodiments, a polypeptide is purified and concentrated from such media using a heparin-Sepharose column (Pharmacia).
In certain embodiments, a polypeptide comprising one or more ABP components or the ABP itself is expressed in an insect system. Certain insect systems for polypeptide expression are well known to those of skill in the art. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. In 10 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 15 example, inactivation of the polyhedrin gene results in the production of virus lacking coat protein.
In certain embodiments, recombinant viruses can be used to infect S. frugiperda cells or Trichoplusia larvae. See, e.g., Smith et al., J. Virol., 46: 584 (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA), 91: 3224-7 (1994).
In certain embodiments, polypeptides comprising one or more ABP components or the ABP itself made in bacterial cells are produced as insoluble inclusion bodies in the bacteria. In certain embodiments, host cells comprising such inclusion bodies are collected by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma, St. Louis, MO) for 15 minutes at room 25 temperature. In certain embodiments, the lysate is cleared by sonication, and cell debris is pelleted by centrifugation for f0 minutes at 12,000 X g. In certain embodiments, the polypeptide-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA; layered over 50% glycerol; and centrifuged for 30 minutes at 6000 X g. In certain embodiments, that pellet can be resuspended in standard phosphate 30 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 5 as a soluble protein. In certain embodiments, such GST fusion protein is purified using a GST Purification Module (Pharmacia).
In certain embodiments, it is desirable to “refold” certain polypeptides, e.g., polypeptides comprising one or more ABP components or the ABP itself. In certain embodiments, such polypeptides are produced using certain recombinant systems 10 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 15 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 20 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 25 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 30 crude fractionation of polypeptide fractionations from non-polypeptide fractions. In certain embodiments, polypeptides are purified using chromatographic and/or
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2020201012 13 Feb 2020 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 5 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 10 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 15 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 20 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 25 different forms of the same general purification scheme. For example, in certain embodiments, cation-exchange column chromatography performed utilizing an HPEC 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 30 polypeptide, or in maintaining binding activity of a polypeptide.
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In certain instances, the electrophoretic migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE. See, e.g., Capaldi et al., Biochem. Biophys. Res. Comm., 76: 425 (1977). It will be appreciated that under different electrophoresis conditions, the apparent molecular weights of purified or partially purified polypeptide can be different.
Exemplary Epitopes
Epitopes to which anti-PCSK9 antibodies useful in the methods provided herein bind are described. In some embodiments, epitopes that are bound by the presently 10 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 Cterminal domain. In some embodiments, the antigen binding protein binds to the substrate-binding groove of PCSK-9 (described in Cunningham et al., incorporated herein in its entirety by reference).
In some embodiments, the domain(s)/region(s) containing residues that are in contact with or are buried by an antibody can be identified by mutating specific residues in PCSK9 (e.g., a wild-type antigen) and determining whether the antigen binding protein can bind the mutated or variant PCSK9 protein. By making a number of individual mutations, residues that play a direct role in binding or that are in 25 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 30 specific example of this general approach utilizes an arginine/glutamic acid scanning protocol (see, e.g., Nanevicz, T., et al., 1995, J. Biol. Chem., 270:37, 21619-21625 and
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Zupnick, A., et al., 2006, J. Biol. Chem., 281:29, 20464-20473). In general, arginine and glutamic acids are substituted (typically individually) for an amino acid in the wildtype 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 5 region of the antigen where the mutation is introduced. Arginines that exist in the wildtype 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 10 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 15 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 20 affinity, EC50, and/or capacity between an antigen binding protein and a mutant PCSK9 antigen is reduced by greater than 10%, greater than 20%, greater than 40 %, greater than 50 %, greater than 55 %, greater than 60 %, greater than 65 %, greater than 70 %, greater than75 %, greater than 80 %, greater than 85 %, greater than 90% or greater than 95% relative to binding between the antigen binding protein and a wild 25 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 30 protein and a wild-type PCSK9 protein (for example, the protein of SEQ ID NO: 1
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2020201012 13 Feb 2020 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 wild5 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, V538R, E539R, T132R, S351R, A390R, A413R, 10 E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E,
H521R, and Q554R as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303. In the shorthand notation used here, the format is: Wild type residue: Position in polypeptide: Mutant residue, with the numbering of the residues as indicated in SEQ ID NO: 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: 20 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: 25 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 30 (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, T132R, S351R, A390R, A413R, E582R, D162R, R164E, E167R, S123R, E129R, A311R, 5 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 10 following mutations: R207E, D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R, and E582R within SEQ ID NO: 1 or SEQ ID NO: 303, as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303). In some embodiments, the binding is reduced. In some embodiments, the reduction in binding is observed as a change in EC50. In some embodiments, the change in EC50 is 15 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 20 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303). In some embodiments, the binding is reduced. In some embodiments, the reduction in binding is observed as a change in Bmax. In some embodiments, the shift in Bmax is a reduction of the maximum signal generated by the ABP. In some embodiments, for an amino acid to be part of an epitope, the Bmax is reduced by at least 10%, for example, reductions of at least any of 25 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 wildtype 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. 30 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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2020201012 13 Feb 2020 embodiments can be compared to an allelic sequence, rather than purely the wild-type sequence shown in FIG. 1A
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 5 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 10 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 15 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 20 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 2IB 12.
In some embodiments, the antigen binding protein binds to a region containing 25 at least one of amino acid 185 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, the ABP competes with ABP 31H4.
In some embodiments, the antigen binding protein binds to a region containing at least one of amino acids 439, 513, 538, and/or 539 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one (e.g., 2, 3, or 4) of the identified residues 30 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 5 some embodiments, the ABP competes with ABP 12H11.
In some embodiments, the antigen binding protein binds to a region containing at least one of amino acid 582, 519, 521, and/or 554 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one (e.g., 2, 3, or 4) of the identified residues are part of the region that is bound by the ABP. In some embodiments, the ABP 10 competes with ABP 3 C4.
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 15 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 20 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 25 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 30 (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 2IB 12 binds to an epitope including residues 162167 (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 5 (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 10 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 15 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 30 parenteral administration (including but not limited to intravenous, intramuscular, subcutaneous, aerosolized, intrapulmonary, intranasal, or intrathecal) to a patient in
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2020201012 13 Feb 2020 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 5 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 10 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 15 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 HPFC (“SEC-HPFC”), cation-exchange HPLC (CEXHPLC), Subvisible Particle Detection by Light Obscuration (“HIAC”) and/or visual inspection.
In certain embodiments, the pharmaceutical formulation comprises any of the antigen binding proteins to PCSK9 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 3IB. In certain other embodiments, the pharmaceutical 25 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 3IB, 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 3 IB. In still other embodiments, the pharmaceutical formulation comprises any of the antigen binding
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2020201012 13 Feb 2020 proteins to PCSK9 depicted in Table 2 and FIGs. 2 and/or 3 and FIGs 31A and 3 IB. 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 5 some embodiments the pharmaceutical formulation comprises any one of 2IB 12, 26H5, 31H4, 8A3, 11F1 or 8A1.
In some embodiments, the pharmaceutical formulation comprises more than one different antigen binding protein to PCSK9. In certain embodiments, pharmaceutical formulations comprise more than one antigen binding protein to PCSK9 wherein the 10 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 15 therapeutic molecule is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of the ABP), and dextran. Such vehicles are described, e.g., in U.S. Application Serial No. 09/428,082, now US Patent No. 6,660,843 and published PCT Application No. WO 99/25044, which are hereby incorporated by reference for any 20 purpose.
In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In some embodiments, the formulation material(s) are for s.c. and/or I.V. administration. In certain embodiments, the pharmaceutical formulation comprises formulation materials 25 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, 30 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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2020201012 13 Feb 2020 phosphate (“NaOAC”), Tris-HCl, Tris buffer, citrates, phosphate buffer, phosphatebuffered 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 5 or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose,, fructose, lactose, mannose, trehelose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counter ions 10 (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates 15 such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants.
(Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack 20 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 25 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 30 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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2020201012 13 Feb 2020 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 2IB 12, 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, 5 about 130 mg/ml, about 140 mg/ml, or about 150 mg/ml. In some embodiments, the concentration of 8A3, 11F1 or 8Al ranges from about 140 mg/ml to about 220 mg/ml, e.g., 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 210 mg/ml, about 220 mg/ml, or about 250 mg/ml.
In another aspect, the pharmaceutical formulation comprises at least one buffering agent such as, for example, sodium acetate, sodium chloride, phosphates, phosphate buffered saline (“PBS”), and/or Tris buffer of about pH 7.0-8.5. The buffer serves to maintain a physiologically suitable pH. In addition, the buffer can serve to enhance isotonicity and chemical stability of the pharmaceutical formulation. In certain 15 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 lOOnM buffering agent, inclusiveof all values in between. In certain embodiments, the buffeming agent is 20 NaOAC. Exemplary pHs of the pharmaceutical formulation include from about 4 to about 6, or from about 4.8 to about 5.8, or from about 5.0 to about 5.2, or about 5, or about 5.2.
In certain embodiments, the pharmaceutical formulation is isotonic with an osmolality ranging from between about 250 to about 350 miliosmol/kg, e.g., about 250 25 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 30 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 5 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% 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 15 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 20 sorbitol, mannitol, glycerol and dulcitol) and/or a disaccharide (including but not limited to sucrose, lactose, maltose and threhalose) and/or an amino acid (including but not limited to proline, arginine, lysine, methionine, and taurine) and or benzyl alcohol; the total of said polyhydroxy hydrocarbon and/or disaccharide and/or amino acid and/or benzyl alchol being about 0.5% to about 10% w/v of the formulation. In certain 25 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 30 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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2020201012 13 Feb 2020 embodiments, the pharmaceutical formulation comprises a stabilizing agent at a concentration of about 9% sorbital. In certain embodiments, the pharmaceutical formulation comprises a a stabilizing agent at a concentration of about 1%, about 2%, about 3%, about 4%, about 5% proline, arginine, lysine, methionine, and/or taurine. In 5 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 10 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 cP=l 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 25 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 sample. In certain embodiments, the pharmaceutical formulation stability is measured using size exclusion HPLC (“SECHPLC”). SEC-HPLC separates proteins based on differences in their hydrodynamic
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2020201012 13 Feb 2020 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 10 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 15 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 iso form 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 20 about a 2% decrease, no more than about a 1% decrease, no more than about a 0.5% decrease in the main isoform peak as compared to a control sample.
In certain embodiments, the pharmaceutical formulation stability is measured using Subvisible Particle Detection by Light Obscuration (“HIAC”). An electronic, liquid-home particle-counting system (HIAC/Royco 9703 or equivalent) containing a 25 light-obscuration sensor (HIAC/Royco HRLD-150 or equivalent) with a liquid sampler quantifies the number of particles and their size range in a given test sample. When particles in a liquid pass between the light source and the detector they diminish or “obscure” the beam of light that falls on the detector. When the concentration of particles lies within the normal range of the sensor, these particles are detected one-by30 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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2020201012 13 Feb 2020 reduced. The changes in the voltage register as electrical pulses that are converted by the instrument into the number of particles present. The method is non-specific and measures particles regardless of their origin. Particle sizes monitored are generally 10 Um, and 25 Um. In the case of HIAC, a stable pharmaceutical formulation should exhibit no more than 6000 I Ogm 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, I Oiim 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 25gm 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 25iim 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 20 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 25 comprises: (i) about 70 mg/ml to about 250 mg/ml of antigen binding protein to PCSK9; (ii) about 0.05 mM to about 40 mM of a buffer such as sodium acetate (“NaOAC”) serves as a buffering agent; (iii) about 1% to about 5% proline, arginine, lysine, methionine, or taurine (also know as 2-aminoethanesulfonic acid) and/or 0.5% to about 5% benzyl alcohol which serves as a stabilizing agent; and (iv) about 0.004% 30 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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2020201012 13 Feb 2020 formulation has a pH in the range of about 4.0 to 6.0. In certain other embodiments, pharmaceutical formulations of this invention comprise (i) at least about 70 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml of an 5 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, 10 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 15 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; 20 (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 25 formulation optionally comprises about 80 mM to about 300 mM proline, arginine, lysine, methionine, or taurine and/or 0.5% to about 5% benzyl alcohol which serves to reduce viscosity. In certain other embodiments, pharmaceutical formulations of this invention comprise (i) at least about 70 mg/ml to about 250 mg/ml of the anti-PCSK9 antibody; (ii) about 10 mM NAOAC; (iii) about 9% sucrose; and (iv) about 0.004% 30 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
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2020201012 13 Feb 2020 mg/ml, about 100 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160 mg/ml, about 180 mg/ml, about 200 mg/ml of an anti-PCSK9 antibody; (ii) about 15 mM NAOAC; (iii) about 9% sucrose; and (iv) about 0.01% polysorbate 20, wherein the formulation has a pH of about 5.2. In certain other embodiments, pharmaceutical 5 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) 10 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 15 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 20 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 25 administered prior to, concurrent with, and subsequent to treatment with a cholesterollowering (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 30 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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2020201012 13 Feb 2020 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 10 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 15 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 20 one additional therapeutic agent, can be formulated as a lyophilizate using appropriate excipients.
In certain embodiments, when parenteral administration is contemplated, a therapeutic formulation can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a desired antigen binding protein to PCSK9, with or 25 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 30 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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2020201012 13 Feb 2020 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 5 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 10 binding protein to PCSK9, with or without at least one additional therapeutic agent, can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in PCT application no. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.
f5 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 20 certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
Additional pharmaceutical formulations will be evident to those skilled in the 25 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 30 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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2020201012 13 Feb 2020 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 5 gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly (2hydroxyethyl-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 10 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, 15 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 20 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) 25 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 readyto-use form
In certain embodiments, kits are provided for producing a single-dose 30 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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2020201012 13 Feb 2020 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 5 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 10 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 15 implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
Dosage and Dosing Regimens
Any of the antigen binding proteins to PCSK9 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 25 and FIGS. 31A and 3IB 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 30 3 and FIGs 31A and 3 IB, 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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2020201012 13 Feb 2020 to PCSK9 depicted in Table 2 and FIGs. 2 and/or 3 and FIGs 31A and 3IB 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 5 13 and/or FIG.31A and 3IB 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 10 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, 15 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 pg/kg to up to about 100 mg/kg or more of antigen binding protein to PCSK9,. In certain embodiments, the 20 dosage can range from 0.1 pg/kg up to about 100 mg/kg; or 1 pg/kg up to about 100 mg/kg; or 5 pg/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 30 700 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 5 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 10 180 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 15 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 20 same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. The formulation can also be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneious delivery, pen delivery devices, as well as autoinjector delivery devices, have applications in delivering a pharmaceutical formulation of the present invention. 25 Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate doseresponse 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 30 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 20 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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2020201012 13 Feb 2020 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, 5 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 10 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 15 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 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 30 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 5 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 10 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 15 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 20 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 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 30 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 5 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 10 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 15 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 20 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 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 30 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 serum LDL cholesterol from an abnormally high level 10 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 15 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 20 level is reduced by at least about 50%. In certain embodiments, the serum LDL cholesterol level is reduced by at least about 55%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 60%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 65%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 70%. In some 25 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.
2020201012 13 Feb 2020
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, embodiments, embodiments, 10 embodiments, embodiments, embodiments, embodiments, embodiments, 15 embodiments, embodiments, embodiments, the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value the serum PCSK9 value is reduced by at least is reduced by at least is reduced by at least is reduced by at least is reduced by at least is reduced by at least is reduced by at least is reduced by at least is reduced by at least is reduced by at least is reduced by at least about 35%. In some about 40%. In some about 45%. In some about 50%. In some about 55%. In some about 60%. In some about 65%. In some about 70%. In some about 75%. In some about 80%. In some 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 high level or even a normal level. In some embodiments, the total cholesterol level is reduced by at least 25 about 20% as compared to a predose level. In some embodiments, the total cholesterol level is reduced by at least about 25%. level is reduced by at least about 30%. level is reduced by at least about 35%. level is reduced by at least about 40%.
level is reduced by at least about 45%. level is reduced by at least about 50%.
In some embodiments, the total cholesterol In some embodiments, the total cholesterol In some embodiments, the total cholesterol In some embodiments, the total cholesterol In some embodiments, the total cholesterol In some embodiments, the total cholesterol
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2020201012 13 Feb 2020 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 nonHDL cholesterol level in a patient diagnosed with homozygous familial 5 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-HDL cholesterol level is reduced by at 10 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 15 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 nonHDL 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, 25 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 30 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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2020201012 13 Feb 2020 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 5 level is reduced by at least about 70%. In some embodiments, the ApoB level is reduced by at least about 75%.
In one aspect, an antigen binding protein to PCSK9 is used to modulate the Lp(a) levels in a patient diagnosed with homozygous familial hypercholesterolemia. In certain embodiments, the antigen binding protein to PCSK9 is neutralizing. In some 10 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, 15 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, 20 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 30 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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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 5 cholesterol) agent. In some embodiments, the agent increases the expression of LDLR, have been observed to increase serum HDL levels, lower LDL levels or lower triglyceride levels. Exemplary agents include, but are not limited to, statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), Nicotinic acid (Niacin) (NIACOR, NIASPAN (slow release 10 niacin), SLO-NIACIN (slow release niacin), CORDAPTIVE (laropiprant)), Fibric acid (LOPID (Gemfibrozil), TRICOR (fenofibrate), Bile acid sequestrants (QUESTRAN (cholestyramine), colesevelam (WELCHOL), COLESTID (colestipol)), Cholesterol absorption inhibitors (ZETIA (ezetimibe)), Combining nicotinic acid with statin (ADVICOR (LOVASTATIN and NIASPAN), Combining a statin with an absorption 15 inhibitor (VYTORIN (ZOCOR and ZETIA) and/or lipid modifying agents. In some embodiments, the ABP is combined with PPAR gamma agonsits, PPAR alpha/gamma agonists, squalene synthase inhibitors, CETP inhibitors, anti-hypertensives, antidiabetic agents (such as sulphonyl ureas, insulin, GLP-1 analogs, DDPIV inhibitors, e.g., metaformin), ApoB modulators, such as mipomersan, MTP inhibitoris and /or 20 arteriosclerosis obliterans treatments. In some embodiments, the ABP is combined with an agent that increases the level of LDLR protein in a subject, such as statins, certain cytokines like oncostatin M, estrogen, and/or certain herbal ingredients such as berberine. In some embodiments, the ABP is combined with an agent that increases serum cholesterol levels in a subject (such as certain anti-psycotic agents, certain HIV 25 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 5 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 XenoMouse® mice (Abgenix, Fremont, 15 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 IgG2K and IgG2X antibodies. Group 1 mice were immunized with human PCSK9. PCSK9 was prepared using standard recombinant techniques using the GenBank 20 sequence as reference (NM_174936). Group 2 involved mice of the XenoMouse® strain XMG4-KL, which produce fully human IgG4K and IgG4X 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 25 total of 10 fig of antigen delivered intraperitoneally into the abdomen. Subsequent boosts are 5ug doses and injection method is staggered between intraperitoneal injections into the abdomen and sub-cutaneous injections at the base of the tail. For intraperitoneal injections antigen is prepared as an emulsion with TiterMax® Gold (Sigma, Cat # T2684) and for subcutaneous injections antigen is mixed with Alum 30 (aluminum phosphate) and CpG oligos. In injections 2 through 8 and 10, each mouse was injected with a total of 5 fig of antigen in the adjuvant alum gel. A final injection
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PCT/US2013/048714 of 5 μg of antigen per mouse is delivered in Phospho buffered saline and delivered into sites 50% IP 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 El
mouse strain XMG2/kl XMG4/kl
# of animals 10 10
immunogen PCSK9-V5/His IP injection 10ug each PCSK9-V5/His IP injection 10ug each
1st boost
Titermax Gold Titermax Gold
2nd boost tail injection 5ug each tail injection 5ug each
Alum/CpG ODN Alum/CpG ODN
3rd boost IP injection 5ug each IP injection 5ug each
Titermax Gold Titermax Gold
4th boost tail injection 5ug each tail injection 5ug each
Alum/CpG ODN Alum/CpG ODN
5th boost IP injection 5ug each IP injection 5ug each
Titermax Gold Titermax Gold
6th boost tail injection 5ug each tail injection 5ug each
Alum/CpG ODN Alum/CpG ODN
7th boost IP injection 5ug each IP injection 5ug each
Titermax Gold Titermax Gold
8th boost tail injection 5ug each tail injection 5ug each
Alum/CpG ODN Alum/CpG ODN
bleed
9th boost IP injection 5ug each IP injection 5ug each
Titermax Gold Titermax Gold
10th boost tail injection 5ug each tail injection 5ug each
Alum/CpG ODN Alum/CpG ODN
11th boost BIP 5ug each BIP 5ug each
PBS PBS
harvest
The protocol used to titer the XenoMouse animals was as follows: Costar 3368 medium binding plates were coated with neutravadin @ 8ug/ml (50ul/well) and incubated at 4°C in lXPBS/0.05% azide overnight. They were washed using TiterTek 3-cycle wash with RO water. Plates were blocked using 250ul of 1XPBS/I%milk and
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2020201012 13 Feb 2020 incubated for at least 30 minutes at RT. Block was washed off using TiterTek 3-cycle wash with RO water. One then captured b-human PCSK9 @ 2ug/ml in lXPBS/l%milk/10mM Ca2+ (assay diluent) 50ul/well and incubated for Ihr at RT. One then washed using TiterTek 3-cycle wash with RO water. For the primary 5 antibody, sera were titrated 1:3 in duplicate from 1:100. This was done in assay diluent 50ul/well and incubated for Ihr at RT. One then washed using TiterTek 3-cycle wash with RO water. The secondary antibody was goat anti Human IgG Fc HRP @ 400 ng/ml in assay diluent at 50ul/well. This was incubated for Ihr at RT. This was then washed using TiterTek 3-cycle wash with RO water and patted dry on paper towels. 10 For the substrate, one-step TMB solution (Neogen, Lexington, Kentucky) was used (50ul/well) and it was allowed to develop for 30 min at RT.
The protocols followed in the ELISA assays were as follows: For samples comprising b-PCSK9 with no V5His tag the following protocol was employed: Costar 3368 medium binding plates (Coming Life Sciences) were employed. The plates were 15 coated with neutravadin at 8 μg/ml in lXPBS/0.05%Azide, (50 μΙ/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 μΐ of 1XPBS/1% milk and incubated approximately 30 minutes at room temperature. The plates were then washed using the M384 plate washer. A 320 cycle wash was performed. The capture was b-hu PCSK9, without a V5 tag, and was added at 2 μg/ml in lXPBS/l%milk/10mM Ca2+ (40 μΙ/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 μΙ/well. The plates were incubated for 1 hour 25 at room temperature. Next, a 3-cycle wash was performed. Goat anti Human IgG Fc HRP at 100 ng/ml (1:4000) in lXPBS/l%milk/10mM Ca2+ (50 μΙ/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 μΙ/well) was added to the plate and 30 was quenched with IN hydrochloric acid (50 μΙ/well) 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 μg/ml in assay diluent. LDLR was detected with goat anti LDLR (R&D Systems, Cat #AF2148) and rabbit 5 anti goat IgGFc HRP at a concentration of 400 ng/ml; the rabbit polyclonal was detected with goat anti-rabbit IgG Fc at a concentration of 400 ng/ml in assay diluent. Negative control was naive XMG2-KL and XMG4-KL sera titrated 1:3 in duplicate from 1:100 in assay diluent.
For samples comprising b-PCSK9 with a V5His tag the following protocol was 10 employed: Costar 3368 medium binding plates (Coming Life Sciences) were employed. The plates were coated with neutravadin at 8 μg/ml in lXPBS/0.05%Azide, (50 μΙ/wcll). 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 μΐ of 1XPBS/1% milk and incubated 15 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 μg/ml in lXPBS/l%milk/10mM Ca2+ (40 μΐ/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 20 sera. The titration was done in assay diluent, at a volume of 50 μΐ/well. The plates were incubated for 1 hour at room temperature. Next, the plates were washed using the M384 plate washer operated using a 3-cycle wash. Goat anti Human IgG Fc HRP at 400 ng/ml in lXPBS/l%milk/10mM Ca2+ was added at 50 μΙ/wcll to the plate and the plate was incubated 1 hour at room temperature. The plates were washed once again, 25 using a 3-cycle wash. The plates were then patted dry with paper towel. Finally, 1 step TMB (Neogen, Lexington, Kentucky) (50 μΐ/well) was added to the plate and the plate was quenched with IN hydrochloric acid (50 μΐ/well) after 30 minutes at room temperature. OD's were read immediately at 450 nm using a Titertek plate reader.
Positive control was LDLR, rabbit anti-PCSK9 titrated 1:3 in duplicate from 3 30 μg/ml in assay diluent. LDLR detect with goat anti-LDLR (R&D Systems, Cat #AF2148) and rabbit anti-goat IgG Fc HRP at a concentration of 400 ng/ml; rabbit poly
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2020201012 13 Feb 2020 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 μg/ml in assay diluent; both detected with goat anti-human IgG Fc HRP at a concentration of 400 ng/ml in assay diluent. Negative control was naive XMG2-KL and XMG4-KL sera 5 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 10 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/ml 2ug/ml
P175807 >72900 @ OD 2.2 68359
P175808 >72900 @ OD 2.3 >72900 @ OD 2.5
P175818 >72900 @ OD 3.2 >72900 @ OD 3.0
Group 1 - P175819 >72900 @ OD 3.4 >72900 @ OD 3.2
P175820 >72900 @ OD 2.4 >72900 @ OD 2.5
lgG2k/l P175821 >72900 @ OD 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
Group 2 - P174509 23380 21628
P174510 15120 9673
lgG4k/l P175773 19407 15973
P175774 54580 44424
P175775 60713 55667
P175776 30871 22899
P175777 16068 12532
Naive
G2 < 100 @ OD 0.54 < 100 @OD 0.48
Naive < 100 @OD 1.57 < 100 @OD 1.32
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Titer Titer
Animal ID b-hu PCSK9 (V5His) @ 2ug/ml b-hu PCSK9 @ 2ug/ml
G4
EXAMPLE 2
Recovery of Lymphocytes, B-cell Isolations, Fusions and Generation of Hybridomas
This example outlines how the immune cells were recovered and the hybridomas were generated. Selected immunized mice were sacrificed by cervical dislocation and the draining lymph nodes were harvested and pooled from each cohort. The B cells were dissociated from lymphoid tissue by grinding in DMEM to release the 10 cells from the tissues, and the cells were suspended in DMEM. The cells were counted, and 0.9 ml DMEM per 100 million lymphocytes was added to the cell pellet to resuspend the cells gently but completely.
Lymphocytes were mixed with nonsecretory myeloma P3X63Ag8.653 cells purchased from ATCC, cat.# CRL 1580 (Kearney et al., (1979)7. Immunol. 123, 154815 1550) at a ratio of 1:4. The cell mixture was gently pelleted by centrifugation at 400 x g 4 min. After decanting of the supernatant, the cells were gently mixed using a 1 ml pipette. Preheated PEG/DMSO solution from Sigma (cat# P7306) (1 ml per million of B-cells) was slowly added with gentle agitation over 1 min followed by 1 min of mixing. Preheated ID MEM (2 ml per million of B cells) (DMEM without glutamine, 20 L-glutamine, pen/strep, MEM non-essential amino acids (all from Invitrogen), was then added over 2 minutes with gentle agitation. Finally preheated IDMEM (8 ml per 106 B-cells) was added over 3 minutes.
The fused cells were spun down 400 x g 6 min and resuspended in 20 ml selection media (DMEM (Invitrogen), 15 % FBS (Hyclone), supplemented with L25 glutamine, pen/strep, MEM Non-essential amino acids, Sodium Pyruvate, 2Mercaptoethanol (all from Invitrogen), HA-Azaserine Hypoxanthine and OPI (oxaloacetate, pyruvate, bovine insulin) (both from Sigma) and IL-6 (Boehringer Mannheim)) per million B-cells. Cells were incubated for 20-30 min at 37C and then
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2020201012 13 Feb 2020 resuspended in 200 ml selection media and cultured for 3-4 days in T175 flask prior to well plating. Thus, hybridomas that produced antigen binding proteins to PCSK9 were produced.
EXAMPLE 3
Selection of PCSK9 Antibodies
The present example outlines how the various PCSK9 antigen binding proteins were characterized and selected. The binding of secreted antibodies (produced from the hybridomas produced in Examples 1 and 2) to PCSK9 was assessed. Selection of 10 antibodies was based on binding data and inhibition of PCSK9 binding to LDLR and affinity. Binding to soluble PCSK9 was analyzed by ELISA, as described below. BIAcore® (surface plasmon resonance) was used to quantify binding affinity.
Primary Screen
A primary screen for antibodies which bind to wild-type PCSK9 was performed. The primary screen was performed on two harvests. The primary screen comprised an ELISA assay and was performed using the following protocol:
Costar 3702 medium binding 384 well plates (Coming Life Sciences) were employed. The plates were coated with neutravadin at a concentration of 4 μg/ml in 20 lXPBS/0.05%Azide, at a volume of 40 μΐ/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 μΐ of 1XPBS/I%milk and incubated approximately 30 minutes at room temperature. The plates were then washed. Again, a 3-cycle wash was performed. The capture sample 25 was biotinylated-PCSK9, without a V5 tag, and was added at 0.9 μg/ml in lXPBS/l%milk/10mM Ca2+ at a volume of 40 μΐ/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 μΐ of supernatant was transferred into 40 μΐ of lXPBS/l%milk/10mM Ca2+ and incubated 1.5 hours at room temperature. Again 30 the plates were washed using the Titertek plate washer operated using a 3-cycle wash.
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2020201012 13 Feb 2020 μΐ/well of Goat anti-Human IgG Fc POD at a concentration of 100 ng/ml (1:4000) in lXPBS/l%milk/10mM 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 μΐ/well of One-step TMB (Neogen, Lexington, Kentucky) was added to the plate and 5 quenching with 40 μΐ/well of IN 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 10 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: 15 Costar 3702 medium binding 384 well plates (Coming Life Sciences) were employed.
The plates were coated with neutravadin at 3 μg/ml in lXPBS/0.05%Azide at a volume of 40 μΐ/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 μΐ of 1XPBS/I%milk and incubated 20 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 bPCSK9, without a V5 tag, and was added at 0.9 μg/ml in lXPBS/l%milk/10mM Ca2+ at a volume of 40 μΐ/well. The plates were then incubated for 1 hour at room temperature. Next, the plates were washed using a 3-cycle wash. 10 μΐ of supernatant 25 was transferred into 40 μΐ of lXPBS/l%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. 40 μΐ/well of Goat anti-Human IgG Fc POD at a concentration of 100 ng/ml (1:4000) in lXPBS/l%milk/10mM Ca2+ was added to the plate, and the plate was incubated 1 hour at room temperature. The plates were washed 30 once again, using the Titertek plate washer operated using a 3-cycle wash. Finally, 40
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2020201012 13 Feb 2020 μΐ/well of One-step TMB (Neogen, Lexington, Kentucky) was added to the plate and was quenched with 40 μΙ/wcll 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 10 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 μg/ml in lXPBS/0.05%Azide at a volume of 40 μΐ/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 15 performed. The plates were blocked with 90 μΐ of 1XPBS/I%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 μg/ml in lXPBS/l%milk/10mM Ca2+ at a volume of 40 μΐ/well. The plates were then incubated for 1 hour at room 20 temperature. Next, the plates were washed using the Titertek plate washer operated using a 3-cycle wash. 50 μΐ of supernatant was transferred to the plates and incubated 1 hour at room temperature. Again the plates were washed using a 3-cycle wash. 40 μΐ/well of Goat anti-Human IgG Fc POD at a concentration of 100 ng/ml (1:4000) in lXPBS/l%milk/10mM Ca2+ was added to the plate and the plate was incubated 1 hour 25 at room temperature. The plates were washed once again, using a 3-cycle wash. Finally, 40 μΐ/well One-step TMB (Neogen, Lexington, Kentucky) was added to the plate and was quenched with 40 μΐ/well of IN hydrochloric acid after 30 minutes at room temperature. OD’s were read immediately at 450 nm using a Titertek plate reader. 579 antibodies were observed to cross-react with mouse PCSK9. These 30 antibodies were then used in the subsequent screenings.
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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 5 hypercholesterolemia in a Utah pedigree”, Hum. Genet. 114: 349-353, 2004). In order to determine if the antibodies were specific for the wild type or also bound to the D374Y form of PCSK9, the samples were then screened for binding to the mutant PCSK9 sequence comprising the mutation D374Y. The protocol for the screen was as follows: Costar 3702 medium binding 384 well plates (Coming Life Sciences) were 10 employed in the screen. The plates were coated with neutravadin at 4 μg/ml in lXPBS/0.05% Azide at a volume of 40 μΐ/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 μΐ of 1XPBS/I%milk and incubated approximately 30 minutes at room temperature. The 15 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 μg/ml in lXPBS/l%milk/10mMCa2+ and incubated for 1 hour at room temperature. The plates were then washed using a Titertek plate washer. A 3cycle wash was performed. Late exhaust hybridoma culture supernatant was diluted 20 1:5 in PBS/milk/Ca2+ (10 ml plus 40 ml) and incubated for 1 hour at room temperature.
Next, 40 μΐ/well of rabbit anti-human PCSK9 (Cayman Chemical) and human anti-His 1.2.3 1:2 at lug/ml in lXPBS/l%milk/10mMCa2+ 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 μΐ/well of Goat anti25 Human IgG Fc HRP at a concentration of 100 ng/ml (1:4000) in lXPBS/l%milk/10mM Ca2+ was added to the plate and the plate was incubated 1 hour at room temperature. 40 μΐ/well of Goat anti-rabbit IgG Fc HRP at a concentration of 100 ng/ml (1:4000) in lXPBS/l%milk/10mM Ca2+ was added to the plate and the plate was incubated 1 hour at room temperature. The plates were then washed using a 30 Titertek plate washer. A 3-cycle wash was performed. Finally, 40 μΐ/well of One-step TMB (Neogen, Lexington, Kentucky) was added to the plate and was quenched with 40
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2020201012 13 Feb 2020 μΐ/well of IN 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 10 receptor ligand blocking screen: Costar 3702 medium binding 384 well plates (Coming Life Sciences) were employed in the screen. The plates were coated with goat anti-LDLR (R&D Cat &AE2148) at 2 μg/ml in lXPBS/0.05%Azide at a volume of 40 μΐ/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 15 performed. The plates were blocked with 90 μΐ of 1XPBS/1% milk and incubated approximately 30 minutes at room temperature. The plates were then washed using the Titertek plate washer. A 3-cycle wash was performed. The capture sample was LDLR (R&D, Cat &2148LD/CE), and was added at 0.4 μg/ml in lXPBS/l%milk/10mM Ca2+ at a volume of 40 μΐ/well. The plates were then incubated for 1 hour and 10 minutes at 20 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 25 wash. 50 μΙ/wcll 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 bPCSK9, 40 μΙ/wcll 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. Linally, 30 40 μΐ/well of One-step TMB (Neogen, Lexington, Kentucky) was added to the plate and was quenched with 40 μΐ/well of IN hydrochloric acid after 30 minutes at room
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2020201012 13 Feb 2020 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 10 inhibition assay. The same protocol was employed in the screen of the 384 member blocker subset assay as was done in the large scale receptor ligand blocking screen. This repeat screen confirmed the initial screening data.
This screen of the 384 member subset identified 85 antibodies that blocked interaction between the PCSK9 mutant enzyme and the LDLR greater than 90%.
Receptor Ligand Binding Assay of Blockers that Bind the Wild Type PCSK9 but not the D374Y Mutant
In the initial panel of 3000 sups there were 86 antibodies shown to specifically bind to the wild-type PCSK9 and not to the huPCSK9(D374Y) mutant. These 86 sups 20 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 (Coming Life Sciences) were employed in the screen. The plates were coated with anti-His 1.2.3 at 10 μg/ml in lXPBS/0.05% Azide at a volume of 40 μΐ/well. The plates were incubated at 4°C overnight. The plates were then washed using a Titertek 25 plate washer (Titertek, Huntsville, AL). A 3-cycle wash was performed. The plates were blocked with 90 μΐ of 1XPBS/I%milk and incubated approximately 30 minutes at room temperature. The plates were then washed using the Titertek plate washer. A 3cycle wash was performed. LDLR (R&D Systems, &2148LD/CF or R&D Systems, &2148LD) was added at 5 μg/ml in lXPBS/l%milk/10mM Ca2+ at a volume of 40 30 μΐ/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 μΐ of hybridoma sup was transferred into 33ul of b-PCSK9 at a concentration of 583 ng/ml in lXPBS/l%milk/10mMCa2+, giving a final b-PCSK9 concentration = 350 ng/ml and 5 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 μΐ/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 μΐ/well streptavidin HRP at 500 ng/ml in 10 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 μΐ/well of One-step TMB (Neogen, Lexington, Kentucky) was added to the plate and was quenched with 40 μΐ/well of IN hydrochloric acid after 30 minutes at room temperature. OD’s were read immediately 15 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 20 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 25 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 30 testing, as described herein. As a result of the screening described herein, the
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EXAMPLE 4.1
Production of Human 31H4 IgG4 Antibodies from Hybridomas
This example generally describes how one of the antigen binding proteins was produced from a hybridoma line. The production work used 50ml exhaust supernatant generation followed by protein A purification. Integra production was for scale up and was performed later. Hybridoma line 31H4 was grown in T75 flasks in 20 ml of media 10 (Integra Media, Table 5). When the hybridoma was nearly confluent in the T75 flasks, it was transferred to an Integra flask (Integra Biosciences, Integra CL 1000, 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 15 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 (IL) 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 20 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 25 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 (0.22pm). Clarified conditioned media was loaded onto a Protein A30 Sepharose column. Optionally, the media can be first concentrated and then loaded onto a Protein A Sepharose column. Non-specific bindings were removed by an
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2020201012 13 Feb 2020 extensive PBS wash. Bound antibody proteins on the Protein A column were recovered by standard acidic antibody elution from Protein A columns (such as 50 mM Citrate, pH 3.0). Aggregated antibody proteins in the Protein A Sepharose pool were removed by size exclusion chromatography or binding ion exchange chromatography on anion exchanger resin such as Q Sepharose resin. The specific IEX conditions for the 31H4 proteins are Q-Sepharose HP at pH 7.8-8.0. Antibody was eluted with a NaCl gradient of 10 mM-500 mM in 25 column volumes.
TABLE 4.1
Composition of Media
INTEGRA MEDIA
HSFM
10% Ultra Low IgG serum
2mmol/L L-glutamine
1% NEAA
4g/L glucose
EXAMPLE 4.2
Production of Recombinant 31H4 Human IgG2
Antibodies From Transfected Cells
The present example outlines how 31H4 IgG2 antibodies were produced from transfected cells. 293 cells for transient expression and CHO cells for stable expression were transfected with plasmids that encode 31H4 heavy and light chains. Conditioned media from transfected cells was recovered by removing cells and cell debris. Clarified conditioned media was loaded onto a Protein A-Sepharose column. Optionally, the 20 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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2020201012 13 Feb 2020 binding ion exchange chromatography on anion exchanger resin such as Q Sepharose resin. The specific IEX conditions for the 31H4 proteins are Q-Sepharose HP at pH 7.8-8.0. The antibody was eluted with a NaCl gradient of 10 mM-500 mM in 25 column volumes.
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EXAMPLE 5
Production of Human 2 IB 12 IgG4 Antibodies from Hybridomas
The present example outlines how antibody 2IB 12 IgG4 was produced from hybridomas. Hybridoma line 2IB 12 was grown in T75 flasks in media (Integra Media, 5 Table 5). When the hybridomas were nearly confluent in the T75 flasks, they were transferred to Integra flasks (Integra Biosciences, Integra CL 1000, 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 10 was placed into the small chamber of an Integra flask in Integra media (see Table 5 for components of Integra media). Integra media alone (IL) 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 15 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 20 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 pm). 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 25 column. Non-specific bindings were removed by an extensive PBS wash. Bound antibody proteins on the Protein A column were recovered by standard acidic antibody elution from Protein A columns (such as 50 mM Citrate, pH 3.0). Aggregated antibody proteins in the Protein A Sepharose pool were removed by size exclusion chromatography or binding ion exchange chromatography on anion exchanger resin 30 such as Q Sepharose resin. The specific IEX conditions for the 2IB 12 proteins are Q150
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Sepharose HP at pH 7.8-8.0. The antibody was eluted with a NaCI gradient of 10 mM500 mM in 25 column volumes.
EXAMPLE 6
Production of Human 2 IB 12 IgG2 Antibodies
From Transfected Cells
The present example outlines how 2IB 12 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 2IB 12 heavy and light chains. Conditioned media from hybridoma cells were recovered by removing cells and cell 10 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 15 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 2IB 12 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 (dideoxy) nucleotide sequencing. 25 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 31H4, 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, 2IB 12, 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 5 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 are disclosed as SEQ ID NOs: 10 12, 23, and 35 respectively, the lambda light chain constant region (SEQ ID NO: 156), the heavy chain variable regions of 31H4, 2IB 12, 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 15 2A-3D and FIG. 3CCC-3JJJ.
Based on the sequence data, the germline genes from which each heavy chain or light chain variable region was derived was determined. The identity of the germline genes are indicated next to the corresponding hybridoma line in FIGs. 2A-3D and 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 2IB 12 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 30 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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2020201012 13 Feb 2020 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 2IB 12 and 5 31H4 variable sequences stop and the lambda sequence starts.
For both 2IB 12 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
SC
KR
GE
GS
QK
IT
ND
KR
PS
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 25 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 30 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 histidine tag and a statement that the ABP sequence “includes the Histidine tag.” 35
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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 KinExA® affinity analysis was performed. The samples and buffers employed in these studies are presented in Table 9.1 below.
TABLE 9.1
sample [sample] mg/ml Buffer [sample] μ in
hPCSK9 1.26 PBS 16.6
mPCSK9-8xHIS 1.44 PBS 18.9
cPCSK9-V5-6xHIS 0.22 PBS 2.9
16F12, anti-PCSK9 huIgG4 4.6 20mM NaOAC, pH 5.2, 50mM NaCI 31.9
2IB 12, anti-PCSK9 huIgG4 3.84 10mM NAOAC, pH 5.2, 9% Sucrose 27.0
31H4, anti-PCSK9 huIgG4 3.3 10mM NAOAC, pH 5.2, 9% Sucrose 22.9
BIAcore® Affinity Measurements
A BIAcore® (surface plasmon resonance device, Biacore, Inc., Piscataway, NJ) affinity analysis of the 2IB 12 antibodies to PCSK9 described in this Example was performed according to the manufacturer’s instructions.
Briefly, the surface plasmon resonance experiments were performed using 15 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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2020201012 13 Feb 2020 immobilized antibody surface (at a flow rate of 100 μΙ/min for 1.5 minutes). Fresh HBS-P buffer (pH 7.4, 0.01 M Hepes, 0.15 M NaCl, 0.005% surfactant P-20, Biacore) supplemented with 0.01% BSA was used as binding buffer. Binding affinities of each anti-PCSK9 antibody were measured in separate experiments against each of the 5 human, mouse, and cynomolgus monkey PCSK9 proteins at pH 7.4 (the concentrations used were 100, 50, 25, 12.5, 6.25, 3.125, and 0 nM).
In addition, the binding affinities of antibody to human PCSK9 were also measured at pH 6.0 with the pH 6.0 HBS-P buffer (pH 6.0, 0.01 M Hepes, 0.15 M NaCl, 0.005% surfactant P-20, Biacore) supplemented with 0.01% BSA. The binding 10 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=l for the 6.0 pH runs). Interestingly, the antibodies appeared to display a tighter binding affinity at the lower 15 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), kd (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 20 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 kon and koff rates.
TABLE 9.3
- Kpn (M-l s-1) K<,ff (s-1) Kd
31H4.1,pH7.4 2.45 e+5 5.348 e-5 210 pM
31H4.1,pH6 5.536 e+6 6.936 e-5 12.5 pM
21B12.1, pH 7.4 3.4918 e+4 6.634 e-6 190 pM
21B12.1,pH6 2.291 e+6 1.676 e-5 7.3 pM
16F12.1, pH 7.4 1.064 e+5 4.983 e-5 470 pM
16F12.1,pH6 2.392 e+6 7.007 e-5 29 pM
KinExA® Affinity Measurements
A KinExA® (Sapidyne Instruments, Inc., Boise, ID) affinity analysis of 16F12 and 31H4 was performed according to the manufacturer’s instructions. Briefly, ReactiGel™ (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 10 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 15 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 20 PCSK9, but approximately 10-250 fold lower affinity to mouse PCSK9. Of the two
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The results of the KinExA® affinity analysis are summarized in Table 9.4, shown below.
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TABLE 9.4
hPCSK9 cPCSK mPCSK
Sample Kd (pM) 95% Cl Kd (pM) 95% Cl 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 Kd of the mAbs to cPCSK9 was adjusted as 50% of the active cPCSK9 in the 15 present.
A BIAcore solution equilibrium binding assay was used to measure the Kd values for ABP 21B12. 21B12.1 showed little signal using KinExA assay, therefore, biacore solution equilibrium assay was applied. Since no significant binding was observed on binding of antibodies to immobilized PCSK9 surface, 2IB 12 antibody was 20 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 O.lmg/ml BSA, 0.005% P20. Binding of the free PCSK9 in the mixed solutions were measured by injecting over the 25 21B12.1 antibody surface. 100% PCSK9 binding signal on 21B12.1 surface was determined in the absence of mAh in the solution. A decreased PCSK9 binding response with increasing concentrations of mAh indicated that PCSK9 binding to mAh in solution, which blocked PCSK9 from binding to the immobilized peptibody surface. Plotting the PCSK9 binding signal versus mAh concentrations, KD was calculated from 30 three sets of curves (0.3, 1 and 3nM fixed PCSK9 concentration) using a one-site homogeneous binding model in KinExA Pro™ software. Although cPCSK9 has lower
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2020201012 13 Feb 2020 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 KD. 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 5 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 Kd (pM) 95% Cl Kd (pM) 95% Cl Kd (pM) 95% Cl
21B12.1 15 9~23 11 7-16 17000 -
EXAMPLE 10
EFFICACY OF 31H4 AND 21B12 FOR BLOCKING D374Y PCSK9/LDLR BINDING
This example provides the IC50 values for two of the antibodies in blocking
PCSK9 D374Y’s ability to bind to LDLR. Clear 384 well plates (Costar) were coated with 2 micrograms/ml of goat anti-LDL receptor antibody (R&D Systems) diluted in buffer A (100 mM sodium cacodylate, pH 7.4). Plates were washed thoroughly with buffer A and then blocked for 2 hours with buffer B (1% milk in buffer A). After washing, plates were incubated for 1.5 hours with 0.4 micrograms/ml of LDL receptor (R&D Systems) diluted in buffer C (buffer B supplemented with 10 mM CaC12). Concurrent with this incubation, 20 ng/ml of biotinylated D374Y PCSK9 was incubated with various concentrations of the 31H4 IgG2, 31H4 IgG4, 2 IB 12 IgG2 or 2IB 12 IgG4 antibody, which was diluted in buffer A, or buffer A alone (control). The
LDL receptor containing plates were washed and the biotinylated D374Y PCSK9/antibody mixture was transferred to them and incubated for 1 hour at room temperature. Binding of the biotinylated D374Y to the LDL receptor was detected by incubation with streptavidin-HRP (Biosource) at 500 ng/ml in buffer C followed by TMB substrate (KPL). The signal was quenched with 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 5xl05 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 μg/ml of D374Y human 15 PCSK9 was incubated with various concentrations of antibody diluted in uptake buffer (DMEM with 1% FBS) or uptake buffer alone (control) for 1 hour at room temperature. After washing the cells with PBS, the D374Y PCSK9/antibody mixture was transferred to the cells, followed by LDL-BODIPY (Invitrogen) diluted in uptake buffer at a final concentration of 6 μg/ml. After incubation for 3 hours at 37°C (5% CO2), cells were 20 washed thoroughly with PBS and the cell fluorescence signal was detected by Safire™ (TECAN) at 480-520nm (excitation) and 520-600nm (emission).
The results of the cellular uptake assay are shown in FIGs. 7A-7D. Summarily, IC50 values were determined for each antibody and found to be 16.7 nM for 31H4 IgG2 (FIG. 7A), 13.3 nM for 31H4 IgG4 (FIG. 7B), 13.3 nM for 21B12 IgG2 (FIG. 7C), and 25 18 nM for 2IB 12 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 antiPCSK9 antibody 31H4 (2 mg/ml in PBS) or control IgG (2 mg/ml in PBS) at a level of lOmg/kg through the mouse’s tail vein at T=0. Naive mice were also set aside as a naive 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 Naive 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 15 allowed to clot at room temperature for 30 minutes. The samples were then spun down in a table top centrifuge at 12,000xg for 10 minutes to separate the serum. Serum total cholesterol and HDL-C were measured using Hitachi 912 clinical analyzer and Roche/Hitachi TC and HDL-C kits.
The results of the experiment are shown in FIGs. 8A-8D. Summarily, mice to 20 which antibody 31H4 was administered showed decreased serum cholesterol levels
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2020201012 13 Feb 2020 over the course of the experiment (FIG. 8A and FIG. 8B). In addition, it is noted that 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 5 total serum cholesterol and HDL levels measured in naive animals at t=0 hrs (*P<0.01, # P<0.05).
In respect to the lowered HDL levels, it is noted that one of skill in the art will appreciate that the decrease in HDL in mice is not indicative that an HDL decrease will occur in humans and merely further reflects that the serum cholesterol level in the 10 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 15 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 31H4 on LDLR Levels in a 6 Day Study
The present example demonstrates that an antigen binding protein alters the level of LDLR in a subject, as predicted, over time. A Western blot analysis was 25 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 30 supernatant was measured using BioRad protein assay reagents (BioRad laboratories). lOOpg of protein was denatured at 70°C for 10 minutes and separated on 4-12% Bis162
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Tris SDS gradient gel (Invitrogen). Proteins were transferred to a 0.45 gm PVDF membrane (Invitrogen) and blocked in washing buffer (50mM Tris PH7.5, 150mM NaCL, 2mM CaCfi 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 5 (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 10 Western blot showed an increase in LDLR protein levels in the presence of antibody 31H4, 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 (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 20 2918) through out the duration of the experiment. Mice were administered either antiPCSK9 antibody 31H4 (2 mg/ml in PBS) or control IgG (2 mg/ml in PBS) at a level of 10 mg/kg through the mouse’s tail vein at T=0. Naive mice were also set aside as naive control group.
Dosing groups and time of sacrifice are shown in Table 14.1. Animals were 25 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 lOmg/kg
2 31H4 72 hr 6 lOmg/kg
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Group Treatment Time point after dosing Number Dose
3 31H4 72 hr 6 1 mg/kg
4 IgG 144 hr 6 lOmg/kg
5 31H4 144 hr 6 lOmg/kg
6 31H4 144 hr 6 1 mg/kg
7 IgG 192 hr 6 lOmg/kg
8 31H4 192 hr 6 lOmg/kg
9 31H4 192 hr 6 1 mg/kg
10 IgG 240 hr 6 lOmg/kg
11 31H4 240hr 6 lOmg/kg
12 31H4 240hr 6 1 mg/kg
13 IgG 312 hr 6 lOmg/kg
14 31H4 312 hr 6 lOmg/kg
15 31H4 312 hr 6 1 mg/kg
16 Naive n/a 6 n/a
When the 6 day experiment was extended to a 13 day study, the same serum cholesterol lowering effect observed in the 6 day study was also observed in the 13 day 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 lOmg/kg dose of 31H4, and with another antibody, 16F12, also at lOmg/kg. Dosing groups and time of sacrifice are shown in 10 Table 14.2.
TABLE 14.2
Group Treatment Time point after dosing Number Dose
1 IgG 24 hr 6 lOmg/kg
2 16F12 24 hr 6 lOmg/kg
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Group Treatment Time point after dosing Number Dose
3 31H4 24 hr 6 lOmg/kg
4 IgG 72 hr 6 lOmg/kg
5 16F12 72 hr 6 lOmg/kg
6 31H4 72 hr 6 lOmg/kg
7 IgG 144 hr 6 lOmg/kg
8 16F12 144 hr 6 lOmg/kg
9 31H4 144 hr 6 lOmg/kg
10 IgG 192 hr 6 lOmg/kg
11 16F12 192 hr 6 lOmg/kg
12 31H4 192 hr 6 lOmg/kg
13 IgG2 240 hr 6 lOmg/kg
14 16F12 240hr 6 lOmg/kg
15 31H4 240hr 6 lOmg/kg
16 IgG2 312 hr 6 lOmg/kg
17 16F12 312 hr 6 lOmg/kg
18 31H4 312 hr 6 lOmg/kg
19 Naive n/a 6 lOmg/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 pre5 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 10 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. 11 A. The results are displayed in FIG. 11B. As can be seen in the graph in FIG. 11B, while both sets of 20 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 25 change is in relation to the naive 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 30 return of total serum cholesterol or HDL cholesterol levels to baseline, observed in the
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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, 2IB 12). At periodic times during the treatment, the human patient is monitored to determine whether the serum cholesterol level has declined. Following treatment, it is 10 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 20 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 30 grown to -90% confluence. The cells were treated with indicated amounts of
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2020201012 13 Feb 2020 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 5 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 Imaged 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 15 were separated by gel electrophoresis and transferred to PVDF membrane.
Immunoblots were performed using rabbit anti-human LDL receptor antibody (Fitzgerald) or rabbit anti-human b-actin antibody. The enhanced chemiluminescent results are shown in the top panels. The intensity of the bands were quantified by Imaged 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 25 greater extent.
Interestingly, as demonstrated by the results in the comparison of FIGs. 12D12F 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 (2IB 12 and 31H4) resulted in a greater increase in LDLR levels, 30 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 5 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 Vh or Vl were converted to FASTA formatting for ease in processing comparative alignments and inferring phylogenies. Next, framework 10 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 15 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 2.0. This program likewise 20 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 25 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 f5 substitutions per 100 residues (see, legend in free illustrations for scale) amongst individual sequences within the group and were used to define consensus sequence 30 collections. The results of the comparisons are depicted in FIGs. 13A-13J and FIGs.
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31A and 3 IB. 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
11F1 Binding Specificity
Results from this assay demonstrate that 11F1 binds to PCSK9 and not to PCSK1, PCSK2, PCSK7, or furin, demonstrating the specificity of 11F1 for PCSK9.
Biotinylated PCSK9, diluted in buffer A (25 mM Tris, 150 mM NaCI, 0.1% BSA, 0.05% tween, pH 7.5) was bound to neutravidin coated 96 well plates at a concentration of 0.2 pg/mL, for one hour incubation at room temperature. Separately, 0.4 pg/mL of 11F1 was incubated for one hour at room temperature with various concentrations (ranging from 0 to 20 pg/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 pg/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-HRP (160 15 ng/mL, diluted in buffer A) (Jackson Laboratories, Bar Harbor, ME) followed by TMB substrate. The reaction was stopped with 1 N HC1 and the absorbance was read at a wavelength of 450 nm on a Spectramax Plus 384 spectrophotometer (Molecular Devices Inc., Sunnyvale, CA).
This assay relied on the ability of proprotein convertase in solution to compete for the 20 binding of 11F1 to plate-captured PCSK9. Pre-incubation of 11F1 and PCSK9 in solution dose dependently and robustly reduced the amount of 1 IF 1 binding to platecaptured PCSK9 detected as reduced OD450 (FIG. 14). All results were expressed as the mean OD450 value ± standard deviation versus concentration of the proprotein convertase. Pre-incubation of 11F1 with PCSK1, PCSK2, PCSK7, or furin, in solution, 25 did not significantly impact the binding of 11F1 to plate-captured PCSK9. Therefore, at the protein concentrations studied, 11F1 binds only to PCSK9 and not to the other proprotein convertase family members tested.
Example 22
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Efficacy of 11F1 Inhibition of LDLR:PCSK9 Binding
The example demonstrates that nanomolar concentrations of 1 IF 1 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 pg/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 pg/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 IN HC1 and the absorbance 20 was read at a wavelength of 450 nm on a SpectraMax Plus 384 Spectrophotometer (Molecular Devices Inc., Sunnyvale, CA). GraphPad Prism (v 4.01) software was used to plot log of antibody concentration versus OD450 to determine IC50 values by nonlinear regression.
11F1 inhibited LDLR:PCSK9 binding. The IC50 values for 11F1 in the D374Y PCSK9 25 assay ranged from 7.3 nM to 10.1 nM with an average (± SD) of 9.1 nM ±1.5 nM (n=3). The IC50 values for 11F1 in the wild-type PCSK9 assay ranged from 4.4 nM to 8.1 nM with an average (± SD) of 5.9 nM ±1.9 nM (n=3). It should be noted that these IC50 values are dependent on the amount of recombinant D374Y PCSK9 or WT
PCSK9 used in the binding assay. A representative dose response curve for both the 30 D374Y and wild-type assays are presented in FIG. 15 and FIG. 16, respectively.
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EXAMPLE 23
Efficacy of 1 IF 1 in Blocking Cell LDL Uptake
11F1 blocks the interaction between PCSK9 and LDLR in vitro and can prevent 5 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 10 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 pm 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 pg/mL) or WT PCSK9 (25 pg/mL) were diluted in uptake 15 buffer (DMEM containing 1% FBS) and incubated with the various concentrations of 11F1 or uptake buffer alone (negative control) for 1 hour at room temperature with shaking. BODIPY-LDL (Invitrogen, Carlsbad, CA) was diluted in uptake buffer to a concentration of 12 pg/mL. Following overnight incubation, HepG2 cells were rinsed twice with DPBS (Mediatech Inc., Herndon, VA). Twenty-five microliters of the 20 D374Y PCSK9 or WT PCSK9 complex with 11F1 and 25 pL 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 pL DPBS. Fluorescent signals were detected using a Safire plate reader (Tecan Systems Inc., San Jose, CA) at 480-520 nm (excitation) and 520-600 nm (emission) and expressed as 25 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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2020201012 13 Feb 2020 recombinant purified D374Y PCSK9 (2 pg/mL) or WT PCSK9 (25 pg/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 5 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 10 D374Y PCSK9 than WT PCSK9 since less D374Y PCSK9 was used in the assay because its binding affinity to the LDLR is 5- to 30-fold greater than that of WT PCSK9 (Cunningham et al, 2007; Fisher et al, 2007; Kwon et al, 2008).
The EC50 values reported here are representative for mean values derived from 3 to 6 separate measurements for 11F1.
EXAMPLE 24
Efficacy of 11F1 and 8A3 in Blocking Human PCSK9 Expressed Via an AdenoAssociated Virus in a mouse model
A single intravenous bolus administration of the anti-PCSK9 antibodies 11F1 or 20 8A3 leads to a significant decrease in serum non-HDL-C and TC in mice expressing human PCSK9 by AAV. This example demonstrates the effectiveness of both antiPCSK9 antibodies in blocking the function of human PCSK9 in vivo.
Briefly, 120 C57BL/6 mice expressing human PCSK9 were generated by infection with an engineered adeno associated virus (AAV) coding for human PCSK9, 25 resulting in elevated levels of circulating low density lipoprotein cholesterol (LDL-C).
Serum cholesterol analysis was performed using the Cobas Integra 400 plus chemistry analyzer (Roche Diagnostics, Indianapolis, IN). Animals were randomized into treatment groups with similar levels of non-HDL-C (LDL-C and VLDL-C), HDL-C and TC. On treatment day 0 (T=0) a subset of mice was euthanized and serum collected 30 to establish that day’s baseline levels. Remaining mice were then administered 1 IF 1,
8A3 or anti-keyhole limpethemocyanin (KLH) IgG2 control antibody at 30 mg/kg via
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2020201012 13 Feb 2020 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 pg/ml of monoclonal antiPCSK9 antibody (31H4) diluted in IX PBS. Plates were washed thoroughly with IX PBS/.05% tween and then blocked for 2 hours with 3% BSA/1XPBS. After washing, plates were incubated for 2 hours with serum diluted in general assay diluents (Immunochemistry Technologies, Bloomington, MN). Recombinant human PCSK9 (1 ng/ml to 500 ng/ml) was assayed concurrently and used to generate a standard curve on each ELISA plate. A rabbit polyclonal biotinylated anti-PCSK9 antibody (D8773, Amgen Inc, CA) was added at 1 ug/ml (in 1%BSA/PBS), followed by neutravidin15 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 IN HC1 and the absorbance measured at 450 nm on a Spectra Max Plus 384 Spectrophotometer (Molecular Devices Inc, Sunnyvale, CA). The standard curve (4-parameter logistic fit) generated with recombinant human PCSK9 was used to determine the corresponding concentration of PCSK9 in the serum samples.
Serum concentrations of antibody were determined using a sandwich ELISA assay. Polyclonal goat anti-human Fc IgG and an HRP-labeled goat anti-human IgG Fey polyclonal reagent (both from Jackson ImmunoResearch Laboratories Inc, West Grove, PA) were used as the capture and the detection antibody, respectively. A
3,3’,5,5’tetramethylbenzidine (TMB) substrate solution reacted with peroxide, and in the presence of horse radish peroxidase (HRP), created a colorimetric signal that was proportional to the amount of the respective anti-PCSK9 antibody bound by the capture reagent. The intensity of the color (optical density, OD) was measured at 450 nm minus 650 nm using a microplate reader (Spectra Max Plus 384). Data was analyzed using Watson version 7.0.0.01 (Thermo Scientific, Waltham, MA) data reduction package with a Logistic (auto-estimate) regression of separately prepared
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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 (t 1/2), 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 25 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 30 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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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, 5 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 10 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 15 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 antiKLH 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 20 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 11F1 and 8A3 when compared to other antibodies dosed in the AAV 25 model because 11F1 and 8A3 CLO-t values are more similar to anti-KLH IgG2 control antibody.
Summary.
Expression of human PCSK9 by AAV in mice (approximately 5 ug/mL) resulted in a serum non-HDL-C level of approximately 33 mg/dL. Following a 30 30 mg/kg injection of 1 IF 1, 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 HDL5 C was observed in animals administered either 11F1 or 8A3. Analysis of serum antibody levels of 1 IF 1 and 8A3 demonstrated a similar profile to anti-KLH IgG2 control antibody.
EXAMPLE 25
Effect of a Single Subcutaneous Dose ofllFl,21B12 and 8 A3 on Serum Lipids in Cynomolgus Monkeys
Single SC administration of 1 IF 1, 8A3 or 2IB 12 to cynomolgus monkeys leads to the significant lowering of serum LDL-C, and TC. This study demonstrated the ability of antiPCSK9 antibodies to lower serum cholesterol in non-human primates.
Briefly, naive male cynomolgus monkeys were acclimated to their environment for at least 2 weeks prior to experimentation. Animals were randomized into treatment groups based on a pre-screen of their serum TC, HDL-C, LDL-C, and triglyceride levels, and their body weight. After 1 week, animals were fasted overnight, and bled from the peripheral vasculature (cephalic or saphenous vein), for measurement of baseline serum lipid levels at a time point designated T = 0. Animals were then injected SC with either anti-KLH IgG2 control antibody, 11F1,21B12, or 8A3 (all in 10 mM NaOAc pH 5.2, 9% sucrose) at 0.5 mg/kg (all at 0.4 mL/kg body weight).
Fasting blood samples were then collected from animals at designated time points over a 45 day period.
Experimental Design
Group No. No Males Route Treatment Dose Level (mg/kg) Cone. (mg/mL) Volume (mL/kg)
1 5 SC Anti-KLH 0.5 1.09 0.4
2 5 SC 21B12 0.5 1.19 0.4
3 5 SC 11F1 0.5 1.11 0.4
4 5 SC 8A3 0.5 1.25 0.4
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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 pL aliquot of the sample (no preparation) was used for analysis with a Hitachi 717 Analyzer using a 6 points standard curve. If the initial value of the sample was higher than the standard curve linearity, then the sample was diluted and repeated with the result multiplied by the appropriate dilution factor. The reagents for the assay (APO-B Reagent Kit # 86071, Antibody Set # 86060, Control Set # 86103) were obtained from DiaSorin (Stillwater, MN).
Antibody concentrations in serum were determined using an enzyme-linked immunosorbent assay (ELISA) with an assay range of 34.4 to 3000 ng/mL (34.4 ng/mL being the lower limit of quantitation [LLOQ]).
Non-compartmental analysis (NCA) was performed on the serum concentrations using the pre-determined nominal time points for each subject using
Watson® LIMS, version 7.0.0.01 (Thermo Scientific, Waltham, MA). Data points for estimating the terminal elimination rate constants and half-lives were chosen by visual inspection of the concentration-time profile and best linear fit (typically from 360 h until the antibody concentrations dropped below the lower limit of quantitation). NCA parameters reported include: terminal half-life (tl/2,z), the maximum serum 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 11F1,21B12, and 8A3 on Serum LDL Cholesterol
Maximal LDL-C lowering for 11F1 was observed 9 days after injection, with a
57% lowering of LDL-C as compared to anti-KLH IgG2 control antibody-treated monkeys (control animals). LDL-C returned to levels similar to those observed in control animals by day 27. Maximal LDL-C lowering for 2IB 12 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 2IB 12 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 11F1,21B12, and 8A3 on Serum HDL Cholesterol and Triglycerides
On average and at each time point, HDL-C or triglyceride levels for animals treated with 11F1 or 8A3 were not significantly different (based on an alpha = 0.05 significance level) from those observed in anti-KLH IgG2 control antibody-treated monkeys. However, 2IB 12 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)
Serum ApoB levels were measured at days 3, 6, 15, 24 and 33, post-injection.
11F1 and 8A3 were associated with ApoB lowering at days 3 to 24, as compared to anti-KLH IgG2 control antibody-treated monkeys (FIG. 28). 2IB 12 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 1 IF 1, 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. 2IB 12 pharmacokinetic behavior with regard to CL/F, Cmax, and AUCO-inf was consistent with that observed in previous studies where 2IB 12 was administered at the same dose. Pharmacokinetics of 1 IF 1 and 8A3 differed significantly from 2IB 12, where lower CL/F was observed (approximately 15%of21B12 CL/F) and longer half-lives were estimated (approximately 200 h compared to 40 h for 2IB 12). 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 1 IF 1 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.
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Summary of Results
Over the course of the 45 day study, statistically significant lowering of TC and LDL-C was observed in animals administered 11F1, 21B12, or 8A3 as compared to anti-KLH IgG2 control antibody. 11F1 was associated with statistically significant 5 LDL-C lowering (vs. anti-KLH IgG2 control antibody) from day 2 to day 24 inclusively. 2IB 12 demonstrated statistically significant LDL-C lowering (vs anti-KLH IgG2 control antibody) from day 1 to day 4 inclusively. 8A3 demonstrated statistically significant LDL-C lowering (vs anti-KLH IgG2 control antibody) from day 1 to day 24 inclusively. Changes in TC and ApoB mirrored changes observed in LDL-C for all 10 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 15 (-54%). 2IB 12 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:591) with expanded enrollment but otherwise identical design to Part A. Both inclusion/exclusion criteria and the Schedule of Assessments is the same for Parts
AandB.
Inclusion Criteria includes:
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Exclusion Criteria includes:
• LDL or plasma apheresis within 8 weeks prior to randomization • New York Heart Failure Association (NYHA) class III or IV or last known left ventricular ejection fraction < 30% • Myocardial infarction, unstable angina, percutaneous coronary intervention (PCI), coronary artery bypass graft (CABG) or stroke within 3 months of randomization • Planned cardiac surgery or revascularization · Uncontrolled cardiac arrhythmia • Uncontrolled hypertension
Schedule of Assessments include, but are not limited to, collection of adverse event (AE) and significant adverse event (SAE) data, vital signs, concomitant medication, laboratory tests, etc.
Subjects who meet inclusion/exclusion criteria 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 2IB 12 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 2IB 12 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
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Table 26.1: Patent Genotypes
Patient Mutation Allele 1 (Estimated LDL-r Function) Mutation Allele 2 (Estimated LDL-r Function) Overall LDL-r Function
Patient 1 Asp266Glu (15%-30%) Asp266Glu (15%-30%) Receptor defective
Patient 2 1187-10 G>AT (Not determined) Asp266Glu (15%-30%) Receptor defective
Patient 3 Asp224Asn (<2%) Cys296Tyr (Not determined) Negative
Patient 4 Deletion Exon 4-18 (Not determined) Cysl97Gly (Not determined) Negative
Patient 5 Asp221Gly (<2%) Asp227Glu (5%-15%) Receptor defective
Patient 6*1 Asp227Glu (5%-15%) Asp227Glu (5%-15%) Receptor defective
Patient 7*1 Asp227Glu (5%-15%) Asp227Glu (5%-15%) Receptor defective
Patient 8 Aspl75Asn (Not determined) Asp227Glu (5%-15%) Receptor defective
*True homozygous patient.
^Mutation at splice acceptor site 10 nucleotides upstream of the first nucleotide of exon 9, 1187.
I Patients share the same genotype.
LDL-r: Low density lipoprotein receptor.
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The 2IB 12 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 5 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 10 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 2week 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. HDLcholesterol and apolipoprotein Al are essentially unchanged with either every 4- or 2week 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).
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Table 26.2: Efficacy Outcomes (Overall).
21B12 formulation (N = 8)
Parameter* Baselin e Value Week 12 Q4W Week 12 Q2W
Value Change from baseline Percentage change from baseline (%) Value Change from baseline Percentage change from baseline (%)
LDL cholesterol (ultracentrifugation), mg/dL
Mean (SE) 441.7 (40.1) 371.1 (50.4) -70.6 (32.3) -16.5(6.7) 380.9 (56.3) 60.8 (43.7) -13.9(9.6)
Range 218 to llllllll 190 to 563 -228 to llllllll -43.6 to 5.2 196 to 614 -217 to 175 -43.3 to 39.9
HDL cholesterol, mg/dL 33.8 (3.2) 34.5 (3.4) 0.8(3.0) 4.7 (7.8) 32.9 (3.7) -0.9 (2.9) -1.4 (7.3)
Apolipoprotei η B, mg/dL 269.1 (18.7) 228.8 (21.3) -40.3 (14.6) -14.9(5.0) 235.3 (24.7) -33.8 (19.0) -12.5(6.7)
Apolipoprotei η A1, mg/dL 99.3 (6.0) 99.3 (4.8) 0.0(4.8) 1.3 (5.0) 103.8 (6.0) 4.5(4.2) 5.2 (4.1)
Triglycerides, mg/dL 110.8 (22.8) 100.8 (17.8) -10.0 (7.0) -5.7 (5.6) 109.1 (14.4) -1.6 (14.8) 5.9(9.1)
Lipoprotein (a), nmol/L 246.5 (61.5, 2 76.0)f 170.6 (41.2) -24.6 (8.2) -11.7(3.8) 168.0 (42.4) -27.3 (7.8) -18.6 (4.3)
Free PCSK9, ng/mL 598.6 (42.8) 447.4 (73.9) -151.3 (81.7) -22.7 (13.1) 73.2 (17.0) -525.4 (43.6) -87.6 (2.8)
Values are mean (SE) unless otherwise stated.
* To convert values for cholesterol to millimoles per liter, multiply by 0.0259. To convert values for Apolipoprotein A1 or Apolipoprotein B to grams per liter, multiply by 0.01. To convert values 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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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.
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Table 26.3: Efficacy Outcomes Based on Mutation Status
Mutation Status Percentage Change from Baseline, %, Mean (SE)
Week12Q4W Week12Q2W
UC LDL Apolipoprotein B Lipoprotein (a) UC LDL Apolipoprotein B Lipoprotein (a)
Total (N = 8) -16.5 (6.7) -14.9 (5.0) -11.7 (3.8) -13.9 (9.6) -12.5 (6.7) -18.6 (4.3)
Defective LDL receptor (N=6) -22.9 (7.2) - 18.3 (6.1) -10.0 (4.7) -23.6 (7.6) -17.9 (7.3) -18.7 (5.7)
Negative LDL receptor (N=2) 2.6 (2.6) -4.5 (2.5) -16.8 (5.7) 15.3 (24.6) 3.4 (9.9) -18.5 (3.7)
Average of Week 4, 8, and 12 Q4W Average of Week 4, 8, and 12 Q2W
UC LDL Apolipoprotein B Lipoprotein (a) UC LDL Apolipoprotein B Lipoprotein (a)
Total (N = 8) -13.3 (6.2) -13.1 (5.1) -11.7 (3.8) -16.9 (9.2) -16.0 (6.9) -20.7 (3.9)
Defective LDL receptor (N=6) -19.3 (6.3) - 18.0 (5.3) -10.0 (4.7) -26.3 (8.3) -22.1 (7.7) -20.0 (5.0)
Negative LDL receptor (N=2) 4.4 (7.3) 1.4 (4.0) -16.8 (5.7) 11.0 (16.7) 2.1 (5.6) -22.7 (7.9)
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 2IB 12 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
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Incorporation by Reference
All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated herein by reference in their entirety. To the extent that 10 any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the present terms and definitions control.
Equivalents
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways 20 and the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims (26)

  1. WHAT IS CLAIMED IS:
    1. 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
    5 about 3000 mg, thereby lowering said serum LDL cholesterol level by at least about 10%.
  2. 2. 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
    10 treating the homozygous familial hypercholesterolemia in said patient.
  3. 3. The method of claim 1, wherein the serum LDL cholesterol level of said patient is lowered by an amount selected from the group consisting of a) at least about 15%, b) at least about 20%, c) at least about 30%, d) at least about 40%, and e) at least about 50%.
    15
  4. 4. The method of claim 3, wherein the anti-PCSK9 antibody comprises,
    a) 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 20 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;
    b) a light chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO :465, a CDRL2 of the CDRL2 sequence in SEQ ID
    25 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;
    30 c) a light chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence in SEQ ID
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    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
  5. 5 in SEQ ID NO:67;
    d) 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
    10 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;
    e) a light chain complementarity region (CDR) of the CDRL1 sequence in SEQ ID NO :485, a CDRL2 of the CDRL2 sequence in SEQ ID
    15 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; or
    20 f) 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
    25 sequence in SEQ ID NO: 583, and a CDRH3 of the CDRH3 sequence in SEQ ID NO:583.
    5. The method of claim 3, wherein the anti-PCSK9 antibody comprises, (a) a light chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 23 and a heavy
    30 chain variable region that comprises and amino acid sequence that is at least 90% identical to that of SEQ ID NO:49;
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    2020201012 13 Feb 2020 (b) a light chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 12 and a heavy chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:67;
    5 (c) a light chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 461 and a heavy chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:459;
    (d) a light chain variable region that comprises an amino acid sequence
    10 that is at least 90% identical to that of SEQ ID NO:465 and a heavy chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:463;
    (e) a light chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 485 and a heavy
    15 chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:483; or (f) a light chain variable region that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO :5 82 and a heavy chain variable region that comprises an amino acid sequence that is at
    20 least 90% identical to that of SEQ ID NO:583.
  6. 6. The method of claim 3, wherein the anti-PCSK9 antibody comprises:
    (a) a light chain variable region that comprises an amino acid sequence, SEQ ID NO: 23, and a heavy chain variable region that comprises and amino acid sequence, SEQ ID NO:49;
    25 (b) a light chain variable region that comprises an amino acid sequence,
    SEQ ID NO: 12, and a heavy chain variable region that comprises an amino acid sequence, SEQ ID NO:67;
    (c) a light chain variable region that comprises amino acid sequence SEQ ID NO: 461 and a heavy chain variable region that comprises
    30 amino acid sequence SEQ ID NO:459;
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    2020201012 13 Feb 2020 (d) a light chain variable region that comprises the amino acid sequence of SEQ ID NO:465 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:463;
    (e) a light chain variable region that comprises the amino acid sequence
    5 of SEQ ID NO: 485 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:483;
    (f) a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 582 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:583; or
    10 (g) a light chain variable region that comprises an amino acid sequence,
    SEQ ID NO:591, and a heavy chain variable region that comprises an amino acid sequence, SEQ ID NO:590;
  7. 7. The method of claim 4, 5 or 6, wherein the anti-PCSK9 antibody further comprises:
    15 (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
    20 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
    25 (h) the light chain constant sequence of SEQ ID NO: 157 and the heavy chain constant sequence of SEQ ID NO: 155.
  8. 8. The method of claim 6, wherein the anti-PCSK9 antibody further comprises a glycine residue at the C-terminal end of the ligh chain variable domain.
  9. 9. The method of claim 6, wherein the at least one anti-PCSK9 antibody is
    30 selected from the group consisting of 2IB 12, 11F1,31H4, 8A3, and 8A1.
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  10. 10. The method of claim 4, wherein the anti-PCSK9 antibody is administered to the patient at a dose selected from the group consisting of: a) 120 mg to about 700 mg, b) about 140 mg to about 600 mg, c) about 140 mg to about 450 mg, d) about 420 mg, e) about 450 mg, f) about 600 mg, g) about 700 mg, h)
    5 about 1400 mg, i) about 1200 mg, j) about 420 mg to about 3000 mg, k) about 1000 mg to about 3000 mg, or 1) about 3000 mg.
  11. 11. The method of claim 10, wherein the anti-PCSK9 antibody is administered to the 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 three
    10 months (5) once every six months and (6) once every twelve months.
  12. 12. The method of claim 10, wherein the administering step comprises administering the at least one anti-PCSK9 antibody parenterally.
  13. 13. The method of claim 12, wherein the administering step comprises administering the at least one anti-PCSK9 antibody intravenously.
  14. 15 14. The method of claim 12, wherein the administering step comprises administering the at least one anti-PCSK9 antibody subcutaneously.
    15. The method of claim 14, wherein the at least one anti-PCSK9 antibody comprises a light chain complementarity determining region (CDR) of the CDRL1 sequence in SEQ ID NO :23, a CDRL2 of the CDRL2 sequence in
    20 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.
  15. 16. The method of claim 14, wherein the at least one anti-PCSK9 antibody
    25 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.
  16. 17. The method of claim 14, wherein the at least one anti-PCSK9 antibody
    30 comprises a light chain variable region that comprises the amino acid
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  17. 18. The method of claim 14, wherein the at least one anti-PCSK9 antibody comprises a light chain variable region that comprises the amino acid
    5 sequence of SEQ ID NO:591 and a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:590
  18. 19. The method of claim 17 or 18, wherein the at least one anti-PCSK9 antibody further comprises:
    (a) the light chain constant sequence of SEQ ID NO: 156;
    10 (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;
    15 (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
  19. 20 heavy chain constant sequence of SEQ ID NO: 155.
    20. The method of claim 19, wherein the anti-PCSK9 antibody further comprises a glycine residue at the C-terminal end of the ligh chain variable domain.
  20. 21. The method of claim 15, wherein the anti-PCSK9 antibody is administered to
    25 the patient at a dose of about 70 mg to about 450 mg subcutaneously once every two weeks, and wherein the serum LDL cholesterol level of the patient is lowered at least about 10 - 50% for about 7-14 days.
  21. 22. The method of claim 15, wherein the anti-PCSK9 antibody is administered to the patient at a dose of about 70 to about 450 mg subcutaneously once every
    30 month, and wherein the serum LDL cholesterol level of the patient is lowered at least about 10 -50% for about 21 to 31 days.
    194
    WO 2014/209384
    PCT/US2013/048714
    2020201012 13 Feb 2020
  22. 23. The method of claim 21, wherein the anti-PCSK9 antibody is administered to the patient at a dose of about 420 mg.
  23. 24. The method of claim 22, wherein the anti-PCSK9 antibody is administered to the patient at a dose of about 420 mg.
    5
  24. 25. A method of lowering serum LDL cholesterol in a patient diagnosed with homozygous familial hypercholesterolemia comprising administering to a patient in need thereof an anti-PCSK9 antibody comprising a light chain that comprises the amino acid sequence of SEQ ID NO:591 and a heavy chain that comprises the amino acid sequence of SEQ ID NO:590 at a dose of about 420
    10 mg, thereby lowering said serum LDL cholesterol level by at least about 10%.
  25. 26. The method of claim 25, wherein the anti-PCSK9 antibody is administered to the patient once every two weeks.
  26. 27. The method of claim 25, wherein the anti-PCSK9 antibody is administered to the patient once every month.
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