JP6639463B2 - Methods of treating homozygous familial hypercholesterolemia - Google Patents

Description

Reference to Sequence Listing This application has been filed with the sequence listing in electronic form. The sequence listing was prepared on June 27, 2013, A-1837-WO-PCT-SequenceList0627713. It is provided as a file titled txt. Sequence listing information in electronic form is incorporated herein by reference in its entirety.

The present invention relates to a method for treating homozygous familial hypercholesterolemia using an antigen-binding protein (eg, an antibody) directed against the proprotein convertase subtilisin / keksin type 9 (PCSK9).

  Homozygous familial hypercholesterolemia is a rare but serious clinical disorder caused by a substantial reduction in low density lipoprotein (LDL) receptor function. As a result, LDL cholesterol levels are significantly elevated, which can lead to cardiovascular disease and often death in childhood (Goldstein JL, Hobbs HH, Brown MS, eds. More than 95% have mutations in the LDL receptor, less than 4% in apolipoprotein B, and less than 0.5% in the proprotein convertase subtilisin / kexin 9 (PCSK9) (Abifadel) M, Varret M, Rabes JP, et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat Genet 2003; 34: 154-6; Rader DJ, Cohen J, Hobbs HH.Monogenic hypercholesterolemia: new insights in pathogenesis and treatment.J Clin Invest 2003; 111: 1975-803. . While truly hereditary homozygous familial hypercholesterolemia is not uncommon, the majority of patients are complex heterozygotes (Usifo E, Leigh SE, Whitall RA, et al. Low-density lipoprotein receptor gene). family hypercholesteromia variant database: update and pathological assessment. Ann Hum Genet 2012; 76: 387-401). LDL receptor residual activity, either negative (less than 2% function) or deficient (2% to 25% function), is associated with the severity of elevated LDL cholesterol and cardiovascular propensity at an early age (Goldstein JL, Hobbs HH, Brown MS, eds. Family hypercholesterolemia. 8th. Edition ed: McGraw-Hill; 2001).

  Responses to conventional treatments, such as statins and ezetimibe, the most commonly used drugs for homozygous familial hypercholesterolemia, are modest, and patients usually undergo LDL apheresis when available. It costs. Reduction of LDL cholesterol by statins tends to correlate with LDL receptor function, but receptor-negative patients show a reduction (Raal FJ, Pappu AS, Illingworth DR, et al. Inhibition of cholesterol synthesis by vasatoratin). homozygous family hypercholesterolemia, Atherosclerosis 2000; 150: 421-8). Improvement of LDL cholesterol by statins appears to reduce morbidity and mortality from cardiovascular disease (Raal FJ, Pilcher GJ, Panz VR, et al. -Lowering therapy. Circulation 2011; 124: 2202-7). In recent years, two drugs, romitapide and mipomersen, both of which reduce hepatic lipoprotein production, have been approved only for the treatment of homozygous familial hypercholesterolemia. Although these two new drugs have been introduced, there is a need to identify new treatments for patients diagnosed with homozygous familial hypercholesterolemia.

Goldstein JL, Hobbs HH, Brown MS, eds. Familial hypercholesterolemia. 8th Edition ed: McGraw-Hill; 2001 Abifadel M, Varret M, Rabes JP, et al. Mutations in PCSK9 Cause automatic dominant hypercholesterolemia. Nat Genet 2003; 34: 154-6. See Radar DJ, Cohen J, Hobbs HH. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment. J Clin Invest 2003; 111: 1795-803. Usifo E, Leigh SE, Whitall RA, et al. Ann Hum Genet 2012; 76: 387-401. Raal FJ, Pappu AS, Illingworth DR, et al. Atherosclerosis 2000; 150: 421-8. Raal FJ, Pilcher GJ, Panz VR, et al. Reduction in mobility in subjects with the homozygos family hypercholesterolatea wisdom associativated associativated associativated associativo esco tieda pioneer stipulations. Circulation 2011; 124: 2202-7

  In some aspects, the invention provided provides a method of reducing serum LDL cholesterol in a patient diagnosed with homozygous familial hypercholesterolemia, wherein the method requires at least one anti-PCSK9 antibody. Administering to the patient at a dosage of about 120 mg to about 3000 mg, wherein the method comprises reducing serum LDL cholesterol levels by at least about 10% compared to the pre-dose levels of serum LDL cholesterol in the patient. In some embodiments of this aspect of the invention, the patient's serum LDL cholesterol level is at least about 20%, at least about 25%, at least about 30%, as compared to the patient's serum LDL cholesterol level prior to administration, 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%, Reduce by at least about 85%, or at least about 90%.

  In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is administered to a patient diagnosed with homozygous familial hypercholesterolemia from about 140 mg to about 3000 mg, about 140 mg to about 2800 mg, about 140 mg to about 2500 mg, about 140 mg to about 2000 mg, about 140 mg to about 1800 mg, about 140 mg to about 1400 mg, about 120 mg to about 1200 mg, about 120 mg to about 1000 mg, about 120 mg to about 700 mg, about 140 mg to about 700 mg, about 140 mg to about 600 mg, About 140 mg to about 450 mg, about 120 mg to about 450 mg, about 120 mg to about 450 mg, about 140 mg to about 450 mg, about 210 mg to about 450 mg, or about 280 mg to about 450 mg, about 210 mg to about 420 mg, about 280 mg to about 4 mg 0 mg, about 420 mg to about 3000 mg, about 700 mg to about 3000 mg, about 1000 mg to about 3000 mg, about 1200 to about 3000 mg, about 1400 mg to about 3000 mg, about 1800 mg to about 3000 mg, about 2000 mg to about 3000 mg, about 2400 mg to about 3000 mg, Or a dosage of about 2800 mg to about 3000 mg is administered. In some embodiments of this aspect, the anti-PCSK9 antibody is administered to the patient in about 35 mg, about 45 mg, about 70 mg, about 105 mg, about 120 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg. About 200 mg, about 210 mg, about 280 mg, about 360 mg, about 420 mg, about 450 mg, about 600 mg, about 700 mg, about 1200 mg, about 1400 mg, about 1800 mg, about 2000 mg, about 2500 mg, about 2800 mg, or about 3000 mg. Administer.

  In some embodiments of this aspect of the invention, the anti-PCSK9 antibodies are administered (1) once a week, (2) once every two weeks, (3) once a month, (4) one month. Every other, (5) once every three months, (6) once every six months, and (7) once every 12 months. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is administered parenterally. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is administered intravenously. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is administered subcutaneously.

  In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is A) (i) CDRH1 from CDRH1 in a sequence selected from the group consisting of SEQ ID NOs: 49, 67, 459, 463, and 483, ( ii) CDRH2 from CDRH2 in a sequence selected from the group consisting of SEQ ID NOs: 49, 67, 459, 463, and 483; (iii) selected from the group consisting of SEQ ID NOs: 49, 67, 459, 463, and 483 CDRH3 from CDRH3 in sequence, and (iv) from CDRH of (i), (ii), and (iii) containing one or more amino acid substitutions, deletions, or insertions of no more than four amino acids. One or more heavy chain complementarity determining regions (CDRHs) selected from the group consisting of: B) (i) SEQ ID NOs: 23, 12 CDRL1 from CDRL1 in a sequence selected from the group consisting of 461, 465, and 485, (ii) CDRL2 from CDRL2 in a sequence selected from the group consisting of SEQ ID NOs: 23, 12, 461, 465, and 485, ( iii) CDRL3 from CDRL3 in a sequence selected from the group consisting of SEQ ID NOs: 23, 12, 461, 465, and 485, and (iv) one or more amino acid substitutions, deletions of no more than four amino acids Or one or more light chain complementarity determining regions (CDRLs) selected from the group consisting of the CDRLs of (i), (ii), and (iii) containing the insertion, or C) one of A). One or more heavy chain CDRHs and one or more light chain CDRLs. 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 CDRHs of A) and at least two CDRLs of B). In some embodiments, the isolated antigen binding protein comprises at least three CDRHs of A) and at least three CDRLs of B).

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 23, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 23, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 23. And the CDRH1 sequence of the CDRH1 sequence of SEQ ID NO: 49, the CDRH2 of the CDRH2 sequence of SEQ ID NO: 49, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 49.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 465, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 465, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 465. And the CDRH2 of the CDRH2 sequence of SEQ ID NO: 463, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 463.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 12, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 12. , A heavy chain complementarity determining region (CDR) of the CDRH1 sequence of SEQ ID NO: 67, CDRH2 of the CDRH2 sequence of SEQ ID NO: 67, and CDRH3 of the CDRH3 sequence of SEQ ID NO: 67.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 461, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 461, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 461. And the CDRH1 of the CDRH1 sequence of SEQ ID NO: 459, the CDRH2 of the CDRH2 sequence of SEQ ID NO: 459, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 459.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 485, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 485, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 485. And the CDRH2 of the CDRH2 sequence of SEQ ID NO: 483, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 483.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 582, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 582, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 582. And the CDRH2 of the CDRH2 sequence of SEQ ID NO: 583, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 583.

  In some embodiments of this aspect of the invention, the anti-PCSK9 antibody has a light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 and at least 90% of the amino acid sequence of SEQ ID NO: 49. A heavy chain variable region comprising an amino acid sequence which is identical to a light chain variable region comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 67. A heavy chain variable region comprising a light chain variable region comprising at least 90% identical to the amino acid sequence of SEQ ID NO: 461 and a heavy chain variable region comprising an amino acid sequence comprising at least 90% identical to the amino acid sequence of SEQ ID NO: 459; Light chain containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 465 A heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 463; a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 485; A heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence or a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 582 and at least 90% to the amino acid sequence of SEQ ID NO: 583 A heavy chain variable region comprising an amino acid sequence that is% identical. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, the amino acid sequence of SEQ ID NO: 12. And a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 461; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 461; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 459; A light chain variable region comprising the amino acid sequence of SEQ ID NO: 463; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 483; A light chain variable region comprising the amino acid sequence of SEQ ID NO: 582 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 583; Comprising a heavy chain variable region comprising the amino acid sequence of the light chain variable region SEQ ID NO: 590 containing. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is selected from the group consisting of 21B12, 31H4, 8A3, 11F1, and 8A1.

  In some aspects, the present invention relates to a method of treating a patient diagnosed with homozygous familial hypercholesterolemia, wherein the at least one anti-PCSK9 antibody is administered to the patient in need thereof from about 120 mg to about 120 mg. A method comprising treating homozygous familial hypercholesterolemia in said patient by administering a dosage of about 3000 mg. In some embodiments of this aspect, the level of serum LDL cholesterol in the patient diagnosed with homozygous familial hypercholesterolemia is at least about 10% compared to the level of the patient prior to administration of serum LDL cholesterol. 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%.

  In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is A) (i) CDRH1 from CDRH1 in a sequence selected from the group consisting of SEQ ID NOs: 49, 67, 459, 463, and 483, ( ii) CDRH2 from CDRH2 in a sequence selected from the group consisting of SEQ ID NOs: 49, 67, 459, 463, and 483; (iii) selected from the group consisting of SEQ ID NOs: 49, 67, 459, 463, and 483 CDRH3 from CDRH3 in sequence, and (iv) from CDRH of (i), (ii), and (iii) containing one or more amino acid substitutions, deletions, or insertions of no more than four amino acids. One or more heavy chain complementarity determining regions (CDRHs) selected from the group consisting of: B) (i) SEQ ID NOs: 23, 12 CDRL1 from CDRL1 in a sequence selected from the group consisting of 461, 465, and 485, (ii) CDRL2 from CDRL2 in a sequence selected from the group consisting of SEQ ID NOs: 23, 12, 461, 465, and 485, ( iii) CDRL3 from CDRL3 in a sequence selected from the group consisting of SEQ ID NOs: 23, 12, 461, 465, and 485, and (iv) one or more amino acid substitutions, deletions of no more than four amino acids Or one or more light chain complementarity determining regions (CDRLs) selected from the group consisting of the CDRLs of (i), (ii), and (iii) containing the insertion, or C) one of A). One or more heavy chain CDRHs and one or more light chain CDRLs. 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 CDRHs of A) and at least two CDRLs of B). In some embodiments, the isolated antigen binding protein comprises at least three CDRHs of A) and at least three CDRLs of B).

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 23, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 23, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 23. And the CDRH1 sequence of the CDRH1 sequence of SEQ ID NO: 49, the CDRH2 of the CDRH2 sequence of SEQ ID NO: 49, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 49.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 465, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 465, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 465. And the CDRH2 of the CDRH2 sequence of SEQ ID NO: 463, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 463.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 12, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 12. , A heavy chain complementarity determining region (CDR) of the CDRH1 sequence of SEQ ID NO: 67, CDRH2 of the CDRH2 sequence of SEQ ID NO: 67, and CDRH3 of the CDRH3 sequence of SEQ ID NO: 67.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 461, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 461, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 461. And the CDRH1 of the CDRH1 sequence of SEQ ID NO: 459, the CDRH2 of the CDRH2 sequence of SEQ ID NO: 459, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 459.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 485, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 485, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 485. And the CDRH2 of the CDRH2 sequence of SEQ ID NO: 483, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 483.

  In some embodiments, the isolated antigen binding protein comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 582, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 582, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 582. And the CDRH2 of the CDRH2 sequence of SEQ ID NO: 583, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 583.

  In some embodiments of this aspect of the invention, the anti-PCSK9 antibody has a light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 and at least 90% of the amino acid sequence of SEQ ID NO: 49. A heavy chain variable region comprising an amino acid sequence which is identical to a light chain variable region comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 67. A heavy chain variable region comprising a light chain variable region comprising at least 90% identical to the amino acid sequence of SEQ ID NO: 461 and a heavy chain variable region comprising an amino acid sequence comprising at least 90% identical to the amino acid sequence of SEQ ID NO: 459; Light chain containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 465 A heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 463; a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 485; A heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence or a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 582 and at least 90 to the amino acid sequence of SEQ ID NO: 583 And a heavy chain variable region comprising an amino acid sequence that is% identical. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, the amino acid sequence of SEQ ID NO: 12. And a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 461; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 461; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 459; A light chain variable region comprising the amino acid sequence of SEQ ID NO: 463; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 483; A light chain variable region comprising the amino acid sequence of SEQ ID NO: 582 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 583; Comprising the amino acid sequence of the light chain variable region SEQ ID NO: 590 comprising comprises a heavy chain variable region. In some embodiments of this aspect of the invention, the anti-PCSK9 antibody is selected from the group consisting of 21B12, 31H4, 8A3, 11F1, and 8A1.

  In certain embodiments, the anti-PCSK9 antibody comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 23, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 23, a CDRL3 of the CDRL3 sequence of SEQ ID NO: 23, 49. It includes the heavy chain complementarity determining region (CDR) of the CDRH1 sequence of No. 49, CDRH2 of the CDRH2 sequence of SEQ ID NO: 49, and CDRH3 of the CDRH3 sequence of SEQ ID NO: 49. In some embodiments, the anti-PCSK9 antibody has a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 23 and an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 49 And a heavy chain variable region comprising: In some embodiments, the anti-PCSK9 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49. In some embodiments, the anti-PCSK9 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 591 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 590. In some embodiments, the anti-PCSK9 antibody is 21B12. In certain embodiments, the anti-PCSK9 antibody comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 23, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 23, a CDRL3 of the CDRL3 sequence of SEQ ID NO: 23, SEQ ID NO: 49 comprises the heavy chain complementarity determining region (CDR) of CDRH1 sequence, CDRH2 of CDRH2 sequence of SEQ ID NO: 49, and CDRH3 of CDRH3 sequence of SEQ ID NO: 49, or at least 90% of the amino acid sequence of SEQ ID NO: 23 An amino acid sequence that is identical and a heavy chain variable region that includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 49, or a light chain variable region that includes the amino acid sequence of SEQ ID NO: 23; Or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 591 The light chain variable region and the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 590, or the antibody is 21B12, and the anti-PCSK9 antibody is administered to the patient in a dosage of about 120 mg to about 450 mg. Administered once weekly subcutaneously, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 3-10 days; the patient is administered subcutaneously once weekly at a dosage of about 120 mg Wherein the patient's serum LDL cholesterol level is reduced by at least about 15 to 50% in about 3 to 10 days; the patient is administered subcutaneously at a dosage of about 140 mg once a week, wherein the patient Lowers serum LDL cholesterol levels by at least about 15 to 50% in about 3 to 10 days; patients are injected subcutaneously at a dosage of about 120 mg to about 450 mg once every other week. Wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 7-14 days; the patient is administered a subcutaneous dose of about 120 mg once every other week, wherein: Reduce the patient's serum LDL cholesterol level by at least about 15-50% in about 7-14 days; give the patient a subcutaneous dose of about 140 mg once every other week, wherein the patient's serum LDL cholesterol is reduced The level is reduced by at least about 15-50% in about 7-14 days; the patient is administered a subcutaneous dose of about 210 mg once every other week, wherein the patient's serum LDL cholesterol level is reduced by about 7 to 14 days. At least about 15-50% in １４14 days; the patient is administered a subcutaneous dose of about 280 mg once every other week, wherein the patient's serum LDL cholesterol The level is reduced by at least about 15-50% in about 7-14 days; the patient is administered a subcutaneous dose of about 350 mg once every other week, wherein the patient's serum LDL cholesterol level is reduced by about 7 to 14 days. Reduces the patient's serum LDL cholesterol level by at least about 15 to 50% once every week at a dosage of about 420 mg, wherein the serum LDL cholesterol level is at least about 14 to 14 days. About 15-50% reduction; the patient is administered a subcutaneous dose of about 280 mg to about 420 mg once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15 to about 31 to 31 days. 50% reduction; patients are administered subcutaneously at a dosage of about 280 mg once every four weeks, wherein the patient's serum LDL cholesterol levels are reduced by about 21-31 days. Also reduces the patient's serum LDL cholesterol level by at least about 15-50% in about 21-31 days at a dosage of about 350 mg once every four weeks. Reduced; patients are administered subcutaneously at a dosage of about 420 mg every four weeks, wherein the patient's serum LDL cholesterol levels are reduced by 15-50% in about 21-31 days.

  In another specific embodiment, the anti-PCSK9 antibody comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 23, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 23, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 23. It comprises the heavy chain complementarity determining region (CDR) of the CDRH1 sequence of SEQ ID NO: 49, the CDRH2 of the CDRH2 sequence of SEQ ID NO: 49, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 49, or at least the amino acid sequence of SEQ ID NO: 23. A light chain variable region comprising an amino acid sequence that is 90% identical to a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 49, or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, or the amino acid sequence of SEQ ID NO: 591 Or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 590, or the antibody is 21B12, and the anti-PCSK9 antibody is administered to the patient in a dosage from about 420 mg to about 3000 mg. Administered intravenously once a week, where the patient's serum LDL cholesterol level is reduced by 15-50% in about 3-10 days; the patient is given an intravenous dose of about 700 mg once a week. Administered, wherein the patient's serum LDL cholesterol level is reduced by 15-50% in about 3-10 days; the patient is administered an intravenous dose of about 1200 mg once a week, wherein the patient Reduces the serum LDL cholesterol level by 15 to 50% in about 3 to 10 days; administered to the patient intravenously once a week at a dosage of about 1200 mg to about 3000 mg. Clear LDL cholesterol levels are reduced by 15-50% in about 3-10 days; patients are dosed intravenously every other week at a dosage of about 420 mg to about 3000 mg, wherein the serum LDL cholesterol levels of the patients are reduced. Reduces the patient's serum LDL cholesterol level by 15 to 50% every other week for about 7 to 14 days, at a dosage of about 700 mg. ５０50% reduction; administered to patients at a dosage of about 1200 mg intravenously every other week, wherein the patient's serum LDL cholesterol level is reduced by 15-50% in about 21-31 days; Administered intravenously every other week at a dosage of about 1200 mg to about 3000 mg, where the patient's serum LDL cholesterol level is increased by 15-50% in about 7-14 days. Reduced; administered to the patient intravenously at a dosage of about 420 mg to about 3000 mg every 4 weeks, wherein the patient's serum LDL cholesterol level is reduced by 15-50% in about 21-31 days. Administered intravenously at a dosage of about 700 mg every 4 weeks, wherein the patient's serum LDL cholesterol level is reduced by 15-50% in about 21-31 days; Administered intravenously every week, wherein the patient's serum LDL cholesterol level is reduced by 15-50% in about 21-31 days; patients are administered intravenously every 4 weeks at a dosage of about 1200 mg to about 3000 mg. Administered, wherein the patient's serum LDL cholesterol level is reduced by 15-50% in about 21-31 days.

  In another specific embodiment, the anti-PCSK9 antibody comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 23, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 23, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 23. It comprises the heavy chain complementarity determining region (CDR) of the CDRH1 sequence of SEQ ID NO: 49, the CDRH2 of the CDRH2 sequence of SEQ ID NO: 49, and the CDRH3 of the CDRH3 sequence of SEQ ID NO: 49, or at least the amino acid sequence of SEQ ID NO: 23. A light chain variable region comprising an amino acid sequence that is 90% identical to a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 49, or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, or the antibody is 21B12, The PCSK9 antibody is administered to the patient subcutaneously at a dosage of about 120 mg once a week, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 7-10 days; Administered subcutaneously once a week at a dosage of about 140 mg, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 7-10 days; Once every other week, where the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 10-14 days; the patient is given a dose of about 140 mg every other week. A single subcutaneous dose, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 10-14 days; Once every other week, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 10-14 days; the patient is given a dose of about 280 mg every other week. A single subcutaneous administration, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 10-14 days; the patient is administered a subcutaneous dose of about 350 mg once every other week. Wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 10-14 days; the patient is administered subcutaneously once every other week at a dosage of about 420 mg; Administered subcutaneously at a dosage of 280 mg to about 450 mg once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days Allowing the patient to be administered subcutaneously at a dosage of about 280 mg once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days; A subcutaneous dose of 350 mg once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days; Administered subcutaneously weekly, where the patient's serum LDL cholesterol level is reduced by 30-50% in about 24-28 days.

  In another specific embodiment, the anti-PCSK9 antibody has a heavy chain variable comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 23 and an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 49. Or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 591 Or the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 590, or the antibody is 21B12, and the anti-PCSK9 antibody is administered to the patient at a dosage of about 420 mg to about 3000 mg once a week. Administered intravenously, where the patient's serum LDL cholesterol level is reduced by 30-50% in about 7-10 days; Once a week, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 7-10 days; the patient is given a dose of about 1200 mg weekly. A single intravenous dose, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 7-10 days; the patient is dosed intravenously once a week with a dosage of more than about 1200 mg to about 3000 mg. Administered intravenously, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 7-10 days; the patient is administered intravenously every other week at a dosage of about 420 mg to about 3000 mg. Reduces the patient's serum LDL cholesterol level by 30-50% in about 10-14 days; the patient is administered an intravenous dose of about 700 mg every other week, wherein the patient's serum L L-cholesterol levels are reduced by 30-50% in about 10-14 days; patients are dosed intravenously every other week at a dosage of about 1200 mg, where the patient's serum LDL cholesterol levels are reduced by about 10-10 days. 30 to 50% reduction over 14 days; patients are dosed intravenously every other week at doses from about 1200 mg to about 3000 mg, wherein the patient's serum LDL cholesterol levels are reduced by 30 to about 10 to 14 days.患者 50% reduced; patients are dosed intravenously at a dosage of about 420 mg to about 3000 mg every 4 weeks, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 24-28 days. Administering to the patient intravenously at a dosage of about 700 mg every 4 weeks, wherein the patient's serum LDL cholesterol level is increased by 30-5 days for about 24-28 days; 0% reduced; the patient is administered intravenously at a dosage of about 1200 mg every 4 weeks, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 24-28 days; Administered intravenously every four weeks at a dosage of greater than about 1200 mg to about 3000 mg, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 24-28 days.

  In certain embodiments of the invention, the anti-PCSK9 antibodies are 8A3, 11F1, and 8A1. In some embodiments, the anti-PCSK9 antibody comprises the light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 465, the CDRL2 of the CDRL2 sequence of SEQ ID NO: 465, the CDRL3 of the CDRL3 sequence of SEQ ID NO: 465, and the sequence SEQ ID NO: 463 comprises the heavy chain complementarity determining region (CDR) of the CDRH1 sequence, SEQ ID NO: 463 CDRH2 sequence, and CDRH3 SEQ ID NO: CDRH3 sequence CDRH3. In some embodiments, the anti-PCSK9 antibody has a light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 465, and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 463. And a heavy chain variable region comprising: In some embodiments, the anti-PCSK9 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 465 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 463. In some embodiments, the anti-PCSK9 antibody is 11F1. In certain embodiments, the anti-PCSK9 antibody comprises the light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 465, the CDRL2 of the CDRL2 sequence of SEQ ID NO: 465, the CDRL3 of the CDRL3 sequence of SEQ ID NO: 465, and the sequence No. 463 comprising the heavy chain complementarity determining region (CDR) of the CDRH1 sequence, SEQ ID NO: 463 CDRH2 sequence, and the CDRH3 sequence of SEQ ID NO: 463 CDRH3, or comprising at least 90 amino acid sequence of SEQ ID NO: 465. An amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 463, or a light chain variable region that includes the amino acid sequence of SEQ ID NO: 465; Or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 463, or 1F1, an anti-PCSK9 antibody is administered to a patient subcutaneously once a week at a dosage of about 150 mg, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 3-10 days. Reducing the patient's serum LDL cholesterol level by at least about 15-50% in about 7-14 days, wherein the patient is administered subcutaneously once a week at a dosage of about 150 mg; A subcutaneous dose of about 150 mg once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 21-31 days; Once a week for 4 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 21-31 days; Administered subcutaneously once every four weeks at a dosage of about 170 mg to about 180 mg, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 21-31 days; Administered subcutaneously once every four weeks at a dosage of 150 mg to about 170 mg, wherein the patient's serum LDL cholesterol level is reduced by at least about 15 to 50% in about 21 to 31 days; Dose once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 21-31 days; Subcutaneously administered, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 31-42 days; A subcutaneous dose of 200 mg once every 6 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 31-42 days; the patient is dosed with about 170 mg to about 180 mg Subcutaneously every six weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 31-42 days; Administered subcutaneously once a week, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 31-42 days; the patient is administered subcutaneously once every 6 weeks at a dosage of about 450 mg Wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 31-42 days; Is administered subcutaneously every 8 weeks, wherein the patient's serum LDL cholesterol level is reduced by 15-50% in about 45-56 days; the patient is administered a dosage of about 170 mg to about 180 mg for 8 weeks. Administered subcutaneously every 8 weeks, wherein the patient's serum LDL cholesterol level is reduced by 15-50% in about 45-56 days; the patient is administered subcutaneously every 8 weeks at a dosage of about 150 mg to about 170 mg; Here, the patient's serum LDL cholesterol level is reduced by 15-50% in about 45-56 days; the patient is administered subcutaneously every 8 weeks at a dosage of about 450 mg, wherein the patient's serum LDL cholesterol level Is reduced by 15-50% in about 45-56 days; administered at a dosage of about 600 mg subcutaneously once every 8 weeks, wherein the patient's serum LDL cholesterol level is reduced. Is reduced by at least about 15-50% in about 45-56 days; a dosage of about 700 mg is administered subcutaneously once every 8 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 45-56 days. About 15-50% reduced; administered at a dosage of about 600 mg subcutaneously once every 12 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 74-84 days; A subcutaneous dose of 700 mg once every 12 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 74-84 days; Subcutaneously administered, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 100-112 days; Dosage is administered subcutaneously once every 16 weeks, where the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 100-112 days.

  In certain embodiments of the invention, the anti-PCSK9 antibody comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 465, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 465, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 465. The CDRH1 sequence of the CDRH1 sequence of SEQ ID NO: 463, CDRH2 of the CDRH2 sequence of SEQ ID NO: 463, and CDRH3 of the CDRH3 sequence of SEQ ID NO: 463. In some embodiments, the anti-PCSK9 antibody has a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 465 and an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 463. Or the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 465 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 463, or the antibody is 11F1 and comprises an anti-PCSK9 antibody. The patient is administered subcutaneously at a dosage of about 150 mg once a week, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 7-10 days; The dosage is administered subcutaneously once every other week, wherein the patient's serum LDL cholesterol level is reduced by about 10-14 days. About 30-50% reduction; the patient is administered subcutaneously at a dosage of about 150 mg once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days. Allowing the patient to be administered subcutaneously at a dosage of greater than about 150 mg to about 200 mg once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days; The patient is administered subcutaneously at a dosage of about 170 mg to about 180 mg once every four weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days; A subcutaneous dose of about 150 mg to about 170 mg once every four weeks, wherein the patient's serum LDL cholesterol level is increased by at least about 3 to about 24 to 28 days. Reducing the patient's serum LDL cholesterol level by at least about 30-50% in about 24-28 days; The patient is dosed subcutaneously at a dosage of about 150 mg once every six weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 40-41 days; Administered subcutaneously once every 6 weeks at a dosage of about 200 mg, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 40-41 days; Once every 6 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 40-41 days; Is administered subcutaneously at a dosage of about 150 mg to about 170 mg once every 6 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 40-41 days; Administered subcutaneously once every six weeks at a dosage of about 450 mg, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 40-41 days; The dosage is administered subcutaneously every eight weeks, where the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 50-56 days; the patient is given 8 mg in a dosage of about 170 mg to about 180 mg. Administered subcutaneously weekly, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 50-56 days; A subcutaneous dose of 50 mg to about 170 mg administered every 8 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 50-56 days; Subcutaneously every 8 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 50-56 days; a subcutaneous dose of about 600 mg once every 8 weeks; Here, the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 50-56 days; a dosage of about 700 mg is administered subcutaneously once every eight weeks, wherein the patient's serum LDL cholesterol level is reduced. Is reduced by at least about 30-50% in about 50-56 days; administered at a dosage of about 600 mg subcutaneously once every 12 weeks, wherein the patient Reduces purified LDL cholesterol levels by at least about 30-50% in about 80-84 days; administered at a dosage of about 700 mg subcutaneously once every 12 weeks, wherein the patient's serum LDL cholesterol levels are reduced by about 80-84 days. Reduced by at least about 30-50% in 84 days; administered at a dosage of about 600 mg subcutaneously once every 16 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 105-112 days. Reduced; administered subcutaneously at a dosage of about 700 mg once every 16 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 105-112 days.

  In certain embodiments of the invention, the anti-PCSK9 antibody comprises a light chain complementarity region (CDR) of the CDRL1 sequence of SEQ ID NO: 465, a CDRL2 of the CDRL2 sequence of SEQ ID NO: 465, and a CDRL3 of the CDRL3 sequence of SEQ ID NO: 465. The CDRH1 sequence of the CDRH1 sequence of SEQ ID NO: 463, CDRH2 of the CDRH2 sequence of SEQ ID NO: 463, and CDRH3 of the CDRH3 sequence of SEQ ID NO: 463. In some embodiments, the anti-PCSK9 antibody has a heavy chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 465 and an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 463. Or comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 465 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 463, or the antibody is 11F1 and comprises an anti-PCSK9 antibody. Administered to a patient, the anti-PCSK9 antibody is administered intravenously to the patient at a dosage of about 420 mg to about 3000 mg once a week, wherein the patient's serum LDL cholesterol level is increased by 30 to about 7 to 10 days. ５０50% reduced; patients are dosed intravenously at a dosage of about 700 mg once a week, where the patient's serum LDL cholesterol The level is reduced by 30-50% in about 7-10 days; the patient is administered an intravenous dose of about 1200 mg once a week, wherein the patient's serum LDL cholesterol level is reduced by about 7-10 30 to 50% per day; patients are dosed intravenously once a week in dosages from about 1200 mg to about 3000 mg, wherein the patient's serum LDL cholesterol levels are reduced by 30 to about 7 to 10 days. ５０50% reduction; patients are dosed intravenously every other week at a dosage of about 420 mg to about 3000 mg, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 10-14 days. Giving the patient an intravenous dose of about 700 mg every other week, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 10-14 days; A dose of about 1200 mg administered intravenously every other week, where the patient's serum LDL cholesterol level is reduced by 30-50% in about 10-14 days; the patient is dosed with more than about 1200 mg to about 3000 mg Iv dose every other week, wherein the patient's serum LDL cholesterol level is reduced by 30-50% in about 10-14 days; the patient is given a dosage of about 420 mg to about 3000 mg every 4 weeks Wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days; the patient is administered intravenously at a dosage of about 700 mg every 4 weeks; Here, the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days; the patient is given a dosage of about 1200 mg for 4 weeks. Administered intravenously, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days; patients are given a dosage of more than about 1200 mg to about 3000 mg every 4 weeks Wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 24-28 days; a iv dose of about 1000 mg to 3000 mg once every 24 weeks; Here, the patient's serum LDL cholesterol level is reduced by at least about 15 to 50% in about 150 to 168 days; a dosage of about 1000 mg to 3000 mg is administered intravenously once every 24 weeks, wherein the patient's serum is reduced. Reduces LDL cholesterol levels by at least about 30-50% in about 160-168 days; about 1000 mg-3000 mg Intravenous administration at a dosage of once every 52 weeks, wherein the patient's serum LDL cholesterol level is reduced by at least about 15-50% in about 350-365 days; at a dosage of about 1000-3000 mg in 52 weeks A single intravenous dose, wherein the patient's serum LDL cholesterol level is reduced by at least about 30-50% in about 360-365 days.

  In another aspect of the invention, at least one anti-PCSK9 antibody is administered to the patient before, after, or simultaneously with the administration of at least one other cholesterol-lowering agent. Cholesterol-lowering agents include statins (eg, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), nicotinic acid (niacin), sustained release niacin (SLO-NIACIN), laropiprant (CORDAPIVE), Fibric acid (LOPID (gemfibrozil), TRICOR (fenofibrate)), bile acid sequestering agents such as cholestyramine (QUESTRAN), colesevelam (WELCHOL), COLESTID (cholestipol)), cholesterol absorption inhibitor (ZETIA (ezetimibe)), Lipid modulator, PPAR-γ agonist, PPAR-α / γ agonist, squalene synthase inhibitor, CETP inhibitor, anti- Blood pressure agents, antidiabetic agents, such as sulfonylureas, insulin, GLP-1 analogs, DDPIV inhibitors, ApoB modulators, MTP inhibitors, and / or treatment of atherosclerosis obliterans, oncostatin M, estrogens, belvin, And therapeutic agents for immune-related diseases.

  In some aspects, the invention includes a method of reducing serum LDL cholesterol in a patient diagnosed with homozygous familial hypercholesterolemia. The method comprises administering to a patient diagnosed with homozygous familial hypercholesterolemia at least one anti-PCSK9 antibody described herein at a dosage of about 120 mg to about 3000 mg. . In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 120 mg to about 450 mg administered once a week (QW). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 140 mg to about 450 mg administered once a week. In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 280 mg to about 450 mg administered once a week. In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 120 mg to about 450 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 140 mg to about 450 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 280 mg to about 420 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 400 mg to about 450 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one out of about 420 mg of the anti-PCSK9 antibody administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 250 mg to about 480 mg administered once every four weeks (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 280 mg to about 420 mg administered once every four weeks (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 350 mg to about 420 mg administered about once every four weeks (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 420 mg to about 3000 mg administered weekly (QW). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 1000 mg to about 3000 mg administered once a week (QW). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 2000 mg to about 3000 mg administered once a week (QW). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 420 mg to about 3000 mg administered bi-weekly (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 1000 mg to about 3000 mg administered bi-weekly (Q2W). In some embodiments, the dosage is about 2000 mg to about 3000 mg of at least one anti-PCSK9 antibody administered bi-weekly (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 420 mg to about 3000 mg administered once a month (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 1000 mg to about 3000 mg administered once a month (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 2000 mg to about 3000 mg administered once a month (Q4W). In some embodiments, the serum LDL cholesterol level is reduced by at least about 10% compared to the serum LDL cholesterol level before administration. In some embodiments, the serum LDL cholesterol level is reduced by at least about 15%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 20%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 25%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 30%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 35%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 40%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 45%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 50%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 55%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 60%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 70%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 80%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 85%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 90%.

  In some aspects, the invention includes a method of reducing serum LDL cholesterol in a patient diagnosed with homozygous familial hypercholesterolemia, wherein the method comprises administering to a patient in need thereof a dosage of Administering at least one anti-PCSK9 antibody at a dosage of: (1) at least about 120 mg weekly (QW), (2) at least about 140 mg weekly (QW), (3 A.) At least about 120 mg every two weeks or biweekly (Q2W); (4) at least about 140 mg every two weeks or biweekly (Q2W); ), (6) at least about 280 mg in biweekly or biweekly (Q2W), (7) at least about 350 mg in biweekly or biweekly manner Q2W), (8) at least about 420 mg every two weeks or biweekly (Q2W), and (9) at least about 150 mg every four weeks (Q4W), (10) at least about 160 mg every four weeks (Q4W), (11) at least about 170 mg every 4 weeks (Q4W), (12) at least about 180 mg every 4 weeks (Q4W), (13) at least about 190 mg every 4 weeks (Q4W) ), (14) at least about 200 mg every 4 weeks (Q4W), (15) at least about 280 mg every 4 weeks (Q4W), (16) at least about 350 mg every 4 weeks (Q4W), (17) At least about 420 mg every 4 weeks (Q4W), (18) At least about 1000 mg every 4 weeks (Q4W), (1 ) 4-week intervals in an amount of at least about 2000 mg (Q4W), and (20) are administered on a schedule selected from the group consisting of 4-week intervals in an amount of at least about 3000 mg (Q4W). In some embodiments, the serum LDL cholesterol level is reduced by at least about 10% compared to the serum LDL cholesterol level before administration. In some embodiments, the serum LDL cholesterol level is reduced by at least about 10% compared to the serum LDL cholesterol level before administration. In some embodiments, the serum LDL cholesterol level is reduced by at least about 15%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 20%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 25%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 30%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 35%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 40%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 45%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 50%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 55%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 60%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 65%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 70%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 80%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 85%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 90%.

  In some aspects, the invention includes a method of reducing serum PCSK9 levels in a patient diagnosed with homozygous familial hypercholesterolemia. The method comprises administering to a patient in need thereof at least one of the dosages of the anti-PCSK9 antibody, wherein the dosage of the anti-PCSK9 antibody is: 1) at least about 120 mg weekly (QW); (2) Weekly (QW) in an amount of at least about 140 mg; (3) Biweekly or biweekly (Q2W) in an amount of at least about 120 mg; (4) Biweekly or biweekly (Q2W) in an amount of at least about 140 mg; 5) Every 2 weeks or every other week (Q2W) in an amount of at least about 150 mg; (6) Every 2 weeks or every other week (Q2W) in an amount of at least about 280 mg; (7) Every 2 weeks or every 2 weeks in an amount of at least about 350 mg (Q2W) Q2W); (8) at least about 420 mg every 2 weeks or every other week (Q2W); and (9) at least about 150 mg every 4 weeks ( (10) at least about 160 mg every 4 weeks (Q4W); (11) at least about 170 mg every 4 weeks (Q4W); (12) at least about 180 mg every 4 weeks (Q4W). (13) at least about 190 mg every 4 weeks (Q4W); (14) at least about 200 mg every 4 weeks (Q4W); (15) at least about 280 mg every 4 weeks (Q4W); 16) Every 4 weeks at least about 350 mg (Q4W); (17) Every 4 weeks at least about 420 mg (Q4W); (18) Every 4 weeks at least about 1000 mg (Q4W); (19) A group consisting of at least about 2000 mg every 4 weeks (Q4W); and (20) at least about 3000 mg every 4 weeks (Q4W). Administered in a schedule to be al selection. In some embodiments, serum PCSK9 levels are reduced by at least about 20% compared to serum PCSK9 levels prior to administration. In some embodiments, the serum PCSK9 value is reduced by at least about 30%. In some embodiments, the serum PCSK9 value is reduced by at least about 40%. In some embodiments, the serum PCSK9 value is reduced by at least about 50%. In some embodiments, the serum PCSK9 value is reduced by at least about 60%. In some embodiments, the serum PCSK9 value is reduced by at least about 65%. In some embodiments, the serum PCSK9 value is reduced by at least about 70%. In some embodiments, the serum PCSK9 value is reduced by at least about 75%. In some embodiments, the serum PCSK9 value is reduced by at least about 80%. In some embodiments, the serum PCSK9 value is reduced by at least about 85%. In some embodiments, the serum PCSK9 value is reduced by at least about 90%.

  In some aspects, the invention includes a method of reducing total cholesterol levels in a patient diagnosed with homozygous familial hypercholesterolemia, the method comprising: Administering at least one anti-PCSK9 antibody comprising: (1)) at least about 120 mg weekly (QW); (2) at least about 140 mg weekly (QW); 3) biweekly or biweekly (Q2W) in an amount of at least about 120 mg; (4) biweekly or biweekly (Q2W) in an amount of at least about 140 mg; (5) biweekly or biweekly in an amount of at least about 150 mg. (6) at least about 280 mg in every 2 weeks or biweekly (Q2W); (7) at least about 350 mg in every 2 weeks or biweekly (8) at least about every 420 weeks every 2 weeks or biweekly (Q2W); and (9) at least about 150 mg every 4 weeks (Q4W); (10) at least about 160 mg every 4 weeks (Q4W); (11) At least about 170 mg every 4 weeks (Q4W); (12) At least about 180 mg every 4 weeks (Q4W); (13) At least about 190 mg every 4 weeks (Q4W) (14) at least about 200 mg every 4 weeks (Q4W); (15) at least about 280 mg every 4 weeks (Q4W); (16) at least about 350 mg every 4 weeks (Q4W); (17) Every 4 weeks at least about 420 mg (Q4W); (18) Every 4 weeks at least about 1000 mg (Q4W); (1 Administered in and (20) of at least about 4 weeks intervals in an amount of 3000 mg (schedule selected from the group consisting of Q4W);) of at least about 4 weeks intervals in an amount of 2000 mg (Q4W). In some embodiments, the total cholesterol level is reduced by at least about 20% compared to the total cholesterol level before administration. In some embodiments, total cholesterol levels are reduced by at least about 25%. In some embodiments, total cholesterol levels are reduced by at least about 30%. In some embodiments, total cholesterol levels are reduced by at least about 35%. In some embodiments, total cholesterol levels are reduced by at least about 40%. In some embodiments, total cholesterol levels are reduced by at least about 45%. In some embodiments, total cholesterol levels are reduced by at least about 50%. In some embodiments, total cholesterol levels are reduced by at least about 55%. In some embodiments, total cholesterol levels are reduced by at least about 60%.

  In some aspects, the invention includes a method of reducing non-HDL cholesterol levels in a patient diagnosed with homozygous familial hypercholesterolemia, the method comprising administering to a patient in need thereof a reduced dosage of non-HDL cholesterol. Administering at least one of the anti-PCSK9 antibodies, wherein the dosage of the anti-PCSK9 antibody is: (1) at least about 120 mg weekly (QW); (2) at least about 140 mg weekly (QW); ( 3) biweekly or biweekly (Q2W) in an amount of at least about 120 mg; (4) biweekly or biweekly (Q2W) in an amount of at least about 140 mg; (5) biweekly or biweekly in an amount of at least about 150 mg. (6) at least about 280 mg in biweekly or biweekly (Q2W); (7) at least about 350 mg in biweekly or (8) every 2 weeks in an amount of at least about 420 mg or every other week (Q2W); and (9) every 4 weeks in an amount of at least about 150 mg (Q4W); (10) 4 in an amount of at least about 160 mg. Weekly (Q4W); (11) at least about 170 mg in 4 weeks (Q4W); (12) at least about 180 mg in 4 weeks (Q4W); (13) at least about 190 mg in 4 weeks (14) at least about 200 mg every 4 weeks (Q4W); (15) at least about 280 mg every 4 weeks (Q4W); (16) at least about 350 mg every 4 weeks (Q4W) (17) At least about 420 mg every 4 weeks (Q4W); (18) At least about 1000 mg every 4 weeks (Q4W); 19) 4-week intervals in an amount of at least about 2000 mg (Q4W); and (20) are administered on a schedule selected from the group consisting of 4-week intervals in an amount of at least about 3000 mg (Q4W). In some embodiments, the level of non-HDL cholesterol is reduced by at least about 30% compared to the level of non-HDL cholesterol prior to administration. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 35%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 40%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 45%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 50%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 55%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 60%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 65%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 70%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 75%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 80%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 85%.

  In some aspects, the invention includes a method of reducing ApoB levels in a patient diagnosed with homozygous familial hypercholesterolemia, the method comprising: Administering at least one anti-PCSK9 antibody, wherein the dosage of the anti-PCSK9 antibody is: (1) at least about 120 mg weekly (QW); (2) at least about 140 mg weekly (QW); (3) (4) Biweekly or biweekly (Q2W) in an amount of at least about 120 mg; (4) Biweekly or biweekly (Q2W) in an amount of at least about 140 mg; (6) at least about 280 mg in biweekly or biweekly (Q2W); (7) at least about 350 mg in biweekly or biweekly (Q2W); (8) at least about 420 mg in every 2 weeks or biweekly (Q2W); and (9) at least about 150 mg in every 4 weeks (Q4W); (10) at least about 160 mg in every 4 weeks (Q4W). (11) at least about 170 mg every 4 weeks (Q4W); (12) at least about 180 mg every 4 weeks (Q4W); (13) at least about 190 mg every 4 weeks (Q4W); 14) Every 4 weeks at least about 200 mg (Q4W); (15) Every 4 weeks at least about 280 mg (Q4W); (16) Every 4 weeks at least about 350 mg (Q4W); (17) At least about 420 mg every 4 weeks (Q4W); (18) at least about 1000 mg every 4 weeks (Q4W); (19) less Also about 4 week intervals in an amount of 2000 mg (Q4W); administered in and (20) of at least about 4 weeks intervals in an amount of 3000mg schedule selected from the group consisting of (Q4W). In some embodiments, the ApoB level is reduced by at least about 10% compared to the ApoB level before administration. In some embodiments, ApoB levels are reduced by at least about 15%. In some embodiments, the ApoB level is reduced by at least about 20%. In some embodiments, ApoB levels are reduced by at least about 25%. In some embodiments, the ApoB level is reduced by at least about 30%. In some embodiments, ApoB levels are reduced by at least about 35%. In some embodiments, the ApoB level is reduced by at least about 40%. In some embodiments, the ApoB level is reduced by at least about 45%. In some embodiments, the ApoB level is reduced by at least about 50%. In some embodiments, the ApoB level is reduced by at least about 55%. In some embodiments, ApoB levels are reduced by at least about 60%. In some embodiments, ApoB levels are reduced by at least about 65%. In some embodiments, the ApoB level is reduced by at least about 70%. In some embodiments, the ApoB level is reduced by at least about 75%.

  In some aspects, the invention includes a method of reducing lipoprotein A ("Lp (a)") levels in a patient diagnosed with homozygous familial hypercholesterolemia, the method comprising the steps of: Administering to the patient at least one of the dosages of an anti-PCSK9 antibody, wherein the dosage of the anti-PCSK9 antibody is: (1) at least about 120 mg weekly (QW); (2) at least about 140 mg (3) biweekly or biweekly (Q2W) in an amount of at least about 120 mg; (4) biweekly or biweekly (Q2W) in an amount of at least about 140 mg; (5) at least about 150 mg. Every two weeks or every other week (Q2W); (6) every two weeks or every two weeks (Q2W) in an amount of at least about 280 mg; (7) two weeks in an amount of at least about 350 mg. (8) at least about 420 mg every two weeks or every other week (Q2W); and (9) at least about 150 mg every four weeks (Q4W); (10) at least about 160 mg (4) every 4 weeks (Q4W); (11) every 4 weeks in an amount of at least about 170 mg (Q4W); (12) every 4 weeks in an amount of at least about 180 mg (Q4W); (13) 4 in an amount of at least about 190 mg. Weekly (Q4W); (14) at least about 200 mg every 4 weeks (Q4W); (15) at least about 280 mg every 4 weeks (Q4W); (16) at least about 350 mg every 4 weeks (Q4W); (17) at least about 420 mg every 4 weeks (Q4W); (18) at least about 1000 mg every 4 weeks Q4W); administered in and (20) of at least about 4 weeks intervals in an amount of 3000 mg (schedule selected from the group consisting of Q4W); (19) at least about 4 weeks intervals in an amount of 2000 mg (Q4W). In some embodiments, Lp (a) levels are reduced by at least about 10% relative to pre-administration Lp (a) levels. In some embodiments, Lp (a) levels are reduced by at least about 15%. In some embodiments, Lp (a) levels are reduced by at least about 20%. In some embodiments, Lp (a) levels are reduced by at least about 25%. In some embodiments, Lp (a) levels are reduced by at least about 30%. In some embodiments, Lp (a) levels are reduced by at least about 35%. In some embodiments, Lp (a) levels are reduced by at least about 40%. In some embodiments, Lp (a) levels are reduced by at least about 45%. In some embodiments, Lp (a) levels are reduced by at least about 50%. In some embodiments, Lp (a) levels are reduced by at least about 55%. In some embodiments, Lp (a) levels are reduced by at least about 60%. In some embodiments, Lp (a) levels are reduced by at least about 65%.

  In some aspects, the present invention includes a method of treating a patient diagnosed with homozygous familial hypercholesterolemia, the method comprising administering to a patient diagnosed with homozygous familial hypercholesterolemia, Administering at least one anti-PCSK9 antibody described herein in a dosage of about 120 mg to about 3000 mg. In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 120 mg to about 450 mg administered once a week (QW). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 140 mg to about 450 mg administered once a week. In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 120 mg to about 450 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 140 mg to about 420 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 105 mg to about 350 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 140 mg to about 280 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 150 mg to about 280 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 150 mg to about 200 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 400 mg to about 450 mg administered once every two weeks (Q2W). In some embodiments, the dosage is at least one out of about 420 mg of the anti-PCSK9 antibody administered once every two weeks (Q2W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 120 mg to about 480 mg administered once every four weeks (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 150 mg to about 420 mg administered once every four weeks (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 400 mg to about 450 mg administered once every four weeks (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 250 mg to about 480 mg administered once every four weeks (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 280 mg to about 420 mg administered once every four weeks (Q4W). In some embodiments, the dosage is at least one anti-PCSK9 antibody from about 350 mg to about 420 mg administered once every four weeks. In some embodiments, the dosage is about 1000 mg every 4 weeks (Q4W). In some embodiments, the dosage is about 2000 mg every 4 weeks (Q4W). In some embodiments, the dosage is about 3000 mg every 4 weeks (Q4W). In some embodiments, the serum LDL cholesterol level is reduced by at least about 10% compared to the serum LDL cholesterol level before administration. In some embodiments, the serum LDL cholesterol level is reduced by at least about 15%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 20%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 25%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 30%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 35%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 40%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 45%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 50%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 55%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 60%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 65%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 70%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 80%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 85%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 90%.

  In some embodiments, the anti-PCSK9 antibody is 21B12, 26H5, 31H4, 8A3, 11F1, and / or 8A1.

  Other embodiments of the invention will be apparent from the disclosure provided herein.

The amino acid sequence of the mature form of PCSK9 is shown, with the prodomain underlined. The amino acid and nucleic acid sequences of PCSK9 are illustrated, the prodomain is underlined, and the signal sequence is in bold. The amino acid and nucleic acid sequences of PCSK9 are illustrated, the prodomain is underlined, and the signal sequence is in bold. The amino acid and nucleic acid sequences of PCSK9 are illustrated, the prodomain is underlined, and the signal sequence is in bold. The amino acid and nucleic acid sequences of PCSK9 are illustrated, the prodomain is underlined, and the signal sequence is in bold. FIG. 4 is a sequence comparison table for various light chains of various antigen binding proteins. FIG. 2C is a continuation of the sequence starting from FIG. 2A. FIG. 2D is a continuation of the sequence starting from FIG. 2B. FIG. 4 is a sequence comparison table for various light chains of various antigen binding proteins. FIG. 2C is a continuation of the sequence starting from FIG. 2A. FIG. 2D is a continuation of the sequence starting from FIG. 2B. FIG. 4 is a sequence comparison table for various light chains of various antigen binding proteins. FIG. 2C is a continuation of the sequence starting from FIG. 2A. FIG. 2D is a continuation of the sequence starting from FIG. 2B. FIG. 4 is a sequence comparison table for various light chains of various antigen binding proteins. FIG. 2C is a continuation of the sequence starting from FIG. 2A. FIG. 2D is a continuation of the sequence starting from FIG. 2B. FIG. 4 is a sequence comparison table for various heavy chains of various antigen binding proteins. FIG. 3C is a continuation of the sequence starting from FIG. 3A. FIG. 3D is a continuation of the sequence starting from FIG. 3B. FIG. 4 is a sequence comparison table for various heavy chains of various antigen binding proteins. FIG. 3C is a continuation of the sequence starting from FIG. 3A. FIG. 3D is a continuation of the sequence starting from FIG. 3B. FIG. 4 is a sequence comparison table for various heavy chains of various antigen binding proteins. FIG. 3C is a continuation of the sequence starting from FIG. 3A. FIG. 3D is a continuation of the sequence starting from FIG. 3B. FIG. 4 is a sequence comparison table for various heavy chains of various antigen binding proteins. FIG. 3C is a continuation of the sequence starting from FIG. 3A. FIG. 3D is a continuation of the sequence starting from FIG. 3B. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. 2 illustrates the amino acid and nucleic acid sequences for the variable domains of some embodiments of the antigen binding protein. Figure 4 depicts the amino acid sequences for various constant domains. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. 4 shows amino acid and nucleic acid sequences for the variable domain of some embodiments of the antigen binding protein. FIG. 4 is a sequence comparison table of various heavy and light chains of some embodiments of an antigen binding protein. FIG. 4 is a sequence comparison table of various heavy and light chains of some embodiments of an antigen binding protein. FIG. 4 is a sequence comparison table of various heavy and light chains of some embodiments of an antigen binding protein. FIG. 4 is a sequence comparison table of various heavy and light chains of some embodiments of an antigen binding protein. FIG. 4 is a sequence comparison table of various heavy and light chains of some embodiments of an antigen binding protein. FIG. 4 is a sequence comparison table of various heavy and light chains of some embodiments of an antigen binding protein. FIG. 4 is a sequence comparison table of various heavy and light chains of some embodiments of an antigen binding protein. FIG. 4 is a sequence comparison table of various heavy and light chains of some embodiments of an antigen binding protein. A series of ABP sequences that specify various differences between human ABP sequences produced in E. coli and ABP sequences (US Pat. No. 8,030,457). It is a binding curve of the antigen binding protein to human PCSK9. It is a binding curve of the antigen binding protein to human PCSK9. Fig. 4 is a binding curve of an antigen-binding protein to cynomolgus monkey PCSK9. Fig. 4 is a binding curve of an antigen-binding protein to cynomolgus monkey PCSK9. It is a binding curve of the antigen binding protein to mouse PCSK9. It is a binding curve of the antigen binding protein to mouse PCSK9. FIG. 4 illustrates the results of an SDSPAGE experiment using PCSK9 and various antigen binding proteins, showing the relative purity and concentration of the protein. FIG. 4 illustrates a graph obtained from a Biacore solution equilibrium assay for 21B12. FIG. 4 illustrates a graph obtained from a Biacore solution equilibrium assay for 21B12. FIG. 4 illustrates a kinetic graph obtained from a Biacore capture assay. FIG. 4 is an inhibition curve of antigen binding protein 31H4 IgG2 for PCSK9 in an in vitro PCSK9: LDLR binding assay. FIG. 4 is an inhibition curve of antigen binding protein 31H4 IgG4 for PCSK9 in an in vitro PCSK9: LDLR binding assay. FIG. 4 is an inhibition curve of antigen binding protein 21B12 IgG2 for PCSK9 in an in vitro PCSK9: LDLR binding assay. FIG. 2 is an inhibition curve of antigen binding protein 21B12 IgG4 for PCSK9 in an in vitro PCSK9: LDLR binding assay. FIG. 4 is an inhibition curve of the antigen binding protein 31H4 IgG2 in a cellular LDL uptake assay showing the effect of ABP in reducing the effect of PCSK9 on blocking LDL uptake. FIG. 9 is an inhibition curve of the antigen binding protein 31H4 IgG4 in a cellular LDL uptake assay showing the effect of ABP in reducing the effect of PCSK9 on blocking LDL uptake. FIG. 4 is an inhibition curve of antigen-binding protein 21B12 IgG2 in a cellular LDL uptake assay showing the effect of ABP in reducing the effect of PCSK9 on blocking LDL uptake. FIG. 4 is an inhibition curve of the antigen-binding protein 21B12 IgG4 in a cellular LDL uptake assay showing the effect of ABP in reducing the effect of PCSK9 on blocking LDL uptake. FIG. 4 is a graph illustrating the serum cholesterol lowering ability of ABP31H4 in mice (change compared to IgG control treated mice) ( ^* p <0.01). FIG. 4 is a graph illustrating the ability of ABP31H4 to lower serum cholesterol in mice (time = change compared to time zero) (#p <0.05). FIG. 2 is a graph illustrating the effect of ABP31H4 on HDL cholesterol levels in C57B1 / 6 mice ( ^* p <0.01). FIG. 4 is a graph illustrating the effect of ABP31H4 on HDL cholesterol levels in C57B1 / 6 mice (#p <0.05). FIG. 5 illustrates Western blot analysis of the ability of ABP31H4 to increase the amount of liver LDLR protein present after various time points. FIG. 4 is a graph illustrating the ability of antigen binding protein 31H4 to reduce total serum cholesterol in wild type mice. Figure 4 is a graph illustrating the ability of antigen binding protein 31H4 to reduce HDL in wild-type mice. Figure 3 is a graph illustrating the serum cholesterol lowering ability of various antigen binding proteins 31H4 and 16F12. Figure 2 illustrates an injection protocol to determine the duration and ability of antigen binding proteins to reduce serum cholesterol. FIG. 11B is a graph illustrating the results of the protocol of FIG. 11A. 1 illustrates LDLR levels in HepG2 cells in response to a combination of statins and ABP21B12. 1 illustrates LDLR levels in HepG2 cells in response to a combination of statins and ABP31H4. FIG. 6 illustrates LDLR levels in HepG2 cells in response to a combination of a statin and a non-neutralizing antibody, ABP25A7.1 (unlike the neutralizing antibody “25A7”). 1 illustrates LDLR levels in HepG2 cells overexpressing PCSK9 in response to a combination of statins and ABP21B12. 1 illustrates LDLR levels in HepG2 cells overexpressing PCSK9 in response to a combination of statins and ABP31H4. FIG. 7 illustrates LDLR levels in PCSK9 overexpressing HepG2 cells in response to a combination of a statin and a non-neutralizing antibody, ABP25A7.1 (unlike the neutralizing antibody “25A7”). 2 illustrates various light chain amino acid sequences of various ABPs for PCSK9. A dot (.) Indicates that no amino acid is present. 1 illustrates the light chain branching diagram for various ABPs for PCSK9. 2 illustrates various heavy chain amino acid sequences of various ABPs for PCSK9. A dot (.) Indicates that no amino acid is present. 1 illustrates heavy chain dendrograms for various ABPs for PCSK9. Shows the comparison of light and heavy chain CDRs and the notation of the groups giving consensus. 3 illustrates the consensus sequence for groups 1 and 2. 5 illustrates the consensus sequence for groups 3 and 4. 3 illustrates the consensus sequence for groups 1 and 2. Points (.) Indicated identical residues. 4 illustrates the consensus sequence for group 2. Points (.) Indicated identical residues. 5 illustrates the consensus sequence for groups 3 and 4. Points (.) Indicated identical residues. 11 is a graph showing the binding specificity of 11F1 in a competition assay using PCSKP, PCSK2, PCSK1, PCSK7, and furin, where OD ₄₅₀ is plotted on the vertical axis and PCSK9 (ug / ml) concentration is plotted on the horizontal axis. In a competitive assay, LDLR by 11f1: D374Y PCSK9 is a graph showing a dose response curve for inhibition of binding, taking the _{OD 450} on the vertical axis, Log [11f1] the horizontal axis represents the (pM). FIG. 4 is a graph showing a dose response curve for inhibition of LDLR: WT PCSK9 binding by 11F1 in a competition assay, with OD ₄₅₀ on the vertical axis and Log [11f1] (pM) on the horizontal axis. FIG. 9 is a graph showing a dose response curve for the ability of 11F1 to block human D374Y PCSK9-mediated reduction of LDL uptake in HepG2 cells, with the relative fluorescence units (× 10 ⁴ ) on the vertical axis and Log [11F1] on the horizontal axis. (NM). FIG. 9 is a graph showing a dose response curve for the ability of 11F1 to block human WT PCSK9-mediated reduction of LDL uptake in HepG2 cells, with the relative fluorescence units (× 10 ⁴ ) on the vertical axis and Log [11F1] on the horizontal axis. (NM). 1 is a bar graph showing the effect of 11F1 and 8A3 on serum non-HDL cholesterol in mice expressing human PCSK9 by AAV, with the non-HDL-C serum concentration (mg / ml) on the vertical axis and the time after injection on the horizontal axis. (Day). 4 is a bar graph showing the effect of 11F1 and 8A3 on serum total cholesterol in mice expressing human PCSK9 by AAV, with the vertical axis taking serum total cholesterol (mg / ml) and the horizontal axis time after injection (day). Is taking. 4 is a bar graph showing the effect of 11F1 and 8A3 on serum HDL cholesterol (HDL-C) in mice expressing human PCSK9 by AAV, with ordinate representing HDL-C (mg / ml) and abscissa representing post-injection I'm taking time (day). 5 is a graph showing the antibody concentration profiles of IgG2, 8A3, and 11F1 in mice expressing human PCSK9 by AAV, wherein the ordinate represents the serum antibody concentration (ng / mL), and the abscissa represents the time after injection (day ) Is taking. FIG. 4 is a table summarizing PK parameters for IgG2, 11F1, and 8A3 in mice expressing human PCSK9 by AAV. FIG. 4 is a graph illustrating the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on serum LDL concentration (LDL-C) in cynomolgus monkeys, with LDL-C (mg / dl) on the vertical axis. , And the time (day) after administration is shown on the horizontal axis. 4 is a graph illustrating the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on serum total cholesterol in cynomolgus monkeys, where the vertical axis represents the total cholesterol concentration (mg / dl) and the horizontal axis represents Take time (day) after administration. 4 is a graph illustrating the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on serum HDL cholesterol in cynomolgus monkeys, where ordinate is HDL-C (mg / dl) and abscissa is Take time (day) after administration. 4 is a graph illustrating the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on serum triglycerides in cynomolgus monkeys, wherein the vertical axis represents serum triglyceride concentration (mg / dl) and the horizontal axis represents The time (day) after administration is taken. FIG. 3 is a graph illustrating the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on apolipoprotein B (ApoB) in cynomolgus monkeys, with the vertical axis taking APOB concentration (mg / dl); The time (day) after administration is shown on the horizontal axis. 5 is a graph illustrating the average pharmacokinetic profiles of anti-KLH antibodies (control), 21B12, 8A3, and 11F1 in cynomolgus monkeys, in which the vertical axis represents antibody concentration (ng / ml), and the horizontal axis represents time after administration (day). Eye). FIG. 4 is a table summarizing PK parameters for anti-KLH antibody (control), 21B12, 8A3, and 11F1 in cynomolgus monkeys. FIG. 4 illustrates a comparison of the light chain amino acid sequences of 8A1, 8A3, and 11F1, and the consensus sequence obtained from the comparison. The CDR sequences are underlined. FIG. 4 illustrates a comparison of the heavy chain amino acid sequences of 8A1, 8A3, and 11F1, and the consensus sequence resulting from the comparison. The CDR sequences are underlined.

  Also provided herein is a method of treating a patient diagnosed with homozygous familial hypercholesterolemia, wherein the method comprises administering at least one of the proprotein convertase subtilisin / keksin 9 (PCSK9). Comprising administering one antigen binding protein (eg, an antibody) to the patient. Furthermore, a method for reducing serum LDL cholesterol in a patient diagnosed with homozygous familial hypercholesterolemia using an antigen-binding protein (eg, an antibody) against a proprotein convertase subtilisin / keksin type 9 (PCSK9) Are also provided herein.

  For convenience, the following sections provide a general overview of the various meanings of the terms used herein. This discussion is followed by general aspects relating to antigen binding proteins, followed by specific examples illustrating the properties of various embodiments of antigen binding proteins and how they can be used. .

Definitions and Embodiments It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. There must be. 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 "comprising" and other forms such as "comprising" and "included" is not limiting. Also, terms such as "element" or "component" include both elements and components that include one unit and elements and components that include two or more subunits, unless otherwise specified. Also, use of the term "part" may include a portion of a component or an entire component.

  The headings provided herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references or parts of references cited in this application, including but not limited to patents, patent applications, editorials, books and articles, are incorporated by reference in their entirety for all purposes. , Which are expressly incorporated herein. As used in this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

  The term "proprotein convertase subtilisin kexin type 9" or "PCSK9" refers to the polypeptide set forth in SEQ ID NOs: 1 and / or 3 or fragments thereof, as well as allelic variants, splice variants, derivative variants, substitutions Refers to variants, deletion variants, and / or insertion variants (including the addition of an N-terminal methionine), fusion polypeptides, and related polypeptides, including but not limited to interspecies homologs. In certain embodiments, the PCSK9 polypeptide comprises a leader sequence residue, a targeting residue, an amino-terminal methionine residue, a lysine residue, a tag residue, and / or a fusion protein residue. (Non-limiting) terminal residues. "PCSK9" has also been referred to as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin / keksin type 9, and neuronal apoptosis control convertase 1. The PCSK9 gene encodes a proprotein convertase protein belonging to the proteinase K subfamily of the secretory subtilase family. The term "PCSK9" describes both the proprotein and the product produced after autocatalysis of the proprotein. If only the autocatalyzed product is mentioned (eg, for an antigen binding protein that selectively binds to cleaved PCSK9), then the protein is "mature", "cleaved", It may be referred to as "processed" or "active" PCSK9. Where only the inactive form is referred to, the protein may be referred to as the "inactive", "pro" or "unprocessed" form of PCSK9. The term PCSK9 as used herein also includes naturally occurring alleles such as mutations D374Y, S127R, and F216L. The term PCSK9 refers to a glycosylated, PEGylated PCSK9 sequence, a PCSK9 sequence whose signal sequence has been cleaved, a PCSK9 sequence that has been cleaved from its pro domain from the catalytic domain but has not been separated from the catalytic domain (eg, PCSK9 molecules that incorporate post-translational modifications of the PCSK9 amino acid sequence, such as FIGS. 1A and 1B).

  The term “PCSK9 activity” includes any biological effect of PCSK9. In certain embodiments, PCSK9 activity comprises the ability of PCSK9 to interact with or bind to a substrate or receptor. In some embodiments, PCSK9 activity is represented by the ability of PCSK9 to bind to the LDL receptor (LDLR). In some embodiments, PCSK9 binds and catalyzes reactions involving LDLR. In some embodiments, PCSK9 activity comprises the ability of PCSK9 to alter (eg, reduce) the availability of LDLR. In some embodiments, PCSK9 activity comprises the ability of PCSK9 to increase the amount of LDL in a subject. In some embodiments, PCSK9 activity comprises the ability of PCSK9 to reduce the amount of LDLR available for binding to LDL. In some embodiments, “PCSK9 activity” includes any biological activity that results from PCSK9 signaling. Typical activities include binding of PCSK9 to LDLR, PCSK9 enzymatic activity that cleaves LDLR or other proteins, binding of PCSK9 to proteins other than LDLR that promotes PCSK9 action, PCSK9 that alters APOB secretion (Sun X -M et al, "Evidence for effect of mutant PCSK9 on apoliprotein B secretion as the cause of unusually severe dominant hypercholesterolemia, Human Molecular Genetics 14: 1161-1169,2005 and Ouguerram K et al," Apolipoprotein B100 metabolis in autosomal-dominant hypercholesterolemia related to mutationsin PCSK9, Arterioscler thromb Vase Biol.24: 1448-1453,2004), the role of PCSK9 in the regeneration and differentiation of nerve cells in the liver (Seidah NG et al, "The secretory proprotein convertase neural apoptosis- regulated convertase 1 (NARC-I): Liver regeneration and neuronal differentiation "PNAS 100: 928-933, 2003), and PCSK in hepatic glucose metabolism. (Costet et al., "Hepatic PCSK9 expression is regulated by nutriental status via insulin and stereoregulatory regulation, e.g. However, the present invention is not limited to these.

  The term "hypercholesterolemia" as used herein refers to a condition in which cholesterol levels are elevated above a desired level. In some embodiments, this is indicative of an elevated serum cholesterol level. In some embodiments, the desired level takes into account various "risk factors" known to those of skill in the art (and described or cited herein).

  As used herein, the term “homozygous familial hypercholesterolemia” or “HoFH” refers to genetic confirmation or clinical diagnosis (eg, untreated LDL cholesterol levels above 13 mmol / L as well as yellow color before 10 years of age). Cholesterol-related disease, as determined by the history of the tumor or any evidence of homozygous familial hypercholesterolemia in the parents.

  The term "polynucleotide" or "nucleic acid" includes both single- and double-stranded nucleotide polymers. Nucleotides, including polynucleotides, can be ribonucleotides or deoxyribonucleotides, or modified forms of any type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2 ', 3'-dideoxyribose, and phosphorothioates, phosphorodithioates, phosphoroselenenoates, phosphoroselenenoates, Internucleotide linkage modifications such as roanilothioate, phosphoraniladate, and phosphoramidate are included.

  The term "oligonucleotide" means a polynucleotide comprising 200 or fewer nucleotides. In some embodiments, the oligonucleotide is 10 to 60 bases in length. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, for example, for use in constructing a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides can include a label, such as a radiolabel, a fluorescent label, a hapten, or an antigenic label for the detection assay. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.

  An "isolated nucleic acid molecule" is one in which the isolated polynucleotide does not have all or a portion of the polynucleotide found in nature therein, or is linked to a polynucleotide that is not naturally linked. It refers to genomic DNA or RNA, mRNA, cDNA or synthetic sources or some combination thereof. It is to be understood that for the purposes of this disclosure, a particular nucleic acid sequence “comprising a nucleic acid” does not encompass intact chromosomes. An isolated nucleic acid molecule "comprising" the specified nucleic acid sequence can comprise, in addition to the specified sequence, up to 10 or up to 20 other coding sequences for other proteins or portions thereof, Alternatively, it can include operably linked regulatory sequences that control the expression of the coding region of the referenced nucleic acid sequence, and / or can include a vector sequence.

  Unless otherwise stated, the left end of any single-stranded polynucleotide sequence discussed herein is the 5 'end, and the left-hand direction of the double-stranded polynucleotide sequence is referred to as the 5' direction. You. The direction of addition of the nascent RNA transcript from 5 'to 3' is referred to as the transcription direction. Sequence regions on the DNA strand having the same sequence as the RNA transcript, 5 'to the 5' end of the RNA transcript, are referred to as "upstream sequences." Sequence regions on the DNA strand having the same sequence as the RNA transcript, 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 a coding sequence to which the control sequence is linked. The nature of such control sequences may depend on the host organism. In certain embodiments, control sequences for prokaryotes may include promoter, ribosome binding site, and transcription termination sequence. For example, control sequences for eukaryotes can include promoters that include one or more of the recognition sites for transcription factors, transcriptional enhancement sequences, and transcription termination sequences. "Control sequences" can include leader sequences and / or fusion partner sequences.

  The term "vector" refers to any molecule or entity (eg, nucleic acid, plasmid, bacteriophage, or virus) used to transmit protein-coding information into a host cell.

  The term “expression vector” or “expression construct” is suitable for the transformation of a host cell, as well as the expression of one or more heterologous coding regions operably linked to a nucleic acid sequence (with the host cell). Together) refers to a vector containing a nucleic acid sequence to induce and / or control. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of the operably linked coding region. Not done.

  As used herein, "operably linked" means that the components to which the term applies are in a relationship capable of performing, under appropriate conditions, its specific function. For example, a control sequence in a vector "operably linked" to the protein coding sequence may be attached to the protein coding sequence such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence. Are linked.

  The term "host cell" refers to a cell that has been transformed with, or can be transformed with, a nucleic acid sequence, thereby expressing the gene of interest. The term refers to the progeny of the parent cell, whether the progeny are morphologically identical to the original parent cell, or genetically identical to the original parent cell, as long as the gene of interest is present. Includes offspring.

  The term "transfection" refers to 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. Numerous transfection techniques are well known in the art and are disclosed herein. See, for example, "Graham et al., 1973, Virology 52: 456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Medicine Cars; , Gene 13: 197 ". Such techniques can be used to introduce one or more foreign DNA portions into a suitable host cell.

  The term "transformation" refers to a change in the genetic properties of a cell, and a cell has been transformed when it has been modified to contain new DNA or RNA. A cell has been transformed when it has been genetically modified from its native state, for example, by the introduction of new genetic material via transfection, transduction, or other techniques. After transfection or transduction, the DNA being transformed can be recombined with the DNA of the cell by physical integration into the chromosome of the cell, or is not replicated and transiently becomes an episomal element. It can be maintained or replicated independently as a plasmid. A cell is considered "stably transformed" when the DNA being transformed is replicated along with cell division.

  The term "polypeptide" or "protein" refers to a naturally occurring protein, i.e., a macromolecule having the amino acid sequence of a protein produced by a naturally occurring non-recombinant cell, or "polypeptide" or "protein". Include molecules produced by genetically engineered or recombinant cells and having the amino acid sequence of a native protein or having deletions, additions, and / or substitutions from one or more amino acids of the native sequence . The term also includes amino acid polymers in which one or more amino acid is a chemical analog of the corresponding naturally occurring amino acid and polymer. Specifically, the terms “polypeptide” and “protein” refer to PCSK9 antigen binding proteins, antibodies, or sequences having deletions, additions, and / or substitutions from one or more amino acids of the antigen binding protein. Is included. The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to a full-length native protein. Such fragments also contain modified amino acids as compared to the native protein. In certain embodiments, fragments are about 5-500 amino acids in length. For example, a fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of an antibody, including the binding domain. In the case of a PCSK9 binding antibody, useful fragments include CDR regions, heavy and / or light chain variable domains, portions of an antibody chain, or only those variable regions comprising two CDRs, and the like. Not limited.

  The term "isolated protein" as referred to means that the protein of interest is (1) free of at least some other proteins normally found together, (2) from the same source (eg, from the same species) (3) expressed by cells from different species, (4) at least one of polynucleotides, lipids, carbohydrates, or other substances that are naturally present together. From about 50%, (5) operably linked (by covalent or non-covalent interactions) with a polypeptide that is not naturally present together, or (6) in its natural state. Means non-existent. Typically, an "isolated protein" comprises at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a sample. Genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof, may encode such an isolated protein. Preferably, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that interfere with its therapeutic, diagnostic, prophylactic research or other uses. Absent.

  The term "amino acid" has its ordinary meaning in the art.

  A “variant” of a polypeptide (eg, an antigen binding protein or antibody) is one or more amino acid residues inserted, deleted, and / or deleted from the amino acid sequence relative to another polypeptide sequence. Includes amino acid sequences that have been replaced. 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, determined by juxtaposing and comparing the sequences. "% Identity" means the percentage of the same residues between amino acids or nucleotides in a molecule being compared and is calculated based on the smallest size of the molecules being compared. For these calculations, a particular mathematical model or computer program (ie, "algorithm") preferably allocates the gaps in the juxtaposition (if any). Methods that can be used to calculate the identity of the juxtaposed nucleic acids or polypeptides include "Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis, H., G., G., G., G., G. Eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press (1987); Sequence Analysis Primer, (Gribskov, M. and Derw. Carillo et al., 1988, SIAMJ. Applied Math., 48: 1073 ".

  In calculating% identity, the sequences being compared are typically aligned to give the largest match between the sequences. One example of a computer program that can be used to determine% identity is the GCG program package containing GAP (Devereux et al., 1984, Nucl. AcidRes. 12: 387; Genetics Computer Group, University of Wisconsin). , Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percent sequence identity is to be determined. The sequences are aligned to obtain the best match for each amino acid or nucleotide ("matched span" as determined by the algorithm). Gap opening penalty (calculated as 3 × average diagonal. “Average diagonal” is the average of the diagonals of the comparison matrix used. “Diagonal” is the sum of each complete amino acid according to this comparison matrix. The score or number assigned to the match) and the gap extension penalty (typically 1/10 of the gap opening penalty) and a comparison matrix such as PAM250 or BLOSum62 are used with the algorithm. In certain embodiments, a standard comparison matrix is also used by the algorithm (for the PAM250 comparison matrix, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5: 345-352; for the BLOSum62 comparison matrix, See Henikoff et al., 1992, Proc.Natl.Acad.Sci USA, 89: 10915-10919).

Examples of parameters that can be used in determining% identity to polypeptide or nucleotide sequences using the GAP program are as follows:
Algorithm: Needleman et al. , 1970, J.A. Mol. Biol. , 48: 443-453.
-Comparison matrix: Henikoff et al. , 1992, BLOSum62 obtained from above.
・ Gap penalty: 12 (No penalty for terminal gap)
・ Gap length penalty: 4
・ Similarity threshold: 0

  Certain alignment schemes for aligning two amino acid sequences may result in only a short region match of the two sequences, even though there is no significant relationship between the two full-length sequences. This small juxtaposed region can have very high sequence identity. Thus, if it is desired to produce an alignment that includes at least 50 or other numbers of consecutive amino acids in the target polypeptide, the selected alignment (GAP program) can be adjusted.

  As used herein, the twenty conventional (eg, naturally occurring) amino acids and their abbreviations follow conventional usage. "Immunology-A Synthesis (2nd Edition, ES Golub and DR Green, Eds., Sinauer Associates, Sunderland, Mass. (1991))" for all purposes, herein by reference. See incorporated). Unnatural amino acids such as the 20 conventional amino acid stereoisomers (eg, D-amino acids), α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids are also included in the invention. It can be a suitable component for a polypeptide. Examples of non-conventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N, N, N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, Includes 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (eg, 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, according to standard usage and convention.

  Similarly, unless otherwise specified, the left end of a single-stranded polynucleotide sequence is the 5 'end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The addition of a nascent RNA transcript in the 5 'to 3' direction is referred to as the transcription direction. Sequence regions on the DNA strand that have the same sequence as the RNA and are 5 'to the 5' end of the RNA transcript are referred to as "upstream sequences." Sequence regions on the DNA strand that have the same sequence as the RNA and are 3 'to the 3' end of the RNA transcript are referred to as "downstream sequences."

  Conservative amino acid substitutions can include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis, rather than by synthesis in a biological system. These include peptidomimetics and other inverted or inverted forms of the amino acid moiety.

  Naturally occurring residues can be classified into classes based on common side chain properties:

1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
3) acidic: Asp, Glu;
4) Basicity: His, Lys, Arg;
5) Residues affecting chain orientation: Gly, Pro; and 6) Aromatic: Trp, Tyr, Phe.

  For example, a non-conservative substitution could involve exchanging a member of one of these classes with a member of another class. Such substituted residues can be introduced, for example, into regions of the human antibody that are homologous to the non-human antibody or into non-homologous regions of the molecule.

  In making changes to the antigen binding protein or PCSK9 protein, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. The hydropathic index is 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), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (- 3.9), and arginine (-4.5).

  The importance of the hydropathic amino acid index in conferring interacting biological functions on proteins is understood in the art. Kyte et al, J. Mol. Mol. Biol. 157: 105-131 (1982). It is known that certain amino acids can be replaced with other amino acids having similar hydropathic indices or scores and still retain similar biological activity. Variations based on the hydropathic index include, in certain embodiments, substitutions of amino acids whose hydropathic index is within ± 2. In certain embodiments, amino acid substitutions that are within ± 1 are included, and in certain embodiments, amino acid substitutions that are within ± 0.5.

  Substitution of similar amino acids is effective on the basis of hydrophilicity, particularly when the biologically functional protein or peptide produced thereby is to be used in an immunological embodiment, as in the present case. Is also understood in the art. In certain embodiments, the local maximum average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, ie, the biological properties of the protein.

  The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+ 3.0 ± 1), glutamic acid (+ 3.0 ± 1), Serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5 ± 1), alanine (-0.5 ), Histidine (-0.5), cysteine (-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), Tyrosine (-2.3), phenylalanine (-2.5), and tryptophan (-3.4). Variations based on similar hydrophilicity values include, in certain embodiments, substitutions of amino acids whose hydrophilicity values are within ± 2, and in certain embodiments, within ± 1. Amino acid substitutions are included, and in certain embodiments, amino acid substitutions that are within ± 0.5 are included. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions."

  Examples of amino acid substitutions are shown in Table 1.

  The term "derivative" refers to a molecule that contains a chemical modification other than an amino acid (or nucleic acid) insertion, deletion, or substitution. In certain embodiments, the derivative comprises a covalent modification, such as, but not limited to, a chemical bond with a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, a chemically modified antigen binding protein can have a longer circulating half-life than an antigen chemically unmodified antigen binding protein. In certain embodiments, a chemically modified antigen binding protein may have improved targeting ability to a desired cell, tissue, and / or organ. In some embodiments, the derivative antigen binding protein is covalently comprising one or more water-soluble polymer attachments, such as, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. Is qualified. For example, US Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and See No. 4,179,337. In certain embodiments, the derivative antigen binding protein is a monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymer, poly- (N-vinylpyrrolidone) -polyethylene glycol, propylene glycol homopolymer, One or more polymers such as, but not limited to, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), and polyvinyl alcohol, and mixtures of such polymers. Including.

  In certain embodiments, the derivative is covalently modified with a polyethylene glycol (PEG) subunit. In certain embodiments, one or more water-soluble polymers are attached at one or more specific positions, for example, at the amino terminus of the derivative. In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In certain embodiments, PEG is used to increase the therapeutic capacity of the antigen binding protein. In certain embodiments, PEG is used to enhance the therapeutic potential for a humanized antibody. Certain such methods are discussed, for example, in US Pat. No. 6,133,426, which is incorporated by reference for all purposes.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptidic compounds have been termed "peptidomimetics" or "peptidomimitics". 15:29 (1986); Vever and Freidinger TINS p.392 (1985); and Evanset al. J. Med. Chem., 30: 1229 (1987). Incorporated herein by reference.) Such compounds are often developed with the aid of computerized molecular modeling. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce a similar therapeutic or prophylactic effect. In general, peptidomimetics are structurally similar to exemplary polypeptides (ie, polypeptides having biochemical or pharmacological activity), such as human antibodies, but by methods well known in the art, _{2 NH -, - CH 2 S} -, - CH 2 -CH 2 -, - CH = CH- ( cis and _{trans), - COCH 2 -, -} CH (OH) CH 2 -, and from _-CH 2 SO- It has one or more peptide bonds optionally substituted by a selected bond. Systematically replacing one or more amino acids of a consensus sequence with a homologous D amino acid (eg, D-lysine in place of L-lysine), in certain embodiments, creates a more stable peptide Can be used to In addition, a constrained peptide containing a consensus sequence or a substantially identical consensus sequence mutation can be purified by methods known in the art, for example, to internal cysteine residues that can form intramolecular disulfide bridges that cyclize the peptide. It can be made by adding a group (Rizo and Gierasch Ann. Rev. Biochem. 61: 387 (1992), incorporated herein by reference for all purposes).

  The term "naturally occurring" as used throughout this specification in connection with biological materials such as polypeptides, nucleic acids, host cells, etc., refers to a substance found in nature or a substance found in nature. Refers to the form.

As used herein, "antigen binding protein"("ABP") means any protein that binds a specified target antigen. In the present application, the identified target antigen is PCSK9 protein or a fragment thereof. "Antigen binding protein" includes, but is not limited to, antibodies and binding portions thereof (such as immunologically functional fragments). Peptibodies are another example of an antigen binding protein. As used herein, the term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen binding protein refers to a fragment of the full-length chain. An antigen binding protein comprising a portion of an antibody that lacks at least some of the amino acids present but is still capable of specifically binding an antigen, regardless of how the portion was obtained or synthesized. Is a seed. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for binding to one epitope. In some embodiments, the fragment is a neutralizing fragment. In some embodiments, fragments can block or reduce the potential for interaction between LDLR and PCSK9. In one aspect, such a fragment retains at least one CDR present in a full-length light or heavy chain, and in some embodiments, a single heavy and / or light chain or one thereof. Including parts. These biologically active fragments can be made by recombinant DNA technology, or by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include Fabs, diabodies (light chains connected via a short peptide linker that is too short to allow pairing between the two domains on the same chain). Heavy chain variable domain on the same polypeptide as the variable domain), Fab ', F (ab') ₂ , Fv, domain antibodies, and single chain antibodies, including but not limited to humans, mice, It can be from any mammalian source, including but not limited to rats, camelids or rabbits. Functional portions of the antigen binding proteins disclosed herein, eg, one or more CDRs, are directed to specific targets in the body, have bifunctional therapeutic properties, or are prolonged. It is also envisioned that a therapeutic agent with a serum half-life could be covalently linked to a second protein or small molecule. As will be appreciated by those skilled in the art, antigen binding proteins can include non-protein components. In some sections of the present disclosure, examples of ABPs are described herein in the form of "numeric / letter / numeric" (eg, 25A7). In these cases, the exact name indicates a particular antibody (eg, 25A7 vs. 21B12). That is, an ABP named 25A7 is not necessarily the same as an antibody named 25A7.1 (unless explicitly taught herein to be the same (eg, 25A7 and 25A7.3)). Not always. Unless otherwise stated, the name of ABP is understood to be the genetic designation for antibody.

  Certain antigen binding proteins described herein are or are derived from antibodies. In certain embodiments, the polypeptide structure of the antigen binding protein may be a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, a synthetic antibody (sometimes referred to herein as an “antibody mimetic”). A), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates") and fragments thereof (including, but not limited to) ) Based on antibodies. In some embodiments, the ABP comprises or consists of an avimer (a tightly binding peptide). These various antigen binding proteins are further described herein. Further, examples of antibodies are described in, for example, US Patent No. 8,030,457 and US Patent No. 8,168,762. Further examples of antibodies include, for example, US Patent No. 8,188,233, US Patent No. 8,188,234, US Patent No. 8,080,243, US Patent No. 8,062,640, WO2008 /. 06332, WO2009 / 055783, WO2011 / 053759, WO2012 / 054438, WO2012 / 088833, WO2012 / 109530, and WO2013 / 039958.

The “Fc” region includes two heavy chain fragments that contain the C _H 1 and C _H 2 domains of an antibody. The two heavy chain fragments, by two or more disulfide bonds, and are coupled together by hydrophobic interactions of the C _{H 3} domain.

A “Fab fragment” comprises one light chain and the C _H 1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

A “Fab ′ fragment” contains one light chain and the VH and C _H1 domains, and forms an interchain disulfide bond between the two heavy chains of the two Fab ′ fragments, resulting in F (ab ′) 2 so as to form _two molecules, comprising a portion of one heavy chain that contains also the region between the C H ₁ and C _{H 2} domain.

"F (ab ') ₂ fragment" as two light chains and interchain disulfide bond is formed between two heavy chains, a portion of the constant region between the C H ₁ and C _{H 2} domains Contains two heavy chains. Thus, an F (ab ') ₂ fragment is composed of two Fab' fragments joined together by a disulfide bond between the two heavy chains.

  An "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 are connected by a flexible linker, forming a single polypeptide chain that forms the antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

"Domain antibodies" are immunologically functional immunoglobulin fragments which contain only the variable regions of the heavy or light chains. In some examples, two or more _VH regions are covalently linked using a peptide linker to make a bivalent domain antibody. The two _VH regions of a bivalent domain antibody can target the same or different antigens.

  A “bivalent antigen binding protein” or “bivalent antibody” contains two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. Bivalent antigen binding proteins and bivalent antibodies can be bispecific. See below. It is understood that bivalent antibodies other than "multispecific" or "multifunctional" antibodies, in certain embodiments, typically have each of their binding sites identical.

  "Multispecific antigen binding proteins" or "multispecific antibodies" are those that target more than one antigen or epitope.

  A "bispecific", "dual specific", or "bifunctional" antigen binding protein or antibody is a hybrid antigen binding protein or antibody, respectively, having two different antigen binding sites. Bispecific antigen binding proteins and antibodies are species of multispecific antigen binding proteins and antibodies and can be made by various methods including, but not limited to, fusion of hybridomas or ligation of Fab 'fragments. it can. See, for example, "Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al, 1992, J. Immunol. 148: 1547-1553". The two binding sites of a bispecific antigen binding protein or antibody bind to two different epitopes that can be on the same or different protein targets.

An antigen binding protein is said to "specifically bind" its target antigen if the dissociation constant ( _Kd ) is ^10-7 M or less. ABP, when _{K d} is of 5 × ^{10 -9} M or less, "high affinity" with specific binding to antigen, when _{K d} is less than or equal to 5 × ^{10 -10} M, "very high It specifically binds to the antigen with "affinity". In one embodiment, the ABP has a _{Kd of} ^10-9M or less. In one embodiment, the off-rate is less than 1 × 10 ⁻⁵ . In other embodiments, ABP is a _{K d} between about ¹⁰ -9 ^{M to -13} M binds to human PCSK9, in yet another embodiment, ABP is 5 × ^{10 -10} or less a _{K d} To join. As will be appreciated by one of skill in the art, in some embodiments, any or all of the antigen binding fragments may specifically bind to PCSK9.

  An antigen binding protein is "selective" if it binds more tightly to one target than to a second target.

  “Antigen binding region” means a protein or a portion of a protein that specifically binds to a particular antigen (eg, paratope). For example, that portion of an antigen binding protein that contains amino acid residues that interact with the antigen and confer its specificity and affinity for the antigen on the antigen binding protein is referred to as an "antigen binding region." An antigen binding region typically includes one or more "complementary binding regions" ("CDRs"). Certain antigen binding regions also include one or more "framework" regions. "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. "Framework" regions can help maintain the proper conformation of the CDRs and promote binding between the antigen-binding region and the antigen. Structurally, the framework regions can be located between CDRs in the antibody. Examples of frameworks and CDR regions are shown in FIGS. 2A-3D, 3CCC-3JJJ. In some embodiments, the sequence of the CDRs for the light chain of antibody 3B6 is as follows: CDR1 TLSSGYSSYEVD (SEQ ID NO: 279), CDR2 VDTGGIVGSKGE (SEQ ID NO: 280), CDR3 GADHGSGTNFVVV (SEQ ID NO: 281), FR It is as follows. FR1 QPVLTQPLFASASLGASVTLTTC (SEQ ID NO: 282), FR2 WYQQRPGKGPRFVMR (SEQ ID NO: 283), FR3 GIPDRFFSVLGSGLNRYLTIKNIQEDESDYHC (SEQ ID NO: 284), and FR4 FGGGTKLT SEQ ID NO: 285.

  In one aspect, there is provided a recombinant antigen binding protein that binds to PCSK9, eg, human PCSK9. In this context, a "recombinant antigen binding protein" is a protein made using recombinant techniques, ie, through the expression of a recombinant nucleic acid described herein. Methods and techniques for making 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 an intact antibody for specific binding to a target antigen, e.g., chimeric, humanized, fully human. , And bispecific antibodies. An “antibody” is a species of an antigen binding protein. An intact antibody generally comprises at least two full-length heavy chains and two full-length light chains, but in some cases may include only heavy chains, a naturally occurring antibody in camelids And may include fewer chains, such as. Antibodies can be obtained from only a single source, or can be "chimeric", ie, different portions of the antibody can be derived from two different antibodies, as further described below. Antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise stated, the term “antibody” includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are: It is described below. Further, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"). ), Chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some embodiments, the term also encompasses peptibodies.

  Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair comprising one full-length “light” chain (in certain embodiments, about 25 kDa) and It has one full-length "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically contains the variable region of about 100-110 or more amino acids required for antigen recognition. The carboxy-terminal portion of each chain typically defines the constant region that may be required for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3 and IgG4. IgM has subclasses such as, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses such as, but not limited to, IgA1 and IgA2. Within full length light and heavy chains, typically the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain being a "J" of about 10 or more amino acids. D "region. For example, “Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (incorporated by reference in its entirety for all purposes)” Please refer to. The variable region of each light / heavy chain pair typically forms the antigen binding site.

  Variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) linked by three hypervariable regions (also called complementarity determining regions or CDRs). The CDRs from the two chains of each pair are typically aligned by framework regions, which may allow binding to specific epitopes. From the N-terminus to the C-terminus, both the 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 done by defining the Kabat sequence of the protein of immunological interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or by "Chothia & Lesk" , J. Mol. Biol., 196: 901-917 (1987); Chothia et al., Nature, 342: 878-883 (1989).

  In certain embodiments, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody light chain binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, an antibody binding region binds an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds an antigen in the absence of an antibody heavy chain. In certain embodiments, each variable region specifically binds to an antigen in the absence of other variable regions.

  In certain embodiments, definitive delineation of the CDRs and identification of the residues comprising the binding site of the antibody is achieved by elucidating the structure of the antibody and / or elucidating the structure of the antibody-ligand complex. You. In certain embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be used to identify or estimate CDR regions. Examples of such methods include, but are not limited to, Kabat definition, Chothia definition, AbM definition, and contact definition.

  The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, for example, "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 the location of certain structural loop regions. See, for example, Chothia et al, J. Mol. Biol., 196: 901-17 (1986); Chothia et al, Nature, 342: 877-83 (1989). The AbM definition uses an integrated package of computer programs created by Oxford Molecular Group that models antibody structure. For example, "Martin et al, Proc Natl Acad Sci (USA), 86: 9268-9272 (1989);" AbM (trademark), A Computer Program for Modeling Variable Regions of Antifoil, Ukd. Please refer to. AbM definitions are described in known databases and in PROTEINS, Structure, Function and Genetics Suppl. , 3: 194-198 (1999), using a combination of the following ab initio methods, as described by "Samudrala et al," Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach, " Modeling the structure Contact definitions are based on the analysis of available complex crystal structures, see, for example, "MacCallum et al, J. Mol. Biol., 5: 732-45 (1996)". I want to.

  By convention, the CDR regions in the heavy chain are typically referred to as H1, H2, and H3, and are numbered sequentially from the amino terminus to the carboxy terminus. CDR regions in the light chain are typically referred to as L1, L2, and L3, and are numbered sequentially from the amino terminus to the carboxy terminus.

The term "light chain" includes full length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, _{V L} and a constant region domain, the _{C L.} The light chain variable region domain is located at the amino terminus of the polypeptide. Light chains include kappa and lambda chains.

The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. The heavy chain of the full length, includes a variable region domain _{V H} and three constant region domains _C H _{1, C} H 2, and _{C H} 3. V _H domain is at the amino terminus of the polypeptide, C _H domain is located at the carboxyl terminus, C H ₃ is closest to the carboxy terminus of a polypeptide. The heavy chain can be of any isotype, such as IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

  Bispecific or bifunctional antibodies are typically artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including but not limited to fusion of hybridomas or ligation of Fab 'fragments. See, for example, "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 with only a single heavy chain.

Each respective immunoglobulin chain is comprised of approximately 90-110 amino acids each and is typically composed of several "immunoglobulin domains" with a characteristic folding pattern. These domains are the basic units that make up the antibody polypeptide. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains, the IgG and IgE isotypes contain two heavy chains and two light chains, and the IgM isotype has five heavy chains and five light chains. Contains light chain. The heavy chain C region typically contains one or more domains that may be required for effector function. The number of heavy chain constant region domains depends on the isotype. IgG heavy chains, for _example, contain three C region domains known as C H _{1, C} H 2, and _{C H} 3. Provided antibodies can have any of these isotypes and subtypes. In certain embodiments of the invention, the anti-PCSK9 antibodies are of the IgG2 or IgG4 subtype.

  The term “variable region” or “variable domain” refers to an antibody that typically comprises about 120-130 amino-terminal amino acids in the heavy chain and about 100-110 amino-terminal amino acids in the light chain. Represents part of a light and / or heavy chain. In certain embodiments, the variable regions of different antibodies differ significantly in amino acid sequence, even between antibodies of the same species. The variable regions of the antibodies typically determine the specificity of an antibody for a target.

The terms "neutralizing antigen binding protein" or "neutralizing antibody" refer to an antigen binding protein or antibody, respectively, that binds a ligand and prevents or reduces the biological effect of the ligand. This alters the binding capacity of the ligand, for example, by directly blocking the binding site on the ligand, or binding to the ligand and through indirect means, such as structural or energetic changes in the ligand. This can be done by: In some embodiments, the term may also refer to an antigen binding protein that prevents the protein to which it binds from performing a biological function. In assessing the binding and / or specificity of an antigen binding protein (eg, an antibody or an immunologically functional fragment thereof), an excess of antibody (as measured in an in vitro competitive binding assay) of at least about 1 -20, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95- When reducing the amount of a binding pair bound to a ligand by 97%, 97-98%, 98-99% or more, the antibody or fragment significantly inhibits binding of the ligand to that binding pair. be able to. In some embodiments, in the case of a PCSK9 antigen binding protein, such a neutralizing molecule can reduce the ability of PCSK9 to bind to LDLR. In some embodiments, the neutralizing ability is characterized and / or described via a competition assay. In some embodiments, neutralizing capacity is described in terms of IC ₅₀ or EC ₅₀ values. In some embodiments, ABP27B2, 13H1, 13B5, and 3C4 are non-neutralizing ABPs, 3B6, 9C9, and 31A4 are weakly neutralizing substances, and the remaining ABPs in Table 2 are strong medium It is a sum substance. In some embodiments, the antibody or antigen binding protein binds to PCSK9 and neutralizes by preventing PCSK9 from binding to LDLR (or reducing the ability of PCSK9 to bind to LDLR). In some embodiments, the antibody or ABP binds to PCSK9 and neutralizes by preventing or reducing PCSK9-mediated degradation of LDLR while binding PCSK9 to LDLR. Thus, in some embodiments, neutralizing ABPs or antibodies prevent (or reduce) the subsequent degradation of PCSK9-associated LDLR while still allowing PCSK9 / LDLR binding.

  The term "target" refers to a molecule or a portion of a molecule that can be bound by an antigen binding protein. In certain embodiments, a target can have one or more epitopes. In certain embodiments, the target is an antigen. The use of "antigen" in the term "antigen binding protein" simply indicates that the protein sequence comprising the antigen can be bound by the antibody. In this context, it is not required that the protein be exogenous or capable of inducing an immune response.

  The term “competing” when used in the context of an antigen binding protein (eg, a neutralizing antigen binding protein or a neutralizing antibody) that competes for the same epitope refers to the antigen binding protein (eg, antibody Alternatively, an immunologically functional fragment thereof prevents or inhibits specific binding of a reference antigen binding protein (eg, a ligand or reference antibody) to a common antigen (eg, PCSK9 or a fragment thereof) (eg, (Reduce) means competition between antigen binding proteins as measured by the assay. To determine whether one antigen binding protein competes with another antigen binding protein, a number of types of competitive binding assays are available, such as solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay ( EIA), a sandwich competition assay (see, eg, Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (eg, Kirkland et al, 1986, J. Immunol. 137: 3614-3619). , Solid phase direct labeling assays, solid phase direct labeling sandwich assays (eg, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Sp.). solid-phase direct labeling RIA using I-125 label (for example, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (for example, Cheung, et. al., 1990, Virology 176: 546-552); and directly labeled RIA (see Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, such assays involve the use of cells having purified antigen or any of them bound to a solid surface, unlabeled test antigen binding protein, and labeled reference antigen binding protein. Including. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the test antigen binding protein. Usually, the test antigen binding protein is present in excess. The antigen binding proteins identified by the competition assay (competitive antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding protein, and epitopes that are bound by the reference antigen binding protein when steric hindrance occurs. Antigen binding proteins that bind to adjacent epitopes that are sufficiently close to E. coli. Further details regarding methods for measuring competitive binding are provided in the Examples herein. Usually, when the competitive antigen binding protein is present in excess, at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% Or inhibits (eg, reduces) the specific binding of a reference antigen binding protein to a common antigen by 75% or more. In some examples, 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 that can be bound by a selective binding agent, such as an antigen binding protein (eg, an antibody or immunologically functional fragment thereof). In some embodiments, an antigen can be used in an animal to produce an antibody that can bind to the antigen. An antigen can have one or more epitopes that can interact with different antigen binding proteins (eg, antibodies).

  The term "epitope" includes any determinant that can be bound by an antigen-binding protein, such as an antibody or a T-cell receptor. An epitope is a region of an antigen that is bound by an antigen-binding protein that targets the antigen and, when the antigen is a protein, includes specific amino acids that directly contact the antigen-binding protein. Most often, the epitope is on a protein, but in some cases can be on other types of molecules, such as nucleic acids. Epitopic determinants can include chemically active surface groups of the molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and have specific three-dimensional structural characteristics and / or specific charge characteristics. Can have. Generally, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

  As used herein, "substantially pure" means that the stated species of the molecule is the predominant species present, i.e., on a molar basis, any other species in the same mixture. Means more abundant than individual species. In certain embodiments, a substantially pure molecule is a composition in which the species of interest accounts for at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition comprises at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, the contaminant species cannot be detected in the composition by conventional detection methods, and therefore, the composition is subject to a substantially homogeneous state consisting of a single detectable macromolecular species. The species is purified.

  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 material.

The terms “label” or “labeled” as used herein include, for example, the incorporation of radiolabeled amino acids or labeled avidin (eg, a fluorescent marker or detectable by optical or chromogenic methods). Refers to the incorporation of a detectable marker by the attachment of a biotin moiety to the polypeptide, which can be detected by streptavidin containing enzymatic activity). In certain embodiments, the label or marker may be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to: Radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I), fluorescent labels (eg, FITC, rhodamine, lanthanide phosphor) ), Enzyme labels (eg, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (eg, leucine zipper pair sequences, secondary antibodies) Binding sites, metal binding domains, epitope tags). In certain embodiments, labels are attached by spacer arms of various lengths to reduce the potential for steric hindrance.

  The term "biological sample" as used herein includes, but is not limited to, any quantity of a substance obtained from an organism or formerly an organism. Such organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such materials include, but are not limited to, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph nodes, and skin.

  The term “pharmaceutical composition” (or drug or drug) as used herein refers to a chemical compound, composition, or composition that, when properly administered to a patient, can induce a desired therapeutic effect. Refers to a composition, drug, or drug. A pharmaceutical composition does not necessarily require more than one type of ingredient.

  The term "therapeutically effective amount" refers to a PCSK9 antigen binding protein that has been determined to produce a therapeutic response in a mammal. Such a therapeutically effective amount is readily ascertained by one skilled in the art.

  The term "modulator" as used herein is a compound that alters or alters the activity or function of a molecule. For example, a modulator can cause an increase or decrease in the magnitude of a particular activity or function of a molecule, as compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor that reduces the magnitude of the activity or function of at least one of the molecules. Certain typical activities and functions of a molecule include, but are not limited to, binding affinity, enzymatic activity, and signal transduction. Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, for example, in US Pat. No. 6,660,843 (corresponding to PCT application WO 01/83525).

  The terms "patient" and "subject" are used interchangeably and refer to human and non-human animal subjects as well as subjects having a previously diagnosed disease, subjects having no previously recognized disease, and receiving medical treatment. Subjects, subjects at risk of developing a disease, and the like.

  The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and applications that reduce the risk that a subject will develop a disease or other risk factor. Treatment does not require complete cure of the disease, but includes embodiments that reduce symptoms or underlying risk factors.

  The term "prevent" does not require that the probability of an event be eliminated by 100%. Rather, the term "prevent" refers to the reduced likelihood of an event occurring in the presence of the compound or method.

  Standard techniques for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection) can be used. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly performed in the art or as described herein. The techniques and procedures are generally performed according to methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. be able to. See, for example, "Sambrook et al, Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989)"), for all purposes herein. Incorporated in). Unless otherwise specified, the nomenclature used in connection with the analytical chemistry, synthetic organic chemistry and medical and pharmaceutical chemistry described herein, and their research techniques and techniques, It is well known in the art and commonly used. Standard techniques for chemical synthesis, analysis, preparation, formulation and delivery of medicaments and treatment of patients can be used.

Antigen binding protein for PCSK9 The proprotein convertase subtilisin kexin type 9 (PCSK9) is a serine protease that is involved in regulating the levels of low density lipoprotein receptor (LDLR) protein (Horton et al, 2007; (Seidah and Prat, 2007). PCSK9 is a prohormone-proprotein convertase of the subtilisin (S8) family of serine proteases (Seidah et al., 2003). Exemplary human PCSK9 amino acid sequences are SEQ ID NO: 1 in FIG. 1A (illustrating the “pro” domain of the underlined protein) and SEQ ID NO: 3 in FIG. 1B (bold signal sequence and underlined). Pro domain is illustrated). A typical human PCSK9 coding sequence is represented as SEQ ID NO: 2 (FIG. 1B). As described herein, the PCSK9 protein can also include fragments of the full-length PCSK9 protein. The structure of the PCSK9 protein was recently solved by two groups (Cunningham et al., Nature Structural & Molecular Biology, 2007, and Piper et al., Structure, 15: 1-8, 2007). , Incorporated herein in its entirety). PCSK9 contains a signal sequence, an N-terminal prodomain, a subtilisin-like catalytic domain, and a C-terminal domain.

  Antigen binding proteins (ABPs) that bind to PCSK9, including human PCSK9, are used in the methods described herein. In some embodiments, the antigen binding protein is a polypeptide comprising one or more complementarity determining regions (CDRs) described herein. In some antigen binding proteins, the CDRs are embedded in a “framework” region that directs the CDRs to achieve the proper antigen binding properties of the CDR. In some embodiments, the antigen binding protein that can be used in the methods provided herein is capable of blocking, blocking, reducing, or modulating the interaction between PCSK9 and LDLR. it can. Such an antigen binding protein is designated as "neutralized". In some embodiments, even when the antigen binding protein is neutralizing and is bound to PCSK9, binding between PCSK9 and LDLR can still occur. For example, in some embodiments, ABPs useful in the methods provided herein prevent or reduce the adverse effects of PCSK9 on LDLR without blocking the LDLR binding site on PCSK9. Thus, in some embodiments, ABP modulates or alters the ability of PCSK9 to cause LDLR degradation without having to suppress the binding interaction between PCSK9 and LDLR. Such ABPs can be specifically described as "non-competitively neutralizing" ABPs. In some embodiments, the neutralizing ABP binds to PCSK9 in a position and / or manner that prevents PCSK9 from binding to LDLR. Such ABPs can be specifically described as "competitively neutralizing" ABPs. Any of the above neutralizing agents can result in a greater amount of free LDLR being present in the subject, thereby resulting in more LDLR binding to LDL (which results in a greater Reduce the amount of LDL). This in turn results in a reduction in the amount of serum cholesterol present in the subject.

  In some embodiments, an antigen binding protein provided herein is capable of inhibiting PCSK9-mediated activity, including binding. In some embodiments, antigen binding proteins that bind to these epitopes inhibit, inter alia, the interaction between PCSK9 and LDLR and other physiological effects mediated by PCSK9. In some embodiments, the antigen binding protein is human, such as a fully human antibody to PCSK9.

  In some embodiments, ABP binds to the catalytic domain of PCSK9. In some embodiments, ABP binds to the mature form of PCSK9. In some embodiments, ABP binds in the prodomain of PCSK9. In some embodiments, the ABP selectively binds to the mature form of PCSK9. In some embodiments, ABP binds to the catalytic domain in a manner that PCSK9 is unable to bind to LDLR, or is unable to bind efficiently. In some embodiments, the antigen binding protein does not bind to the C-terminus of the catalytic domain. In some embodiments, the antigen binding protein does not bind to the N-terminus of the catalytic domain. In some embodiments, 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 measured by a competition assay between the antibodies disclosed herein and other antibodies. In some embodiments, the ABP binds to an epitope bound by one of the antibodies listed in Table 2. In some embodiments, the antigen binding protein binds to a specific conformational state of PCSK9 and prevents PCSK9 from interacting with LDLR. In some embodiments, the ABP binds to the V domain of PCSK9. In some embodiments, ABP binds to the V domain of PCSK9 and prevents (or reduces) PCSK9 from binding to LDLR. In some embodiments, the ABP binds to the V domain of PCSK9 and does not prevent (or reduce) binding of PCSK9 to LDLR, but does prevent deleterious activity on LDLR mediated through PCSK9 on LDLR; Or reduce.

  The disclosed antigen binding proteins useful in the methods provided herein have a variety of uses. Some of the antigen binding proteins are useful, for example, in specific binding assays, in the affinity purification of PCSK9, especially human PCSK9 or its ligand, and in screening assays to identify other antagonists of PCSK9 activity. Some of the antigen binding proteins are useful for inhibiting PCSK9 binding to LDLR or inhibiting PCSK9-mediated activity.

  In some embodiments, an antigen binding protein useful in the methods provided herein comprises one or more CDRs (eg, 1, 2, 3, 4, 5, or 6 CDRs). )including. In some embodiments, the antigen binding protein comprises one or more CDRs inserted and / or linked into (a) a polypeptide structure and (b) a polypeptide structure. The polypeptide structure can take a variety of different forms. For example, the polypeptide structure can be or include a naturally occurring antibody framework or a fragment or variant thereof, or be completely synthetic in nature. Examples of various polypeptide structures are described further below.

In certain embodiments, the polypeptide structure of the antigen binding protein is an antibody, or a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, a synthetic antibody (herein "antibody mimic"). ), Chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and portions (or fragments) of each. , But not limited to) antibodies. In some examples, the antigen binding protein is an immunological fragment of an antibody (eg, Fab, Fab ′, F (ab ′) ₂ , or scFv). Various structures are further described and defined herein.

  Some of the antigen binding proteins 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 a human PCSK9 protein having and / or consisting of residues 153-692 of SEQ ID NO: 3. In some embodiments, the ABP specifically and / or selectively binds to human PCSK9 having and / or consisting of residues 31-152 of SEQ ID NO: 3. In some embodiments, ABP selectively binds to the human PCSK9 protein (SEQ ID NO: 1) illustrated in FIG. 1A. In some embodiments, the antigen binding protein specifically binds to at least a fragment of the PCSK9 protein and / or to a full-length PCSK9 protein with or without a signal sequence.

In embodiments where the antigen binding protein is used for therapeutic applications, the antigen binding protein can inhibit, block, or modulate one or more biological activities of PCSK9. In one embodiment, the antigen binding protein specifically binds to human PCSK9 and / or at least about 20% -40%, 40-60%, e.g., by measuring binding in an in vitro competitive binding assay. 60-80%, 80-85% or more substantially inhibits the binding of human PCSK9 to LDLR. Some of the antigen binding proteins provided herein are antibodies. In some embodiments, ABP ^is 10 ^{-7, 10 -8,} 10 ^-9, 10 ^{-10, 10 -11,} 10 ^-12, has a ^{10 -13} M less than _{K d} (more strongly bound ). In some embodiments, the ABP is less than 1 μM, 1000 nM-100 nM, 100 nM-10 nM, 10 nM-1 nM, 1000 pM-500 pM, 500 pM-200 pM, less than 200 pM, 200 pM-150 pM, for blocking binding of LDLR to PCSK9. having 200pM~100pM, 100pM~10pM, the _{IC 50} of 10pM~1pM (D347Y, high affinity variant).

  One example of the IgG2 heavy chain constant domain of the anti-PCSK9 antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 154, FIG. 3KK.

  One example of the IgG4 heavy chain constant domain of the anti-PCSK9 antibody of the present invention has the amino acid sequence 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 shown in SEQ ID NO: 157, FIG. 3KK.

  One example of the λ light chain constant domain of the anti-PCSK9 antibody has the amino acid sequence shown in SEQ ID NO: 156, FIG.

  The variable regions of the immunoglobulin chains are relatively conserved framework regions (FR) linked by three hypervariable regions, more often referred to as "complementarity determining regions" or CDRs. Generally exhibit the same overall structure including The CDRs from the two chains of each heavy / light chain pair are typically aligned by framework regions to form a structure that specifically binds to a specific epitope on the target protein (eg, PCSK9). Is done. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions are generally consistent with the following order of these elements. FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to amino acids occupying positions in each of these domains. This numbering system is described in “Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD)” or “Chothia & Lesk, 1987, J. Mol. Biol. , 1989, Nature 342: 878-883 ".

  Various heavy and light chain variable regions are provided herein and are illustrated in FIGS. 2A-3JJ and 3LL-3JJJ and 3LLL. In some embodiments, each of these variable regions can be attached to the heavy and light chain constant regions to form a complete antibody heavy and light chain, respectively. Furthermore, it is possible to combine each of the heavy and light chain sequences so produced to form a complete antibody structure.

  Some specific examples of the light and heavy chain variable regions of the provided antibodies and their corresponding amino acid sequences are summarized in Table 2.

  Similarly, each of the exemplary variable heavy chains listed in Table 2 can be combined with any of the exemplary variable light chains shown in Table 2 to form antibodies. Table 2 shows typical light and heavy chain pairs found in some of the antibodies disclosed herein. In some examples, the antibody comprises at least one variable heavy chain and one variable light chain obtained from those listed in Table 2. In another example, the antibody contains two identical light chains and two identical heavy chains. As an example, an antibody or antigen binding protein can include a heavy and light chain, two heavy chains or two light chains. In some embodiments, the antigen binding protein comprises one, two, and / or three heavy and / or light CDRs (CDRs for these sequences) obtained from at least one of the sequences listed in Table 2. (And / or consist of) as outlined in FIGS. 2A-3D and FIGS. 3CCC-3JJJ and 3KKK and other embodiments in 13A-13J. In some embodiments, all six CDRs (CDR1-3 (CDRL1, CDRL2, CDRL3) from the light chain and CDR1-3 (CDRHI, CDRH2, and CDRH3) from the heavy chain are all part of the ABP. In some embodiments, one, two, three, four, five, or more CDRs are included in the ABP, hi some embodiments, from CDRs in the sequences in Table 2. One resulting heavy and one light CDR is included in the ABP (the CDRs for the sequences in Table 2 are outlined in FIGS. 2A-3D). Additional compartments (as illustrated in 2A-2D, 3A-3D, 3CCC-3JJJ and 3LLL and other embodiments in 13A-13J) are also included in the ABP. Examples of CDRs and FRs for the heavy and light chains described in Table 2 are outlined in Figures 2A-3D (and other embodiments in Figures 3CCC-3JJJ and 3LLL and 13A-13J). The optionally present light chain variable sequences (including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4) can be selected from: 5, 7, 9, 10, 12, 13, , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46 , 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, and 485. The optionally present heavy chain variable sequence (CDR , CDR2, CDR3, FR1, FR2, FR3, and FR4) can be selected from: 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. Some of the entries in FIGS. Other boundaries of the FR have been identified, these alternatives are identified by the “vl” after the ABP name. Only compartments with differences are shown in the table. It is understood that the remaining compartments of the light or heavy chains are the same as shown for the base ABP in the other panels. Thus, in FIG. 2C, only the differences are described, for example, 19H9vl in FIG. 2C has the same FR1, CDR1, and FR2 as 19H9 in FIG. 2A. Additional nucleic acid sequences are provided in the figure for three of the nucleic acid sequences (ABP26E10, 30B9, and 31B12). As will be appreciated by those skilled in the art, the production of antibodies or ABPs actually requires the use of one or less of such sequences. Indeed, in some embodiments, only one of the specific heavy or light chain nucleic acids need be present, or need not be present at all.

  In some embodiments, antibodies useful in the methods described herein include the antibodies described in US Patent No. 8,030,457 or US Patent No. 8,168,762. included. Further examples of antibodies useful in the methods described herein include, for example, US Patent Nos. 8,188,233, 8,188,234, 8,080,243. U.S. Patent No. 8,062,640, WO2008 / 06332, WO2009 / 055783, WO2011 / 053759, WO2012 / 054438, WO2012 / 088833, WO2012 / 109530, and WO2013 / 039958.

  In some embodiments, the ABP is encoded by a nucleic acid sequence capable of encoding any of the protein sequences in Table 2.

  In some embodiments, ABP selectively binds to a form of PCSK9 that binds to LDLR (eg, an autocatalyzed form of the molecule). In some embodiments, the antigen binding protein is c-terminal to the catalytic domain (eg, 5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40 in the c-terminus). Does not bind to many amino acids). In some embodiments, the antigen binding protein comprises the n-terminus of the catalytic domain (eg, 5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40 in the n-terminus). Does not bind to many amino acids). In some embodiments, ABP binds to amino acids within amino acids 1-100 of the mature form of PCSK9. In some embodiments, the ABP comprises amino acids 31-100, 100-200, 31-152, 153-692, 200-300, 300-400, 452-683, 400-500, 500-600, 31-692. , 31-449, and / or 600-692 (and / or an amino acid sequence consisting thereof). 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, ABP binds to both the catalytic and pro domains. In some embodiments, ABP binds to the catalytic domain and blocks a region on the catalytic domain that interacts with the pro domain. In some embodiments, the ABP is selected from Piper et al. (Structure 15: 1-8 (2007), including the stereoscopic representation therein, incorporated herein by reference in its entirety) the position or surface at which the prodomain interacts. In some embodiments, ABP binds to the catalytic domain and constrains the mobility of the pro domain.In some embodiments, ABP binds without binding to the pro domain In some embodiments, the ABP binds to the catalytic domain without binding to the pro domain, preventing the pro domain from reorienting to bind PCSK9 to LDLR. In some embodiments, the ABP comprises residues around the prodomain in Piper et al. Binds in the same epitope as 149-152.In some embodiments, ABP binds to the groove on the V domain (as outlined in Piper et al.). ABP binds to a histidine-rich patch near the groove on the V domain, hi some embodiments, such an antibody (binding to the V domain) is not neutralizing. Antibodies that bind to the domain are neutralizing.In some embodiments, neutralizing ABP prevents PCSK9 binding to LDLR In some embodiments, neutralizing ABP inhibits PCSK9 degradation of LDLR Does not prevent binding of PCSK9 to LDLR (eg, ABP 31A4). P binds or blocks at least one of the histidines depicted in Figure 4 of the article of Piper et al .. In some embodiments, ABP binds the catalytic triad in PCSK9. Cut off.

In some embodiments, the antibody selectively binds to a variant PCSK9 protein (eg, D374Y) as compared to wild-type PCSK9. In some embodiments, these antibodies bind the variant at least twice as strongly as the wild-type (as measured via _Kd ), preferably from 2-5, 5-10, 10-100, It binds more strongly to the mutant than the wild type 100-1000, 1000-10,000-fold or more. In some embodiments, the antibody selectively inhibits variant D374Y PCSK9 from interacting with LDLR over its ability to inhibit wild-type PCSK9 from interacting with LDLR. In some embodiments, these antibodies are more potent than the wild-type (as measured via the IC ₅₀ ) binding to LDLR and block the ability of the variant to bind to the LDLR (eg, less than the wild-type). To a mutant that is at least twice as strong, preferably 2-5, 5-10, 10-100, 100-1000 times or more stronger than wild-type). In some embodiments, the antibody binds and neutralizes both wild-type PCSK9 and a variant form of PCSK9 (such as D374Y) at similar levels. In some embodiments, the antibody binds to PCSK9 and prevents a variant of LDLR from binding to PCSK9. In some embodiments, a variant of LDLR is at least 50% identical to human LDLR. It is noted that variants of LDLR are known to those of skill in the art (eg, BrownMS et al., "Calcium cages, acid baths and recycling receptors" Nature 388: 629-630, 1997). In some embodiments, ABP can increase the level of effective LDLR in heterozygous familial hypercholesterolemia (where there is a loss-of-function variant of LDLR).

  In some embodiments, the ABP is at least 50%, 50-60, 60-70, 70-80, 80-90, 90 relative to the form of PCSK9 illustrated in FIGS. 1A and / or 1B. Binds to (but does not block) variants of PCSK9 that are ９５95, 95-99, or more percent identical. In some embodiments, the ABP is at least 50%, 50-60, 60-70, 70-80, 80-90, at least 50% of the mature form of PCSK9 illustrated in FIG. 1A and / or 1B. Binds (but does not block) variants of PCSK9 that are 90-95, 95-99 or more% identical. In some embodiments, the ABP is at least 50%, 50-60, 60-70, 70-80, 80-90, 90 relative to the form of PCSK9 illustrated in FIGS. 1A and / or 1B. Binds to variants of PCSK9 that are ９５95, 95-99, or more percent identical and prevent interacting with LDLR. In some embodiments, the ABP is at least 50, 50-60, 60-70, 70-80, 80-90, 90-95, 95-95 relative to the mature form of PCSK9 illustrated in FIG. It binds to variants of PCSK9 that are 99% or greater in percent identity and prevent it from interacting with LDLR. In some embodiments, the variant of PCSK9 is a variant at position 474, a human variant such as E620G and / or E670G. In some embodiments, the amino acid at position 474 is valine (as in other humans) or threonine (as in cynomolgus monkeys and mice). In view of the cross-reactivity data presented herein, it is likely that the present antibodies will readily bind to the above variants.

  In some embodiments, the ABP binds to an epitope bound by one of the antibodies listed in Table 2. In some embodiments, the antigen binding protein binds to a specific conformational state of PCSK9 and prevents PCSK9 from interacting with LDLR.

Humanized antigen binding proteins (eg, antibodies)
As described herein, an antigen binding protein for PCSK9 can include a humanized antibody and / or a portion thereof. An important practical application of such a strategy is the "humanization" of the mouse humoral immune system.

  In certain embodiments, a humanized antibody is substantially non-immunogenic in a human. In certain embodiments, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody was derived. See, for example, U.S. Patent Nos. 5,530,101, 5,693,761, 5,693,762, and 5,585,089.

  In certain embodiments, amino acids of the antibody variable domain are identified that can be modified without diminishing their immunogenicity while reducing the natural affinity of the antigen binding domain. See, for example, U.S. Patent Nos. 5,766,886 and 5,869,619.

  In certain embodiments, modification of the antibody by methods known in the art typically achieves increased binding affinity for the target and / or reduces the immunogenicity of the antibody in the recipient. Designed to reduce. In certain embodiments, a humanized antibody is modified to remove glycosylation sites to increase the affinity of the antibody for its cognate antigen. See, for example, "Co et al., Mo 1. Immunol 30: 1361-167 (1993)." In certain embodiments, techniques such as "reshaping," "superchimerization," or "venering / resurfacing" are used to generate humanized antibodies. See, for example, "Vaswami et al., Annals of Allergy, Asthma, & Immunol 81: 105 (1998); Roguska et al., Prot. Engineer., 9: 895-904 (1996); No. 035 ". In certain such embodiments, such techniques typically reduce the immunogenicity of the antibody by reducing the number of foreign residues, but after repeated administration of the antibody, the anti-idiotype and anti-idiotype Does not suppress allotype responses. For example, "Gilliland et al., J. Immunol 62 (6): 3663-71 (1999)" describes some other methods for reducing immunogenicity.

  In certain instances, a humanized antibody results in a loss of antigen binding capacity. In certain embodiments, a humanized antibody is "backmutated." In such embodiments, the humanized antibody is mutated to include one or more of the amino acid residues found in the donor antibody. See, for example, "Saldanha et al., Mol Immunol 36: 709-19 (1999)".

  In certain embodiments, the complementarity determining regions (CDRs) of the light and heavy chain variable regions of the antibody to PCSK9 can be grafted onto framework regions (FR) from the same or another species. In certain embodiments, the CDRs of the light and heavy chain variable regions of an antibody to PCSK9 can be grafted into consensus human FRs. To create a consensus human FR, in certain embodiments, FRs from several human heavy or light chain amino acid sequences are juxtaposed to identify a consensus amino acid sequence. In certain embodiments, the FRs of the antibody to the PCSK9 heavy or light chain are replaced with FRs from a different heavy or light chain. In certain embodiments, rare amino acids in the FRs of the heavy and light chains of the antibody to PCSK9 are not substituted, but the rest of the FR amino acids are substituted. Unusual amino acids are specific amino acids at positions not normally found in FRs. In certain embodiments, an implanted variable region derived from an antibody against PCSK9 may be used with a constant region that is different from the constant region of the antibody against PCSK9. In certain embodiments, the implanted variable region is part of a single-chain Fv antibody. CDR grafting is described, for example, in U.S. Patent Nos. 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761, 5th. Nos. 5,565,332, 5,585,089 and 5,530,101 and "Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323. 327 (1988); Verhoeyen et al., Science, 239: 1544-1536 (1988), Winter, FEBS Letts., 430: 92-94 (1998), which are hereby incorporated by reference for all purposes. Incorporated herein by reference.

Human antigen binding protein (eg, antibody)
As described herein, an antigen binding protein that binds to PCSK9 can include a human (ie, fully human) antibody and / or a portion thereof. In certain embodiments, provided are nucleotide sequences encoding heavy and light chain immunoglobulin molecules and amino acid sequences comprising heavy and light chain immunoglobulin molecules, particularly those corresponding to the variable regions. In certain embodiments, sequences corresponding to complementarity determining regions (CDRs), particularly CDR1 through CDR3, are provided. According to certain embodiments, there is provided a hybridoma cell line expressing such an immunoglobulin molecule. According to one particular embodiment, there is provided a hybridoma cell line expressing such a monoclonal antibody. In certain embodiments, the hybridoma cell line is selected from at least one of the cell lines listed in Table 2, for example, 21B12, 16F12, and 31H4. In certain embodiments, a purified human monoclonal antibody against human PCSK9 is provided.

  With the expectation that mice will produce human antibodies in the absence of mouse antibodies, large fragments of the human Ig locus can be used to modify mouse strains deficient in mouse antibody production. Large human Ig fragments can preserve large variable gene diversity and appropriate control of antibody production and expression. By exploiting the murine machinery for antibody diversification and selection and lack of immunological tolerance to human proteins, the regenerated human antibody repertoire in these mouse strains will be able to target any antigen of interest (human High-affinity fully human antibodies (including antigens). Using hybridoma technology, antigen-specific human MAbs with the desired specificity can be generated and selected. Certain exemplary methods are described in WO 98/24893, U.S. Patent No. 5,545,807, EP546073, and EP546073.

  In certain embodiments, constant regions from non-human species can be used with human variable regions.

  Due to the ability to clone and reconstruct megabase-sized human loci in the yeast artificial chromosome (YAC) and to introduce them into the mouse germline, the functionality of extremely large or roughly mapped loci An approach is provided to elucidate the components and create a useful model of human disease. Furthermore, the use of such techniques to replace mouse loci with their human equivalents has led to the expression and regulation of developing human gene products, their interaction with other systems, and the induction and progression of disease. Insight into their involvement can be given.

  Human antibodies avoid some of the problems associated with antibodies having mouse or rat variable and / or constant regions. The presence of such a mouse or rat-derived protein may result in rapid elimination of the antibody, or may result in the generation of an immune response to the antibody by the patient. In order to avoid the use of antibodies derived from mice or rats, it may be functional in rodents, other mammals or animals so that rodents, other mammals or animals produce fully human antibodies. Through the introduction of various human antibody loci, fully human antibodies can be produced.

  Humanized antibodies are antibodies that have at least some of these non-human antibody amino acid sequences replaced with human antibody sequences, starting with the first containing a non-human antibody amino acid sequence. This is different from human antibodies, in which the antibody is (or can be) encoded by genes that humans carry.

Variants of Antigen Binding Proteins Other antibodies provided are formed by the variable heavy and variable light chain combinations or subparts shown in Table 2 and FIGS. 2A-3JJ and 3LL-JJJ and 3LLL, wherein 2A-3JJ and 3LL-JJJ and the amino acid sequence of the sequence of 3LLL, at least 50%, 50-60%, respectively, of the entire sequence or subparts of the sequence (eg, one or more CDRs) , 60-70, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or variable light chains having greater than 99% identity and / or An ABP variant as described above comprising a variable heavy chain. In some cases, such an antibody comprises at least one heavy chain and one light chain, while in other cases, the variant form has two identical light chains and two identical heavy chains. Chains (or subparts thereof). In some embodiments, to identify compartments of an antibody that can be modified by observing variations that affect binding and variations that do not appear to affect binding, FIGS. 13A-13J, and other embodiments in FIGS. 31A and 31B) can be used. For example, by comparing similar sequences, compartments that can be modified (eg, specific amino acids) are identified and how they are modified while still retaining (or improving) the functionality of ABP Can be specified. In some embodiments, variants of ABP include consensus groups and sequences illustrated in FIGS. 13A, 13C, 13F, 13G, 13H, 131, 13J, and / or 31A and 31B, wherein the variation is It is possible at positions specified as variable therein. The CDRs shown in FIGS. 13A, 13C, 13F, 13G, 31A, and 31B are based on the Chothia method (based on the position of the structural loop region. For example, “Standard transformations for the canonical structures of immunoglobulin's Alzheimer's Algis-Branches” See M. Lesk and Cyrus Chothia, Journal of Molecular Biology, 273 (4): 927-948, 7 November (1997) and the Kabat method (based on sequence variability, for example, Sequence esto siego estro estro estro estro estro estro estro estro estro sero s fo s e s fo r s s e s i s e s i s s e s s o f s e s s e s i s i s o f e s e s i s s o s e s i s o f e) Fifth Edition.NIH Pub ication No.91-3242, Kabat et al., have been defined based on a hybrid combination of (1991)). Each residue determined by either method was included in the final list of CDR residues (described in FIGS. 13A, 13C, 13F, 13G, 31A, and 31B). The CDRs in FIGS. 13H, 13I, and 13J were obtained only by the Kabat method. Unless otherwise stated, the defined consensus sequences, CDRs, and FRs in FIGS. 13H-13J define and control the notation CDRs and FRs for the ABP referenced in FIG.

  In certain embodiments, the antigen binding protein is 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 comprising a heavy chain comprising a variable region comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from at least one of the sequences. In certain embodiments, the antigen binding protein is 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, A heavy chain comprising a variable region comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from at least one of the sequences 467, 471, 475, 479, and 483. In certain embodiments, the antigen binding protein is 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, A heavy chain comprising a variable region comprising an amino acid sequence at least 99% identical to an amino acid sequence selected from at least one of the sequences of 467, 471, 475, 479, and 483.

  In some embodiments, the antigen binding protein is 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, At least 90%, 90-95%, and / or 95-99% identical to one or more CDRs obtained from CDRs in at least one of the sequences of 467, 471, 475, 479, and 483; Contains an array. In some embodiments, there are 1, 2, 3, 4, 5, or 6 CDRs (each being at least 90%, 90-95%, and / or 95-99% identical to the sequence above). .

  In some embodiments, the antigen binding protein is 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, At least 90%, 90-95%, and / or 95-99% identical to one or more FRs obtained from the FRs in at least one of the sequences of 467, 471, 475, 479, and 483. Contains an array. In some embodiments, there are 1, 2, 3, or 4 FRs (each being at least 90%, 90-95%, and / or 95-99% identical to the sequence above).

  In certain embodiments, the antigen binding protein comprises SEQ ID NOs: 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, A light chain comprising a variable region comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from at least one of the sequences 469, 473, 477, 481, and 485. In certain embodiments, the antigen binding protein comprises SEQ ID NOs: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, An amino acid sequence that is at least 95% identical to an amino acid sequence selected from at least one of the sequences 30, 30, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. Includes a light chain that includes the variable region. In certain embodiments, the antigen binding protein comprises SEQ ID NOs: 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, A light chain comprising a variable region comprising an amino acid sequence at least 99% identical to an amino acid sequence selected from at least one of the sequences of 469, 473, 477, 481, and 485.

  In some embodiments, the antigen binding protein is SEQ ID NOs: 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, At least 90%, 90-95%, and / or 95-99% identical to one or more CDRs obtained from CDRs in at least one of the sequences of 469, 473, 477, 481, and 485. Contains an array. In some embodiments, there are 1, 2, 3, 4, 5, or 6 CDRs (each being at least 90%, 90-95%, and / or 95-99% identical to the sequence above). .

  In some embodiments, the antigen binding protein is SEQ ID NOs: 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, At least 90%, 90-95%, and / or 95-99% identical to one or more FRs obtained from FRs in at least one of the sequences of 469, 473, 477, 481, and 485. Contains an array. In some embodiments, there are 1, 2, 3, or 4 FRs (each being at least 90%, 90-95%, and / or 95-99% identical to the sequence above).

  In light of the present disclosure, one of skill in the art would be able to determine the appropriate variant of ABP using well-known techniques, as described herein. In certain embodiments, one of skill in the art can identify appropriate regions of the molecule that can be altered without destroying activity by targeting regions that are not believed to be important for activity. In certain embodiments, residues and portions of the molecule that are conserved between similar polypeptides can be identified. In certain embodiments, it is possible to make conservative amino acid substitutions in regions that may be important for biological activity, or even for structure, without destroying the biological activity or adversely affecting the polypeptide structure. it can.

  In addition, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acids that are important for activity or structure in similar proteins. One of skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

  One of skill in the art can also analyze the three-dimensional structure and amino acid sequence for similar structures in ABP. In view of such information, one skilled in the art can predict the alignment of the amino acid residues of the antibody with respect to its three-dimensional structure. In certain embodiments, one skilled in the art would recognize that amino acid residues predicted to be present on the surface of a protein are likely to be present on the surface of the protein, as those amino acid residues may be involved in important interactions with other molecules. Can be selected so as not to cause significant changes to certain amino acid residues. Furthermore, one of skill in the art can also create test variants that contain a single amino acid substitution at each desired amino acid residue. This variant can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about the appropriate variant. For example, if it is found that a change to a particular amino acid residue results in impaired activity, undesirably reduced activity, or inappropriate activity, variants having such changes may be avoided. . In other words, based on information gathered from such routine experimentation, one of ordinary skill in the art, alone or in combination with other mutations, can readily determine which amino acids should avoid further substitution. .

  Numerous scientific publications deal with the prediction of secondary structure. "Moult J., Curr. Op. In Biotech., 7 (4): 422-427 (1996), Chou et al., Biochemistry, 13 (2): 222-245 (1974); Chou et al., Biochemistry. Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148 (1978); Chou et al., Ann. Rev. Biochem., 113 (2): 211-222 (1974); , 47: 251-276 and Chou et al., Biophys. J., 26: 367-384 (1979). In addition, computer programs can now be used to assist in predicting secondary structure. One method for predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins that have greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. Recent enhancements in the protein structure database (PDB) have improved the predictability of secondary structure, such as the number of possible folds in the structure of a polypeptide or protein. See Holm et al., Nucl. Acid. Res., 27 (1): 244-247 (1999). It suggests that there is a limited number of folds in a given polypeptide or protein, and that if a certain number of structures are determined, structural predictions can be dramatically increased in accuracy. (Brenner et al., Curr. Op. Struct. Biol., 7 (3): 369-376 (1997)).

  Further methods of predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol., 7 (3): 377-87 (1997); Sippl et al., Structure, 4 ( 1): 15-19 (1996)), "Profile analysis" (Bowie et al., Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym., 183: 146-159 (1990)). Gribskov et al., Proc. Nat. Acad. Sci., 84 (13): 4355-4358 (1987)) and "Evolutionary linkage" (Holm, supra (1999), and Brenner, supra (1997)). See).

  In certain embodiments, antigen binding protein variants include glycosylation variants that have altered numbers and / or types of glycosylation sites relative to the amino acid sequence of the parent polypeptide. In certain embodiments, the protein variant comprises more or less N-linked glycosylation sites than the native protein. N-linked glycosylation sites are characterized by the sequence: Asn-X-Ser or Asn-X-Thr (the amino acid residue denoted as X can be any amino acid residue except proline). Substitution of amino acid residues to create this sequence provides a new site at which N-linked carbohydrate chains can be added. Alternatively, substitutions that remove this sequence will remove an existing N-linked carbohydrate chain. An N-linked carbohydrate, wherein one or more N-linked glycosylation sites (typically naturally occurring) have been removed to create one or more new N-linked sites. Strand rearrangement is also provided. Other preferred antibody variants include cysteine variants in which one or more cysteine residues have been deleted or replaced with another amino acid (eg, serine) relative to the parent amino acid sequence. Cysteine variants may be useful when the antibody has to be refolded into a biologically active conformation, such as after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues compared to the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

  According to certain embodiments, the amino acid substitutions (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) binding affinity for forming a protein complex. Amino acid substitutions that alter the sex, (4) alter the binding affinity, and / or (4) impart or modify other physicochemical or functional properties to such polypeptides. . According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) are made in the naturally occurring sequence (in certain embodiments, intermolecular contacts). (In a portion of the polypeptide that is outside the domain that forms). In certain embodiments, conservative amino acid substitutions typically cannot substantially alter the structural properties of the parent sequence (eg, the replacement amino acid cleaves a helix present in the parent sequence). There should be no propensity to exist or a tendency to disrupt other types of secondary structure that characterize the parent sequence). Examples of polypeptide secondary and tertiary structures recognized in the art are described in "Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York, Canada) (1984); Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Hornton et al., Nature 354: 105 (1991) "(by reference, respectively). Which will be incorporated in the textbook).

  In some embodiments, the variant is a variant of the nucleic acid sequence of ABP disclosed herein. One skilled in the art will appreciate that the above discussion can be used to identify, evaluate, and generate ABP protein variants, and for nucleic acid sequences that can encode these protein variants. Thus, nucleic acid sequences encoding protein variants (and nucleic acid sequences encoding ABP in Table 2, but different from the nucleic acid sequences explicitly disclosed herein) are envisioned. For example, ABP variants have 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 nucleic acid sequence or SEQ ID NO: 152, 153; 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, and 1 1, 296, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, At least one to six (and various combinations thereof) of the CDRs encoded by the nucleic acid sequences in 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, and 484. It can have 80, 80-85, 85-90, 90-95, 95-97, 97-99, or more identity.

  In some embodiments, under stringent conditions, the nucleic acid sequence encoding a particular ABP (or the nucleic acid sequence itself) is any of the nucleic acid sequences encoding the proteins in Table 2 (eg, 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, but are not limited thereto, provided that the antibody (or nucleic acid encoding the antibody) can be selectively hybridized. Sequence) is a variant. In one embodiment, suitable moderately stringent conditions are 5X SSC; pre-wash in a solution of 0.5% SDS, 1.0 mM EDTA (pH 8.0), 50 ° C, -65 ° C, 5x SSC. Hybridization overnight, or in the case of interspecies homology, hybridization at 45 ° C. in 0.5 × SSC, then 2 ×, 0.5 × containing 0.1% SDS, and Includes two washes at 65 ° C. for 20 minutes with each of 0.2 × SSC. Such hybridizing DNA sequences are also within the scope of the invention, and because of code degeneracy, nucleotides encoding the antibody polypeptides encoded by the hybridizing DNA sequences and the amino acid sequences encoded by these nucleic acid sequences The same applies to arrays. In some embodiments, the variants of the CDRs include one or more of the CDRs in the sequence (each CDR in light of the other embodiments in FIGS. 2A-3D and FIGS. 3CCC-3JJJ and 15A-15D). Can be readily determined) and the amino acid sequence encoded by the nucleic acid sequence and the hybridizing nucleic acid sequence. The term "selectively hybridize" as referred to in this context means to detectably and selectively bind. The polynucleotides, oligonucleotides, and fragments thereof of the present invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize the amount of detectable binding to non-specific nucleic acids. . High stringency conditions can be used to achieve selective hybridization conditions, as is known in the art, and as discussed herein. Generally, nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the invention and the nucleic acid sequence of interest is at least 80%, more typically at least 85%, 90%, 95%, It has a preferably increasing homology of 99% and 100%. Two amino acid sequences are homologous if there is a partial or complete identity between the two amino acid sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned, such that a maximal match is obtained. Gaps (in either of the two sequences being matched) are allowed in maximizing matches. A gap length of 5 or less is preferred, and 2 or less is more preferred. Alternatively and preferably, as the term is used herein, a value of 5 (in units of standard deviation) using a mutation data matrix and the program ALIGN using a gap penalty of 6 or more is used. Given many alignment scores, two protein sequences (or polypeptide sequences derived therefrom, at least 30 amino acids in length) are homologous. See Dayhoff, MO, in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, p. . If, when optimally aligned using the ALIGN program, the amino acids of the two sequences or portions thereof are greater than or equal to 50%, the two sequences or portions thereof are more preferably homologous. As used herein, the term "corresponding" refers to a polynucleotide sequence that is homologous (ie, identical and not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence. ) Or that the polypeptide sequence is identical to the reference polypeptide sequence. In contrast, the term "complementary" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. By way of example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

Preparation of Antigen-Binding Proteins (eg, Antibodies) In certain embodiments, antigen-binding proteins (such as antibodies) are produced by immunization with an antigen (eg, PCSK9). In certain embodiments, the antibody is immunized with full-length PCSK9, a soluble form of PCSK9, only the catalytic domain, the mature form of PCSK9 shown in FIG. 1A, a splice variant form of PCSK9, or fragments thereof. It can be produced by chemical conversion. In certain embodiments, the antibodies of the invention can be polyclonal or monoclonal, and / or can be recombinant antibodies. In certain embodiments, an antibody of the invention is a human antibody prepared, for example, by immunization of a transgenic animal capable of producing human antibodies (see, eg, PCT Application Publication WO 93/12227). ).

  In certain embodiments, certain strategies can be used to manipulate the unique properties of the antibody, such as the affinity of the antibody for its target. Such strategies include, but are not limited to, using site-specific or random mutagenesis of a polynucleotide molecule encoding the antibody to generate the antibody variant. In certain embodiments, such production is followed by screening for antibody variants that exhibit the desired change (eg, increased or decreased affinity).

  In certain embodiments, the amino acid residue targeted in the mutagenesis strategy is an amino acid residue in a CDR. In certain embodiments, amino acids in the framework regions of the variable domain are targeted. In certain embodiments, such framework regions have been shown to contribute to the target binding properties of certain antibodies. See, for example, Hudson, Curr. Opin. Biotech., 9: 395-402 (1999) and references therein.

  In certain embodiments, by restricting random or site-directed mutagenesis to hypermutation sites in the CDRs (sites corresponding to regions susceptible to mutation during the somatic affinity maturation process), A smaller and more effectively screened library of antibody variants is created. See, for example, "Chodhury & Pastan, Nature Biotech., 17: 568-572 (1999)" and references therein. In certain embodiments, identifying hypermutation sites such as, but not limited to, certain direct and inverted repeats, certain consensus sequences, certain secondary structures, and certain palindromes To do so, certain types of DNA elements can be used. For example, such DNA elements that can be used to identify hypermutation sites include pyrimidines (C or T) followed by a guanine (G) followed by a quaternary sequence containing purines (A or G). Subsequent, but not limited to, either adenosine or thymidine (A or T) (i.e., A / GGC / TA / T). Another example of a DNA element that can be used to identify hypermutation sites is the serine codon AGC / T.

Preparation of Fully Human ABPs (eg, Antibodies) In certain embodiments, phage display technology is used to generate monoclonal antibodies. In certain embodiments, such techniques produce fully human monoclonal antibodies. In certain embodiments, a polynucleotide encoding a single Fab or Fv antibody fragment is expressed on the surface of a phage particle. See, for example, Hoogenboom et al. , J .; Mol. Biol. , 227: 381 (1991); Marks et al. , J MoI Biol 222: 581 (1991); U.S. Patent No. 5,885,793. In certain embodiments, phage are "screened" to identify antibody fragments that have an affinity for a target. Thus, certain such processes mimic immunoselection through display of the antibody fragment repertoire on the surface of the filamentous bacteriophage and subsequent binding of the phage to the target by binding of the antibody fragment repertoire. In certain such approaches, 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 made by cloning the naturally rearranged human V gene from peripheral blood lymphocytes. . See, for example, "Mullinax et al, Proc Natl Acad Sci (USA), 87: 8095-8099 (1990)."

  According to certain embodiments, the antibodies of the invention are prepared through the use of transgenic mice that have a significant portion of the inserted human antibody-producing genome but lack the production of endogenous mouse antibodies. . Such mice are then capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of mouse immunoglobulin molecules and antibodies. The techniques used to achieve this result are disclosed in the patents, applications, and references disclosed herein. In certain embodiments, disclosed in PCT published application WO 98/24893 or "Mendez et al, Nature Genetics, 15: 146-156 (1997)", which is hereby incorporated by reference for all purposes. Methods such as the method performed can be used.

  Generally, fully human monoclonal ABPs (eg, antibodies) specific for PCSK9 can be made as follows. A transgenic mouse containing a human immunoglobulin gene is immunized with an antigen of interest (eg, PCSK9), and lymphocytes (such as B cells) are obtained from the mouse expressing the antibody. To prepare an immortalized hybridoma cell line, such harvested cells are fused with a myeloid cell line to identify a hybridoma cell line that produces antibodies specific to the antigen of interest. Such hybridoma cell lines are screened and selected. In certain embodiments, provided is the production of a hybridoma cell line that produces an antibody specific to PCSK9.

  In certain embodiments, fully human antibodies expose human splenocytes (B or T cells) to an antigen in vitro and then in an immunodeficient mouse (eg, SCID or nod / SCID). It is produced by reconstituting cells that have been lost. See, for example, "Brams et al, J. Immunol. 160: 2051-2058 (1998); Carballido et al., Nat. Med., 6: 103-106 (2000)". In certain such approaches, transplantation of human fetal tissue into SCID mice (SCID-hu) results in long-term hematopoiesis and growth of human T cells. See, for example, "McCune et al., Science, 241: 1532-139 (1988); Ifversen et al., Sem. Immunol., 8: 243-248 (1996)". In certain cases, the humoral immune response in such chimeric mice is dependent on the co-growth of human T cells in the animal. See, for example, Martensson et al., Immunol., 83: 1271-179 (1994). In one particular approach, human peripheral blood lymphocytes are transplanted into SCID mice. See, for example, Mosier et al., Nature, 335: 256-259 (1988). In certain such embodiments, higher such transplanted cells are treated when treated with either a stimulant, such as staphylococcal enterotoxin A (SEA), or with an anti-human CD40 monoclonal antibody. A level of B cell production is detected. See, for example, "Martensson et al., Immunol., 84: 224-230 (1995); Murphy et al, Blood, 86: 1946-1953 (1995)".

  Thus, in certain embodiments, fully human antibodies can be produced by expression of recombinant DNA in host cells or by expression in hybridoma cells. In other embodiments, antibodies can be produced using the phage display technology described herein.

  The antibodies described herein were prepared through the use of XenoMouse® technology, as described herein. Such mice are then capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of mouse immunoglobulin molecules and antibodies. The techniques used to achieve that are disclosed in the patents, applications, and references disclosed in the Background section of this specification. However, in particular, preferred embodiments of transgenic production of mice and antibodies derived therefrom are described in US patent application Ser. No. 08 / 759,620, filed Dec. 3, 1996 and published Jun. 11, 1998 International Patent Application Nos. WO 98/24893 and WO 00/76310 published December 21, 2000, the disclosures of which are incorporated herein by reference. See also "Mendez et al, Nature Genetics, 15: 146-156 (1997)", the disclosure of which is incorporated herein by reference.

  Through the use of such techniques, fully human monoclonal antibodies to various antigens have been produced. Basically, a mouse XenoMouse® strain is immunized with the antigen of interest (eg, PCSK9) and lymphocytes (such as B cells) are recovered from the highly immunized mouse, and the recovered lymphocytes are Is fused with a myeloid cell line to prepare an immortalized hybridoma cell line. These hybridoma cell lines are screened and selected to identify those that produce antibodies specific to the antigen of interest. Provided herein are methods for generating a plurality of hybridoma cell lines that produce antibodies specific to PCSK9. Further provided herein is the characterization of the antibodies produced by such cell lines, such as nucleotide and amino acid sequence analysis of the heavy and light chains of such antibodies.

  Generation of the XenoMouse® strain of mice is described in US patent application Ser. No. 07 / 466,008, filed Jan. 12, 1990, and Ser. No. 07 / 610,515, filed Nov. 8, 1990. No. 07 / 919,297 filed on Jul. 24, 1992, No. 07 / 922,649 filed on Jun. 30, 1992, and No. 08 / filed on Mar. 15, 1993. 031,801; 08 / 112,848 filed on August 27, 1993; 08 / 234,145 filed on April 28, 1994; filed on January 20, 1995. No. 08 / 376,279, 08 / 430,938 filed on Apr. 27, 1995, and 08 / 464,584 filed on Jun. 5, 1995, Jun. 5, 1995. Filed on Nos. 08 / 464,582, 08 / 463,191 filed Jun. 5, 1995, and 08 / 462,837, filed Jun. 5, 1995, Jun. 5, 1995. 08 / 486,853, filed on Jun. 5, 1995, 08 / 486,857, filed on Jun. 5, 1995, and 08 / 486,859, filed on Jun. 5, 1995. 08 / 462,513 filed on June 5, 1996; 08 / 724,752 filed on October 2, 1996; 08/759, filed on December 3, 1996; No. 620, U.S. Publication No. 2003/0093820, filed Nov. 30, 2001, and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, No. 6,075,181, No. 5,939,598 and Japanese Patent No. 3068180B2, the first 3068506B2, and are discussed in the first 3068507B2, is described. European Patent Application EP 0463151B1 published on June 12, 1996, International Patent Application WO 94/02602 published on February 3, 1994, International Patent Application WO 96/34096, 1998 published on October 31, 1996. See also WO 98/24893, published June 11, 2000, and WO 00/76310, published December 21, 2000. The entire disclosures of each of the above patents, applications, and references are incorporated herein by reference.

In another approach, GenPharm International, Inc. Others, such as, used the "minilocus" approach. In the minilocus approach, foreign Ig loci are mimicked through the inclusion of fragments (each gene) from the Ig locus. Thus, one or more _VH genes, one or more _DH genes, one or more _JH genes, a mu constant region, and usually a second constant region (preferably a gamma constant region) ) Are formed in the construct for insertion into animals. This approach is described in Surani et al. U.S. Pat. Nos. 5,545,807 and 5,545,806, 5,625,825, 5,625,126, and 5,633, respectively to Lonberg & Kay. No. 425, No. 5,661,016, No. 5,770,429, No. 5,789,650, No. 5,814,318, No. 5,877,397, No. Nos. 5,874,299 and 6,255,458; U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfort and Berns; Berns et al. U.S. Patent Nos. 5,612,205; 5,721,367 and 5,789,215; and U.S. Patents 5,643,763, 1990 to Choi & Dunn, and GenPharm. International U.S. Patent Application No. 07 / 574,748 filed on Aug. 29, 1990; 07 / 575,962 filed on Aug. 31, 1990; 07 filed on Dec. 17, 1991. No./810,279, filed on Mar. 18, 1992, No. 07 / 853,408, filed on June 23, 1992, No. 07 / 904,068, filed on Dec. 16, 1992. No. 07 / 05990,860 filed on Aug. 26, 1993, No. 08 / 053,131 filed on Jul. 26, 1993, No. 08 / 096,762 filed on Jul. 22, 1993, No. 08 / 155,301 filed on Nov. 18, 993, No. 08 / 161,739 filed on Dec. 3, 1993, and No. 08/165, filed Dec. 10, 1993. No. 699, 08 / 209,741, filed March 9, 1994, the disclosures of which are incorporated herein by reference. EP 0546073 B1, International Patent Applications WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, and See also U.S. Patent No. 5,981,175, the entire disclosure of which is incorporated herein by reference. 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 Tuailon et al. , (1995), Fishwild et al. (1996), the entire disclosures of which are incorporated herein by reference.

  Kirin has also shown the production of human antibodies from mice into which microcell fusions, large pieces of chromosomes, or entire chromosomes have been introduced. See European Patent Applications 773288 and 843961, the disclosures of which are incorporated herein by reference. In addition, KM ™ mice have been produced that are the result of crosses between Kirin's Tc mice and Medarex's minilocus (Humab) mice. These mice have the human IgH transchromosome of Kirin mice and the kappa chain transgene of Genpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4: 91-102).

  Human antibodies can also be derived by in vitro methods. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast display, and the like. .

In some embodiments, the antibodies described herein have human IgG4 and IgG2 heavy chains. Antibodies can also be of other human isotypes, including IgG1. Antibodies have high affinity, as measured by various techniques, and typically have a Kd of about 10 ⁻⁶ to about 10 ⁻¹³ M or less.

  As will be appreciated, the antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used to transform appropriate mammalian host cells. Transformation can be accomplished, for example, by packaging the polynucleotide into a virus (or into a viral vector) and then using the virus (or vector) or as described in U.S. Patent Nos. 4,399,216; 4,912,040; No. 4,740,461 and U.S. Pat. No. 4,959,455 (these patents are incorporated herein by reference). Obtained by any known method for introducing a polynucleotide into a host cell, including transducing a cell. The transformation technique used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, Includes encapsulation of polynucleotides in liposomes and direct microinjection of DNA into the nucleus.

  Mammalian cell lines that can be used as expression hosts are well known in the art and include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human liver Many immortalizations available from the American Type Culture Collection (ATCC), such as, but not limited to, cell carcinoma cells (eg, HepG2), human epithelial kidney 293 cells, and numerous other cell lines Cell lines. Particularly preferred cell lines can be selected through the determination of which cells have high expression levels and produce antibodies with constitutive PCSK9 binding properties.

In certain embodiments, the antibody and / or ABP is produced by at least one of the following hybridomas: 21B12, 31H4, 16F12, listed in Table 2 or disclosed in the Examples. Any other hybridoma. In certain embodiments, the antigen binding protein has a dissociation constant (K _D ) of less than about 1 nM, for example, 1000 pM-100 pM, 100 pM-10 pM, 10 pM-1 pM, and / or 1 pM-0.1 pM or less. To join.

  In certain embodiments, the antigen binding protein comprises an immunoglobulin molecule of at least one of the following types: IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM. In certain embodiments, the antigen binding protein comprises a human kappa light chain and / or a human heavy chain. In certain embodiments, the heavy chain is a heavy chain of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, the antigen binding protein has been cloned for expression in a mammalian cell. In certain embodiments, the antigen binding protein comprises a constant region other than any of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotype constant regions.

  In certain embodiments, the antigen binding protein comprises a human λ light chain and a human IgG2 heavy chain. In certain embodiments, the antigen binding protein comprises a human λ light chain and a human IgG4 heavy chain. In certain embodiments, the antigen binding protein comprises a human lambda light chain and a human IgG1, IgG3, IgE, IgA, IgD, or IgM heavy chain. In another embodiment, the antigen binding protein comprises a human kappa light chain and a human IgG2 heavy chain. In certain embodiments, the antigen binding protein comprises a human kappa light chain and a human IgG4 heavy chain. In certain embodiments, the antigen binding protein comprises a human kappa light chain and a human IgG1, IgG3, IgE, IgA, IgD, or IgM heavy chain. In certain embodiments, the antigen binding protein comprises an antibody variable region linked to a constant region that is neither a constant region for the IgG2 isotype nor a constant region for the IgG4 isotype. In certain embodiments, the antigen binding protein has been cloned for expression in a mammalian cell.

  In certain embodiments, the conservative modifications to the heavy and light chains (and corresponding modifications to the encoding nucleotides) of the antibodies obtained from at least one of the hybridoma strains: 21B12, 31H4, and 16F12 are: Hybridoma strains: will produce antibodies to PCSK9 with similar functional and chemical properties to those obtained from 21B12, 31H4, and 16F12. In addition, certain other modifications (and corresponding modifications to the encoding nucleotides) to the heavy and light chains of the antibodies obtained from at least one of the hybridoma strains: 21B12, 31H4, and 16F12 are hybridoma strains. : 21B12, 31H4, and 16F12 (e.g., SEQ ID NOs: 592-593, etc.) will produce antibodies to PCSK9 that have similar functional and chemical properties as the antibodies obtained. In contrast, in certain embodiments, a substantial modification of the functional and / or chemical properties of the antibody to PCSK9 comprises: (a) the structure of the molecular backbone in the region of substitution (eg, sheet or helical) Substitutions in the amino acid sequences of the heavy and light chains that differ significantly from (b) the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining the bulk of the side chains. Can be achieved by:

  For example, a `` conservative amino acid substitution '' refers to the replacement of a natural amino acid residue with a non-natural residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. May be included. In addition, any natural residue in the polypeptide can be replaced with alanine, as described previously for "alanine scanning mutagenesis."

  Desired amino acid insertions or substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such insertions or substitutions are desired. In certain embodiments, amino acid substitutions are used to identify key residues of the antibody to PCSK9, or to increase or decrease the affinity of the antibody for PCSK9, as described herein. be able to.

  In certain embodiments, the antibodies of the invention can be expressed in cell lines other than hybridoma cell lines. In certain embodiments, sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell. According to certain embodiments, transformation comprises packaging the polynucleotide into the host cell by any known method, for example, packaging the polynucleotide into a virus (or into a viral vector), Host cells are transduced with the virus (or vector) or are disclosed in U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 ( These patents can be made for any purpose, such as by transduction acquisition methods known in the art, as exemplified in (hereby incorporated by reference). In certain embodiments, the transformation technique used may depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, , And direct microinjection of DNA into the nucleus.

  Mammalian cell lines that can be used as expression hosts are well known in the art and include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human liver Includes many immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, cell carcinoma cells (eg, HepG2), and numerous other cell lines But not limited to these. In certain embodiments, cell lines can be selected through determining which cell lines have high expression levels and produce antibodies with constitutive HGF binding properties. Expression vectors suitable for mammalian host cells are well known.

  In certain embodiments, the antigen binding protein comprises one or more polypeptides. In certain embodiments, any of a variety of expression vector / host systems may be used to express a polynucleotide molecule that encodes a polypeptide comprising one or more ABP components or ABP itself. Such systems include microorganisms such as bacteria transformed with a recombinant bacteriophage, plasmid, or cosmid DNA expression vector; yeast transformed with a yeast expression vector; infected with a viral expression vector (eg, baculovirus). An insect cell line; a plant cell line transfected with a viral expression vector (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with a bacterial expression vector (eg, Ti or pBR322 plasmid); Or animal cell lines, including, but not limited to.

  In certain embodiments, a polypeptide comprising one or more ABP components or ABP itself is recombinantly expressed in yeast. Certain such embodiments use a commercially available expression system, eg, Pichia Expression System (Invitrogen, San Diego, CA), according to the manufacturer's instructions. In certain embodiments, such a system relies on a prepro-α sequence to induce secretion. In certain embodiments, transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction with methanol.

  In certain embodiments, a secreted polypeptide comprising one or more ABP components or ABP itself is purified from a yeast growth medium. In certain embodiments, the method used to purify the polypeptide from the yeast growth medium is the same as the method used to purify the polypeptide from bacterial and mammalian cell supernatants.

  In certain embodiments, a nucleic acid encoding a polypeptide comprising one or more ABP components or the ABP itself is cloned into a baculovirus expression vector, such as pVL1393 (PharMingen, San Diego, CA). In certain embodiments, such vectors are used according to the manufacturer's instructions (PharMingen) to infect Spodoptera frugiperda cells and produce recombinant polypeptides in media without the sF9 protein. Can be. In certain embodiments, polypeptides are purified and concentrated from such media using a heparin-Sepharose column (Pharmacia).

  In certain embodiments, a polypeptide comprising one or more ABP components or 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 calforica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. In certain embodiments, a nucleic acid molecule encoding a polypeptide can be inserted into a non-essential gene of a virus (eg, within a polyhedrin gene) and placed under the control of a promoter for that gene. In certain embodiments, successful insertion of a nucleic acid molecule will render non-essential genes inactive. In certain embodiments, the inactivation results in a detectable characteristic. For example, inactivation of the polyhedrin gene results in the production of a virus lacking coat protein.

  In certain embodiments, the S.D. Recombinant virus can be used to infect frugiperda cells or Trichoplusia larvae. See, for example, "Smith et al., J. Virol., 46: 584 (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA) 91: 3224-7 (1994)".

In certain embodiments, a polypeptide comprising one or more ABP components or ABP itself, produced in bacterial cells, is produced as insoluble inclusion bodies in bacteria. In certain embodiments, host cells containing such inclusion bodies are collected by centrifugation, washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA, and 0.1 mg / mL lysozyme (Sigma). , St. Louis, MO) for 15 minutes at room temperature. In certain embodiments, the lysate is cleaned by sonication and the cell lysate is sedimented by centrifugation at 12,000 × g for 10 minutes. In certain embodiments, the pellet containing the polypeptide is resuspended in 50 mM Tris, pH 8 and 10 mM EDTA; layered on 50% glycerol and centrifuged at 6000 × g for 30 minutes. In certain embodiments, the pellet can be resuspended in standard phosphate buffered saline (PBS) without Mg ⁺⁺ and Ca ⁺⁺ . In certain embodiments, the polypeptide is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (see, eg, Sambrook et al., Supra). In certain embodiments, such gels can be soaked in 0.4 M KCl to visualize proteins, and the soaked gels can be cut out and run in gel running buffer lacking SDS. Can be electroeluted. According to certain embodiments, a glutathione-S-transferase (GST) fusion protein is produced in bacteria as a soluble protein. In certain embodiments, such a GST fusion protein is purified using a GST purification module (Pharmacia).

  In certain embodiments, it may be desirable to “refold” certain polypeptides, eg, polypeptides that include one or more ABP components or the ABP itself. In certain embodiments, such polypeptides are produced using certain recombinant systems discussed herein. In certain embodiments, the polypeptide is “refolded” and / or “oxidized” to form a desired tertiary structure and / or create disulfide bonds. In certain embodiments, such structures and / or linkages are associated with some biological activities of the polypeptide. In certain embodiments, refolding is accomplished using any of a number of techniques known in the art. A typical method includes, but is not limited to, exposing the solubilized polypeptide agent to a pH typically above 7 in the presence of a chaotropic agent. A typical chaotropic agent is guanidine. In certain embodiments, the refolding / oxidation solution also contains a reducing agent and an oxidized form of the reducing agent. In certain embodiments, the reducing agent and its oxidized form are present in a ratio that can produce a particular redox potential that allows disulfide shuffling to occur. In certain embodiments, such shuffling allows for the formation of cysteine bridges. Typical redox couples include, but are not limited to, cysteine / cystamine, glutathione / dithiobis GSH, cupric chloride, dithiothreitol DTT / dithiane DTT, and 2-mercaptoethanol (bME) / dithio-bME. Not limited. In certain embodiments, co-solvents are used to increase the efficiency of refolding. Typical co-solvents include, but are not limited to, glycerol, polyethylene glycols of various molecular weights, and arginine.

  In certain embodiments, a polypeptide comprising one or more ABP components or ABP itself is substantially purified. Certain protein purification techniques are known to those skilled in the art. In certain embodiments, protein purification comprises crude fractionation of a polypeptide fraction from a non-polypeptide fraction. In certain embodiments, the polypeptide is purified using chromatography and / or electrophoresis techniques. Typical purification methods include precipitation with ammonium sulfate, precipitation with PEG, immunoprecipitation, centrifugation after heat denaturation, chromatography (affinity chromatography (eg, Protein-A-Sepharose), ion exchange chromatography, exclusion chromatography, Including, but not limited to, chromatography and reverse phase chromatography), gel filtration, hydroxylapatite chromatography, isoelectric focusing, polyacrylamide gel electrophoresis, and combinations of such and other techniques. But not limited to these. In certain embodiments, the polypeptide is purified by high performance protein liquid chromatography or high pressure liquid chromatography (HPLC). In certain embodiments, the purification steps can be varied or some steps can be omitted, but still provide a method suitable for the preparation of a substantially purified polypeptide.

  In certain embodiments, the purity of the polypeptide preparation is quantified. Certain methods for quantifying the degree of purification are known to those skilled in the art. Certain exemplary methods include, but are not limited to, measuring the specific binding activity of a preparation and assessing the amount of polypeptide in the preparation by SDS / PAGE analysis. Absent. One particular method for assessing the amount of purification of a polypeptide preparation involves calculating the binding activity of the preparation and comparing it to the binding activity of the original extract. In certain embodiments, the results of such calculations are expressed as "fold purifications." The unit used to represent the amount of binding activity depends on the particular assay being performed.

  In certain embodiments, a polypeptide comprising one or more ABP components or ABP itself is partially purified. In certain embodiments, partial purification can be achieved by using fewer purification steps or by using different forms of the same general purification scheme. For example, in certain embodiments, cation exchange column chromatography performed using an HPLC apparatus may generally result in a greater "fold purification" than the same technique using a low pressure chromatography system. In certain embodiments, methods that result in a lower degree of purification may have advantages over total recovery of the polypeptide or maintaining polypeptide binding activity.

  In certain cases, the electrophoretic migration of a polypeptide may vary (sometimes significantly) with different conditions of SDS / PAGE. See, for example, Capaldi et al., Biochem. Biophys. Res. Comm., 76: 425 (1977). It is understood that under different electrophoretic conditions, the apparent molecular weight of the purified or partially purified polypeptide may vary.

Exemplary Epitopes Provided are epitopes to which the anti-PCSK9 antibodies useful in the methods provided herein bind. In some embodiments, the epitopes bound by the antibodies disclosed herein are particularly useful. In some embodiments, antigen binding proteins that bind to any of the epitopes bound by the antibodies described herein are useful. In some embodiments, epitopes bound by any of the antibodies listed in Table 2 and FIGS. 2 and 3 are particularly useful. In some embodiments, the epitope is on the catalytic domain PCSK9.

  In some embodiments, the antigen binding proteins disclosed herein specifically bind to an N-terminal prodomain, a subtilisin-like catalytic domain, and / or a C-terminal domain. In some embodiments, the antigen binding protein binds to the substrate binding groove of PCSK-9 (described in Cunningham et al., Incorporated herein by reference in its entirety).

  In some embodiments, the domain / region containing residues that are in contact with or buried by the antibody mutates certain residues in PCSK9 (eg, a wild-type antigen) and the antigen binding protein It can be identified by measuring whether it can bind to a mutated or variant PCSK9 protein. By creating a number of individual mutations, residues that play a direct role in binding or residues that are sufficiently close to the antibody so that the mutations can affect the binding between the antigen-binding protein and the antigen. The group can be identified. From the knowledge of these amino acids, one can elucidate the domain or region of the antigen that contains the residues that contact the antigen binding protein or that are covered by the antibody. Such a domain can include a binding epitope of an antigen binding protein. One example of this general approach uses an arginine / glutamic acid scanning protocol (eg, Naneviczs, T., et al., 1995, J. Biol. Chem., 270: 37, 21719-21625 and Zupnick). , A., et al, 2006, J. Biol. Chem., 281: 29, 20644-20473). In general, arginine and glutamic acid are charged and bulky, and therefore have the potential to disrupt the binding between the antigen binding protein and the antigen in the region of the mutated antigen. Amino acids in the wild-type polypeptide are substituted (typically individually) for arginine and glutamic acid. Arginine present in the wild-type antigen is replaced with glutamic acid. A variety of such individual variants are obtained and the collected binding results are analyzed to determine which residues affect binding.

Altered binding (e.g., reduced or increased) between the antigen binding protein and the variant PCSK9 used herein (e.g., the Biacore test or bead-based assays described below in the Examples) known as measured by the method) binding affinity, such as a change in EC _50, and / or (e.g., become apparent by a decrease of Bmax in a plot of the antigen binding protein concentrations against the original concentration) the total binding of the antigen binding protein It means that there is a change in performance (eg, decline). Significant changes in binding suggest that the mutated residue is directly involved in binding to the antigen binding protein or is close to the binding protein when the binding protein is bound to the antigen.

  In some embodiments, a significant decrease in binding is due to the fact that the binding affinity, EC50, and / or ability between the antigen binding protein and the mutant PCSK9 antigen increases when the antigen binding protein and wild-type PCSK9 (eg, SEQ ID NO: 1 and And / or greater than 10%, greater than 20%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70% as compared to binding between , More than 75%, more than 80%, more than 85%, more than 90%, or more than 95%. In certain embodiments, binding is reduced below a detectable limit. In some embodiments, the binding of the antigen binding protein to the variant PCSK9 protein is 50% of the binding observed between the antigen binding protein and the wild-type PCSK9 protein (eg, the protein of SEQ ID NO: 1 and / or SEQ ID NO: 303). % (E.g., less than 40%, 35%, 30%, 25%, 20%, 15%, or 10%) demonstrates a significant decrease in binding. Such binding measurements can be performed using various binding assays known in the art.

  In some embodiments, an antigen binding protein that exhibits significantly lower binding to a variant PCSK9 protein in which residues in the wild-type PCSK9 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 303) have been replaced with arginine or glutamic acid. Is provided. In some embodiments, the following mutations are compared to the wild-type PCSK9 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 303): R207E, D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, Any one or more of A413R, E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 244) has significantly reduced or increased binding of the antigen binding protein to the variant PCSK9 protein. In abbreviated notation used herein, the format is: wild type residue: position in polypeptide: variant residue, with residues as shown in SEQ ID NO: 1 or SEQ ID NO: 303. Numbering has been done.

  In some embodiments, compared to a wild-type PCSK9 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 303), the following positions as shown in SEQ ID NO: 207, 208, 185, 181, 439, One or more of 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 (e.g., 1, The binding of the antigen binding protein to the mutant PCSK9 protein having the mutation (2, 3, 4, 5, or more) is reduced or increased. In some embodiments, compared to a wild-type PCSK9 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 303), the following positions as shown in SEQ ID NO: 207, 208, 185, 181, 439, One or more of 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521 and 554 (e.g., (3, 4, 5, or more) mutant PCSK9 proteins with reduced or increased binding of the antigen binding protein. In some embodiments, the following positions in SEQ ID NO: 207, 208, 185, 181, 439, 513, 538, 539 as compared to a wild-type PCSK9 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 303). , 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 (e.g., 1, 2, 3, 4 The binding of the antigen binding protein to the mutant PCSK9 protein having a mutation (5, 5 or more) is substantially reduced or increased.

  In some embodiments, the following mutations relative to the wild-type PCSK9 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 303): R207E, D208R, R185E, R439E, E513R, V538R within SEQ ID NO: 1 or SEQ ID NO: 303. , E539R, T132R, S351R, A390R, A413R, E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and one or more of Q554R (eg, 1, 2, 3, 4, 5, etc.), the binding of ABP to the mutant PCSK9 protein is significantly reduced or increased.

  In some embodiments, the following mutations relative to the wild-type PCSK9 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 303): R207E, D208R, R185E, R439E, E513R, V538R within SEQ ID NO: 1 or SEQ ID NO: 303. , E539R, T132R, S351R, A390R, A413R, and a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, etc.) of E582R significantly reduced ABP binding or To increase. In some embodiments, binding is reduced. In some embodiments, reduced binding is observed as a change in EC50. In some embodiments, the change in EC50 is an increase in the EC50 value (and thus a decrease in binding).

  In some embodiments, the following mutations are compared to wild-type PCSK9 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 303): D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R in SEQ ID NO: 1. , R519E, H521R, and Q554R have significantly reduced or increased binding of ABP to a mutant PCSK9 protein having one or more (eg, 1, 2, 3, 4, 5, etc.). In some embodiments, binding is reduced. In some embodiments, reduced binding is observed as a change in Bmax. In some embodiments, the shift in Bmax is a reduction in the maximum signal caused by ABP. In some embodiments, for amino acids that are part of an epitope, the Bmax is reduced by at least 10%, for example, by the following amounts: 20, 30, 40, 50, 60, 70, 80, 90, 85 , 98, 99, or 100%, in some embodiments, may indicate that the residue is part of an epitope.

  Although the variant forms listed immediately above are referred to with respect to the wild-type sequence shown in SEQ ID NO: 1 or SEQ ID NO: 303, it is understood that in the allelic variants of PCSK9, the amino acids at the indicated positions may differ. You. Antigen binding proteins that exhibit significantly lower binding to such allelic forms of PCSK9 are also envisioned. Thus, in some embodiments, any of the above embodiments can be compared to an allelic sequence rather than the purely wild-type sequence shown in FIG. 1A.

  In some embodiments, for a variant PCSK9 protein in which a residue at a selected position in a wild-type PCSK9 protein has been mutated to any other residue, the binding of the antigen binding protein is significantly reduced. In some embodiments, for the specified positions, arginine / glutamic acid substitutions described herein are used. In some embodiments, alanine is used for the location specified.

  As described above, residues directly involved in binding or covered by the antigen binding protein can be identified from scanning results. Thus, these residues may provide an indication of the domain or region of SEQ ID NO: 1 (or SEQ ID NO: 303 or SEQ ID NO: 3) that contains the binding region to which the antigen binding protein binds. In some embodiments, the antigen binding protein comprises amino acids 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162 of SEQ ID NO: 1 or SEQ ID NO: 303. Binds to a domain containing at least one of 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554. In some embodiments, the antigen binding protein comprises amino acids 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162 of SEQ ID NO: 1 or SEQ ID NO: 303. 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 bind to a region containing at least one of them.

  In some embodiments, the antigen binding protein binds to a region containing at least one of amino acids: 162, 164, 167, 207, and / or 208 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, two or more (eg, 2, 3, 4, or 5) of the identified residues are part of a region bound by ABP. In some embodiments, ABP competes with ABP21B12.

  In some embodiments, the antigen binding protein binds to a region containing at least one of amino acids 185 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, ABP competes with ABP31H4.

  In some embodiments, the antigen binding protein binds to a region containing at least one of the amino acids 439, 513, 538, and / or 539 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, two or more (eg, 2, 3, or 4) of the identified residues are part of a region bound by ABP. In some embodiments, ABP competes with ABP31A4.

  In some embodiments, the antigen binding protein comprises at least one of the following amino acids: 123, 129, 311, 313, 337, 132, 351, 390, and / or 413 of SEQ ID NO: 1 or SEQ ID NO: 303. To the area you want. In some embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) of the identified residues are part of a region bound by ABP. . In some embodiments, ABP competes with ABP12H11.

  In some embodiments, the antigen binding protein binds to a region containing at least one of the amino acids 582, 519, 521, and / or 554 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, two or more (eg, 2, 3, or 4) of the identified residues are part of a region bound by ABP. In some embodiments, ABP competes with ABP3C4.

  In some embodiments, the antigen binding protein binds to the region within the fragment or full length sequence of SEQ ID NO: 1 or SEQ ID NO: 303. In another embodiment, the antigen binding protein binds to a polypeptide consisting of these regions. The notation "SEQ ID NO: 1 or SEQ ID NO: 303" indicates that one or both of these sequences may be used or may be appropriate. This term does not imply that only one should be used.

  As mentioned above, the above description refers to specific amino acid positions with reference to SEQ ID NO: 1. However, throughout this specification, reference is made to the Pro / Cat domain starting at position 31 as set forth in SEQ ID NO: 3. As described below, SEQ ID NO: 1 and SEQ ID NO: 303 lack the PCSK9 signal sequence. Therefore, any comparison between these various disclosures should take into account this difference in numbering. In particular, any amino acid position in SEQ ID NO: 1 corresponds to the amino acid at position 30 in the protein of SEQ ID NO: 3. For example, position 207 of SEQ ID NO: 1 corresponds to position 237 of SEQ ID NO: 3 (full-length sequence and generally the numbering system used herein). Table 39.6 summarizes how the above positions with reference to SEQ ID NO: 1 (and / or SEQ ID NO: 303) correspond to SEQ ID NO: 3 (including the signal sequence). Thus, any of the above embodiments described with respect to SEQ ID NO: 1 (and / or SEQ ID NO: 303) are described with reference to SEQ ID NO: 3 by the corresponding positions described.

  In some embodiments, ABP21B12 binds to an epitope comprising residues 162-167 (eg, residues D162-E167 of SEQ ID NO: 1). In some embodiments, ABP12H11 binds to an epitope comprising residues 123-132 (eg, S123-T132 of SEQ ID NO: 1). In some embodiments, ABP12H11 binds to an epitope comprising residues 311 to 313 (eg, A311 to D313 of SEQ ID NO: 1). In some embodiments, the ABP may bind to an epitope comprising any one of these chains of the sequence.

In another aspect, there is provided an antigen binding protein that competes with one of the exemplified antibodies or functional fragments that binds to an epitope described herein for specific binding to PCSK9. You. Such an antigen binding protein may also bind to the same or overlapping epitope as one of the antigen binding proteins exemplified herein. Antigen binding proteins and fragments that compete with or bind to the same epitope as the exemplified antigen binding proteins are expected to exhibit similar functional properties. Exemplary antigen binding proteins and fragments include those described above, such as those having the heavy and light chain variable region domains and the CDRs contained in Table 2 and / or FIGS. Thus, as a specific example, provided antigen binding proteins include:
(A) all six of the CDRs listed for the antibodies listed in FIGS. 2-3;
(B) VH and VL listed for the antibodies listed in Table 2; or (c) two light chains and two heavy chains specified for the antibodies listed in Table 2. Includes those that compete with the antibody or antigen binding protein having a chain.

Therapeutic Pharmaceutical Formulations and Administration Provided herein are pharmaceutical formulations comprising an antigen binding protein to PCSK9 useful in the methods described above. As used herein, “pharmaceutical formulation” refers to parenteral administration to a patient in need thereof (including intravenous, intramuscular, subcutaneous, spray, intrapulmonary, intranasal, or intrathecal) A sterile composition of a pharmaceutically active drug suitable for (but not limited to), ie, at least one antigen binding protein to PCSK9, which was considered safe by the U.S. Food and Drug Administration or other foreign national authority, Only pharmaceutically acceptable excipients, diluents and other additives are included. Pharmaceutical formulations include liquids, for example, aqueous solutions which can be administered directly, lyophilized powders which can be reconstituted into a solution by adding a diluent prior to administration. Specifically excluded from the scope of the term "pharmaceutical formulation" are compositions for topical administration to a patient, compositions for oral ingestion, and compositions for parenteral nutrition.

  In certain embodiments, the pharmaceutical formulation is a stable pharmaceutical formulation. As used herein, the phrase “stable pharmaceutical formulation,” “stable formulation,” or “stable pharmaceutical formulation” refers to at least one month, or two months, or three months, or Biologically exhibiting no more than 5% increase in aggregation and / or loss of biological activity when stored at 2-8 ° C. for 6 months, or 1 year, or 2 years Refers to a pharmaceutical formulation of the active protein. The stability of the formulation may be determined by size exclusion HPLC ("SEC-HPLC"), cation exchange HPLC (CEX-HPLC), detection of invisible particulates by light shading ("HIAC"), and / or visual inspection (but , And can be readily determined by one of skill in the art using any number of standard assays, including, but not limited to,

  In certain embodiments, the pharmaceutical formulation comprises one or more heavy chain complementarity determining regions (CDRHs), as shown in Table 2 and FIGS. 2 and / or 3 and FIGS. And any antigen binding protein to PCSK9, including one or more light chain complementarity determining regions (CDRLs). In certain other embodiments, the pharmaceutical formulation comprises an amino acid that is at least 90% identical to the amino acid sequence of the antigen binding protein for PCSK9, as illustrated in Table 2 and FIGS. 2 and / or 3 and FIGS. 31A and 31B. An amino acid sequence that is at least 90% identical to the amino acid sequence of any antigen binding protein to PCSK9, as shown in Table 2 and FIGS. 2 and / or 3 and FIGS. 31A and 31B. And any heavy chain variable region, including any antigen binding protein to PCSK9. In yet other embodiments, the pharmaceutical formulation comprises any of the antigen binding proteins for PCSK9 illustrated in Table 2 and FIGS. 2 and / or 3 and FIGS. 31A and 31B. In certain other embodiments, the pharmaceutical formulation may include other antigen binding proteins to PCSK9, ie, an antibody consisting of the light chain variable domain of SEQ ID NO: 588 and the heavy chain variable domain of SEQ ID NO: 589. In some embodiments, the pharmaceutical formulation comprises any one of 21B12, 26H5, 31H4, 8A3, 11F1, or 8A1.

  In some embodiments, the pharmaceutical formulation comprises two or more different antigen binding proteins to PCSK9. In certain embodiments, the pharmaceutical formulation comprises two or more antigen binding proteins for PCSK9 that bind to two or more epitopes. In some embodiments, the various antigen binding proteins do not compete with each other for binding to PCSK9. In some embodiments, any of the antigen binding proteins illustrated in Table 2 and FIGS. 2 and / or 3 can be combined together in a pharmaceutical formulation.

  In certain embodiments, the antigen binding protein and / or therapeutic molecule for PCSK9 is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, polyethylene glycol, glycogen (eg, glycosylation of ABP), and dextran. Such vehicles are described, for example, in US patent application Ser. No. 09 / 428,082 (currently US Pat. No. 6,660,843) and published PCT application WO 99/25044, which are incorporated by reference for all purposes. , Incorporated herein by reference.

  In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed. In some embodiments, the formulation material is for subcutaneous and intravenous administration. In certain embodiments, the pharmaceutical formulation modifies, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release, rate of adsorption or penetration of the composition Formulation material to store, maintain, or retain.

In certain embodiments, suitable formulation materials include amino acids (such as proline, arginine, lysine, methionine, taurine, glycine, glutamine, or asparagine), antimicrobial agents, antioxidants (ascorbic acid, sodium sulfite, or sulfite). Sodium hydrogen), buffers (borate, bicarbonate, sodium phosphate (“NaOAC”), Tris-HCl, Tris buffer, citrate, phosphate buffer, phosphate buffered saline (ie, PBS buffer) or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (caffeine, polyvinylpyrrolidone, β-cyclodextrin, or Hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and Other carbohydrates (such as glucose, sucrose, fructose, lactose, mannose, trehalose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulin), proteins (such as serum albumin, gelatin, or immunoglobulin), coloring agents, Flavoring agents and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, Methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide, etc., solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or Bitols, etc., suspending agents, surfactants or wetting agents (pluronics, PEG, sorbitan esters, polysorbates 20, such as polysorbate 20, polysorbate 80, tritons, tromethamine, lecithin, cholesterol, tyloxapearl, etc.), stability enhancers (sucrose or Such as sulobitol), tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical auxiliaries. Not limited. ^{(Remington's Pharmaceutical Sciences, 18 th} Edition, A.R.Gennaro, ed., Mack Publishing Company (1995).

  In certain embodiments, the optimal pharmaceutical formulation will be determined by one skilled in the art, for example, depending on the intended route of administration, mode of delivery, and desired dosage. See, for example, "Remington's Pharmaceutical Sciences, supra." In certain embodiments, such formulations can affect 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 a high concentration of an antigen binding protein for PCSK9. In certain embodiments, the ABP concentration ranges from about 70 mg / ml to about 250 mg / ml, for example, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 100 mg / ml. ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml, about 210 mg / ml ml, about 220 mg / ml, about 230 mg / ml, about 240 mg / ml, or about 250 mg / ml, and all values in between. In some embodiments, the concentration of 21B12, 26H5, or 31H4 ranges from about 100 mg / ml to about 150 mg / ml, for example, 100 mg / ml, about 100 mg / ml, about 120 mg / ml, about 130 mg / ml. ml, about 140 mg / ml, or about 150 mg / ml. In some embodiments, the concentration of 8A3, 11F1, or 8A1 ranges from about 140 mg / ml to about 220 mg / ml, for example, 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml. 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 embodiment, the pharmaceutical formulation comprises, for example, sodium acetate, sodium chloride, phosphate, phosphate buffered saline ("PBS"), and / or Tris buffer at about pH 7.0 to 8.5. At least one buffer is included. Buffering agents help maintain a physiologically relevant pH. In addition, buffers serve to enhance the isotonicity and chemical stability of the pharmaceutical formulation. In certain embodiments, the buffering agent ranges from about 0.05 mM to about 40 mM, for example, about 0.05 mM, about 0.1 mM, about 0.5 mM, about 1.0 mM, about 5.0 mM, A buffer comprising 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 100 nM, and all values in between. In certain embodiments, the buffer is NaOAC. Typical pH of the pharmaceutical formulation includes about 4 to about 6, or about 4.8 to about 5.8, or about 5.0 to about 5.2, or about 5, or about 5.2. .

  In certain embodiments, the pharmaceutical formulation ranges from about 250 to about 350 mOsmol / kg, for example, about 250 mOsm / kg, about 260 mOsm / kg, about 270 mOsm / kg, about 280 mOsm / kg, about 290 mOsm / kg. About 300 mOsm / kg, about 310 mOsm / kg, about 320 mOsm / kg, about 330 mOsm / kg, about 340 mOsm / kg, or about 350 mOsm / kg, and isotonic with an osmolality including all values in between. As used herein, "osmolarity" is a measure of the ratio of solute to volume fluid. In other words, the number of molecules and ions (or molecules) per kilogram of solution. Osmolality may be measured with an analytical instrument called an osmometer, such as the Advanced Instruments 2020 multi-sample osmometer, Norwood, MA. The Advanced Instruments 2020 multi-sample osmometer measures osmolality using the freezing point depression method. The higher the osmolyte in the solution, the lower the temperature at which the solution freezes. Osmolality may be measured using any other method and in any other unit known in the art, such as linear extrapolation.

  In yet 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 in an amount ranging from about 0.004% to about 10% by weight of the formulation ("w / v"), for example, about 0.004% of the formulation. %, About 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, At a concentration of about 0.5%, about 1%, about 5%, or about 10% w / v. In certain embodiments, the pharmaceutical formulation comprises polysorbate 80 at a concentration ranging from about 0.004% to about 0.1% w / v of the formulation. In certain embodiments, the pharmaceutical formulation comprises polysorbate 20 at a concentration ranging from about 0.004% to about 0.1% w / v of the formulation.

  In certain embodiments, the pharmaceutical formulation comprises a polyhydroxy hydrocarbon (including, but not limited to, sorbitol, mannitol, glycerol, and dulcitol), and / or a disaccharide (including sucrose, lactose, maltose, and trehalose). And / or amino acids (including, but not limited to, proline, arginine, lysine, methionine, and taurine), and / or at least one stabilizing agent such as benzyl alcohol. The total amount of hydroxy hydrocarbons, and / or disaccharides, and / or amino acids, and / or about 0.5% to about 10% w / v of the formulation, such as benzyl alcohol. In certain embodiments, the pharmaceutical formulation comprises a stabilizing agent comprising about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8% of sucrose, Contains at a concentration of about 9%, or about 10%. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of about 5% of sucrose. In certain embodiments, the pharmaceutical formulation comprises a stabilizing agent comprising about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8% of sorbitol, Contains at a concentration of about 9%, or about 10%. In certain embodiments, the pharmaceutical formulation comprises a stabilizing agent at a concentration of about 9% of sorbitol. In certain embodiments, the pharmaceutical formulation comprises a stabilizing agent at a concentration of about 1%, about 2%, about 3%, about 4%, about 5% of proline, arginine, lysine, methionine, and / or taurine. Including. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of about 2-3% of proline. In certain embodiments, the pharmaceutical formulation comprises a stabilizing agent at a concentration of about 1%, about 2%, about 3%, about 4%, about 5% of benzyl alcohol. In certain embodiments, the pharmaceutical formulation comprises the stabilizer at a concentration of about 1-2% of benzyl alcohol.

  In one aspect, the pharmaceutical formulation has a viscosity level of less than about 30 centipoise (cP) when measured at room temperature (ie, 25C). As used herein, "viscosity" is the flow resistance of a fluid, which may be measured in centipoise (cP) or millipascal seconds (mPa-s), where a given shear rate And 1 cP = 1 mPa-s. The viscosity may be measured using a viscometer, for example, a Brookfield Engineering Dial Reading Viscometer model LVT. Viscosity may be measured using any other method in any other unit known in the art (eg, 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 embodiment, the pharmaceutical formulation is as measured by at least one stability assay known to those of skill in the art, such as an assay that examines the biophysical or biochemical characteristics of a biologically active protein over time. It is stable. As mentioned above, the stable pharmaceutical formulations of the present invention are stored at 2-8 ° C. for at least one month, or two months, or three months, or six months, or one year, or two years. , A pharmaceutical formulation of a biologically active protein that exhibits no more than 5% increase in aggregation and / or loss of biological activity as compared to a control formulation sample. In certain embodiments, the stability of the pharmaceutical formulation is measured using size exclusion HPLC ("SEC-HPLC"). SEC-HPLC separates proteins based on differences in hydrodynamic volume. Molecules with large hydrodynamic protein volumes elute faster than molecules with small volumes. For SEC-HPLC, a stable pharmaceutical formulation should exhibit no more than about 5% increase in high molecular weight species compared to the control sample. In certain other embodiments, the pharmaceutical formulation has no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, no more than about 0.1%, as compared to a control sample. It should exhibit an increase in high molecular weight species not exceeding 5%.

  In certain embodiments, the stability of the pharmaceutical formulation is measured using cation exchange HPLC (CEX-HPLC). CEX-HPLC separates proteins based on differences in surface charge. At a given pH, the charged isoform of anti-PCSK9 ABP was separated on a cation exchange column and eluted using a salt gradient. The eluate is monitored by UV absorbance. The charged isoform distribution is evaluated by determining the peak area of each isoform as a percentage of the total peak area. For CEX-HPLC, a stable pharmaceutical formulation should exhibit no more than about 5% reduction in the isoform major peak compared to the control sample. In certain other specific embodiments, a stable pharmaceutical formulation should exhibit a reduction in the major isoform peak of about 3% to about 5% or less compared to a control sample. In certain embodiments, the pharmaceutical formulation has a major isoform reduction of about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less as compared to a control sample. Should be presented.

  In certain embodiments, the stability of a pharmaceutical formulation is measured using light shielding ("HIAC") to detect invisible microparticles. An electronic liquid particle counting system (HIAC / Royco 9703 or equivalent) that includes a light-shielded sensor (HIAC / Royco HRLD-150 or equivalent) with a liquid collection instrument can determine the number and size range of particles in a given test sample. Quantify. As the particles in the liquid pass between the light source and the detector, they reduce or "blurr" the luminous flux impinging on the detector. If the concentration of the particles is within the normal range of the sensor, these particles will be detected one by one. The passage of each particle through the detection area reduces the light incident on the photodetector, and the voltage output of the photodetector decreases instantaneously. The change in voltage is registered as an electrical pulse and converted to the number of particles present by the instrument. The method is non-specific and measures particles regardless of their origin. The particle sizes to be monitored are generally 10 μm and 25 μm. In the case of HIAC, a stable pharmaceutical formulation should exhibit no more than 6000 10 μm particles per container (or unit) as compared to a control sample. In certain embodiments, the stable pharmaceutical formulation has no more than 5000, no more than 4000, no more than 3000, no more than 2000, no more than 1000 μm per container (or unit) compared to a control sample. Should exhibit particles. In yet other embodiments, a stable pharmaceutical formulation should exhibit no more than 600 25 μm particles per container (or unit) as compared to a control sample. In certain embodiments, the stable pharmaceutical formulation has no more than 500, no more than 400, no more than 300, no more than 200, no more than 100, per container (or unit), compared to a control sample. It should exhibit no more than 50 25 μm particles.

  In certain embodiments, the stability of the pharmaceutical formulation is measured using a visual assessment. Visual evaluation is a qualitative method used to describe the visible physical properties of a sample. Depending on the feature being evaluated (eg, color, clarity, presence of particles or foreign matter), the sample is illuminated against the black and / or white background of the test booth. The sample is also illuminated with a milky white reference and a color reference. For visual evaluation, a stable pharmaceutical formulation should not exhibit a significant change in color, clarity, presence of particles or foreign matter, as compared to a control sample.

  One aspect of the invention is: (i) about 70 mg / ml to about 250 mg / ml of the antigen binding protein for PCSK9, and (ii) about 0.05 mM to about 40 mM of sodium acetate acting as a buffer ("NaOAC And (iii) about 1% to about 5% of proline, arginine, lysine, methionine, or taurine (also known as 2-aminoethanesulfonic acid), and / or 0.5% From about 0.004% to about 10% of a non-ionic surfactant (polysorbate 80, polysorbate 60, polysorbate 40, w / v of the formulation). And the polysorbate 20, including but not limited to, wherein the formulation has a pH in the range of about 4.0-6.0. It is a drug formulation. In certain other embodiments, the pharmaceutical formulation of the invention comprises (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 anti-PCSK9 antibody, (ii) about 10 mM NAOAC, (iii) about 0.01% polysorbate 80, and (iv) about 2 % To 3% proline (or about 250 mM to about 270 mM proline), wherein the formulation has a pH of about 5. In certain other embodiments, the pharmaceutical formulation of the invention comprises (i) at least about 70 mg / ml, about 100 mg / ml, about 120 mg / ml, about 140 mg / ml of the anti-PCSK9 antibody, 21B12, 26H5, and / or Or 31H4, (ii) about 10 mM NAOAC, (iii) about 0.01% polysorbate 80, and (iv) about 2% to 3% proline (or about 250 mM to about 270 mM proline), wherein: The formulation has a pH of about 5. In certain other embodiments, the pharmaceutical formulation of the invention comprises (i) at least about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml. An anti-PCSK9 antibody, 8A3, 11F1, and / or 8A1, (ii) about 10 mM NAOAC, (iii) about 0.01% polysorbate 80, and (iv) about 2% to 3% proline (or about 250 mM to about 250%). 270 mM proline), wherein the formulation has a pH of about 5.

  One aspect of the invention is: (i) at least about 70 mg / ml to about 250 mg / ml of the anti-PCSK9 antibody; (ii) about 5 mM to about 20 mM of a buffer (such as NAOAC); and (iii) w / about 1% to about 10% by weight of a polyhydroxy hydrocarbon (such as sorbitol) or disaccharide (such as sucrose), and (iv) about 0.004% to about 10% surfactant (w / v) of the formulation ( Such as polysorbate 20 or polysorbate 80), wherein the formulation is a pharmaceutical formulation having a pH in the range of about 4.8-5.8, wherein the pharmaceutical formulation optionally comprises about 80 mM About 300 mM proline, arginine, lysine, methionine, or taurine, and / or 0.5% to about 5% of benzyl alcohol, which acts to reduce viscosity. In certain other embodiments, the pharmaceutical formulation of the invention comprises (i) at least about 70 mg / ml to about 250 mg / ml of the anti-PCSK9 antibody, (ii) about 10 mM NAOAC, and (iii) about 9% sucrose. And (iv) about 0.004% polysorbate 20, wherein the formulation has a pH of about 5.2. In certain other embodiments, the pharmaceutical formulation of the invention comprises (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 anti-PCSK9 antibody, (ii) about 15 mM NAOAC, (iii) about 9% sucrose, and (iv) about 0.01% polysorbate 20, where the formulation comprises about 5. It has a pH of 2. In certain other embodiments, the pharmaceutical formulation of the invention comprises (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 anti-PCSK9 antibody, (ii) about 20 mM NAOAC, (iii) about 9% sucrose, and (iv) about 0.01% polysorbate 20, where the formulation comprises about 5% It has a pH of 0.2. In certain other embodiments, the pharmaceutical formulation of the invention comprises (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 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.

  The pharmaceutical formulations of the present invention can be administered in combination therapy (ie, in combination with other drugs). In certain embodiments, the combination therapy comprises an antigen binding protein capable of binding PCSK9 in combination with at least one anticholesterol agent. Agents include, but are not limited to, chemicals, antibodies, antigen-binding regions, and combinations and complexes thereof, prepared synthetically in vitro. In certain embodiments, an agent can act as an agonist, antagonist, allosteric modulator, or toxin. In certain embodiments, the agent promotes increased expression of LDLR by acting to inhibit or stimulate its target (eg, activation or inhibition of a receptor or enzyme), or increases serum cholesterol levels. Can be reduced.

  In certain embodiments, the antigen binding protein for PCSK9 can be administered before, concurrently with, and after treatment with a cholesterol-lowering (serum and / or total cholesterol) agent. In certain embodiments, the antigen binding protein to PCSK9 can be administered prophylactically to prevent or reduce the onset of hypercholesterolemia, heart disease, diabetes, and / or any cholesterol-related disease. it can. In certain embodiments, an antigen binding protein to PCSK9 can be administered for the treatment of pre-existing hypercholesterolemic conditions. In some embodiments, the ABP delays the onset of the disease and / or symptoms associated with the disease. In some embodiments, ABP is provided to a subject lacking any symptom or any part of any one of the cholesterol-related diseases.

  In certain embodiments, the antigen binding protein for PCSK9 is used with certain 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 an agent and the antigen binding protein for PCSK9 can be provided together by inclusion in the same formulation. In certain embodiments, such agents can be separately formulated and provided together by inclusion in a therapeutic kit. In certain embodiments, such agents and the antigen binding protein to PCSK9 can be formulated separately and provided together by inclusion in a therapeutic kit. In certain embodiments, such agents can be given separately.

  In certain embodiments, mixing the selected formulation having the desired purity with the optionally used formulation agent (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. Thereby, a formulation comprising an antigen binding protein for PCSK9, with or without at least one additional therapeutic agent, can be prepared for storage. Further, in certain embodiments, a formulation comprising an antigen binding protein for PCSK9, with or without at least one additional therapeutic agent, may be formulated as a lyophilized material using appropriate excipients.

  In certain embodiments, where parenteral administration is contemplated, the therapeutic formulation may comprise the desired antigen binding to PCSK9 in a pharmaceutically acceptable vehicle, with or without additional therapeutic agents. It can be in the form of a parenterally acceptable pyrogen-free aqueous solution containing the protein. In certain embodiments, the parenteral injection vehicle is a sterile, non-sterile vehicle wherein the antigen binding protein to PCSK9 is formulated as a sterile, isotonic solution, with or without the addition of at least one additional therapeutic agent. It is distilled water. In certain embodiments, the preparation comprises a sustained or sustained release of the product, such as injectable microspheres, bioerodable particles, polymeric compounds (such as polylactic or polyglycolic acid), beads or liposomes. The product can then include a formulation of the desired molecule with the addition of an agent that can provide it via depot injection. In certain embodiments, hyaluronic acid may also be used and may have the effect of promoting a sustained period in the circulation. In certain embodiments, an implantable drug delivery device can be used to introduce a desired molecule.

  In certain embodiments, a pharmaceutical formulation can be formulated for inhalation. In certain embodiments, the antigen binding protein to PCSK9, with or without at least one additional therapeutic agent, can be formulated as a dry inhalable powder. In certain embodiments, an inhalation solution comprising an antigen binding protein for PCSK9, with or without at least one additional therapeutic agent, may be formulated with a propellant for aerosol delivery. In certain embodiments, the solution may be nebulized. Pulmonary administration is further described in PCT application PCT / US94 / 001875 which describes pulmonary delivery of chemically modified proteins.

  In certain embodiments, the pharmaceutical formulation comprises an effective amount of an antigen binding protein for PCSK9 in a mixture with non-toxic excipients suitable for the manufacture of tablets, with or without at least one additional therapeutic agent. Can be included. In certain embodiments, solutions can be prepared in unit dosage form by dissolving the tablets in sterile water or another appropriate vehicle. In certain embodiments, suitable excipients include calcium carbonate, sodium or sodium bicarbonate, an inert diluent such as lactose or calcium phosphate, or a binder such as starch, gelatin or gum arabic, or stearin. Lubricants such as, but not limited to, magnesium acid, stearic acid or talc.

  Additional pharmaceutical formulations will be apparent to those skilled in the art, such as formulations comprising an antigen binding protein to PCSK9, with or without at least one additional therapeutic agent in a sustained or controlled delivery formulation. In certain embodiments, techniques for formulating liposomal carriers, bioerodible microparticles, or various other sustained or controlled delivery means, such as porous beads and depot injection, are also known to those of skill in the art. See, for example, PCT application PCT / US93 / 00829 which describes the sustained release of porous polymeric microparticles for the delivery of pharmaceutical formulations. In certain embodiments, a sustained release preparation may include a semi-permeable polymer matrix in the form of a molded product, for example, a film or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. Nos. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamic acid (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech. , 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al., Supra), and poly-D (-)-3-hydroxybutyric acid (EP 133,988). In certain embodiments, sustained release formulations can also include liposomes, which can be prepared by any of the methods known in the art. See, for example, Epstein et al. Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); EP 036,676; EP 088,046, and EP 143,949.

  Pharmaceutical formulations to be used for in vivo administration are typically sterile. In certain embodiments, this can be achieved by filtration through a sterile filtration membrane. In certain embodiments, if the formulation is lyophilized, sterilization using this method may be performed either before or after lyophilization and reconstitution. In certain embodiments, formulations for parenteral administration may be stored in lyophilized form or in solution. In certain embodiments, parenteral formulations are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. .

  In certain embodiments, after the pharmaceutical formulation has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations may be stored either in a ready-to-use form or in a form (eg, lyophilized) that is reconstituted prior to administration.

  In certain embodiments, after the pharmaceutical formulation has been formulated, it may be stored in a ready-to-use form as a solution or suspension in a pre-filled syringe.

  In certain embodiments, a kit is provided for making a single dosage unit. In certain embodiments, a kit may contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and / or multiple chamber pre-filled syringes (eg, liquid syringes and lyophilized syringes) are included.

  In certain embodiments, the effective amount to be used in the treatment of a pharmaceutical formulation comprising an antigen binding protein to PCSK9, with or without at least one additional therapeutic agent, will depend, for example, on the therapeutic context and purpose. Dependent. Thus, one of ordinary skill in the art, according to certain embodiments, will use the antigen binding protein for PCSK9 with the molecule to be delivered, with or without at least one additional therapeutic agent. The appropriate dosage level for treatment will vary, depending in part on the disease, the route of administration, and the size (body weight, body or organ size), and / or the patient's symptoms (age and general health). Will be understood. In certain embodiments, a physician may titrate the dosage and modify the route of administration for optimal therapeutic effect.

  In certain embodiments, the formulation may be administered locally via implantation of a membrane, sponge, or another suitable material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, if an implant device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, sustained release bolus, or continuous administration. It can be carried out.

Dosage and Dosage Schedule One or more heavy chain complementarity determining regions (CDRHs) and one or more light chain complementations as depicted in Table 2 and FIGS. 2 and / or 3 and FIGS. 31A and 31B Any of the antigen binding proteins for PCSK9 that include a sex-determining region (CDRL) can be administered to a patient diagnosed with homozygous familial hypercholesterolemia according to the methods of the present invention. In certain other embodiments, the light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of the antigen binding protein for PCSK9 illustrated in Tables 2 and 2 and / or 3 and FIGS. 31A and 31B. A variable region and a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of any antigen binding protein to PCSK9 illustrated in Tables 2 and 2 and / or 3 and FIGS. 31A and 31B. An antigen binding protein to PCSK9, comprising: can be administered to a patient diagnosed with homozygous familial hypercholesterolemia according to the methods of the present invention. In yet other embodiments, any of the antigen binding proteins for PCSK9, illustrated in Table 2 and FIGS. 2 and / or 3 and / or 13 and / or FIGS. It can be administered to patients diagnosed with zygotic familial hypercholesterolemia. In certain other embodiments, the antibody consisting of another antigen binding protein to PCSK9; ie, the light chain variable domain of SEQ ID NO: 588 and the heavy chain variable domain of SEQ ID NO: 589, is homozygous for familial hypercholesterolemia. Can be administered to patients diagnosed with the disease. 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 (eg, an anti-PCSK9 antibody) that is administered to a patient according to the methods of the present invention is generally a therapeutically effective amount. The amount of ABP is expressed in milligrams of antibody (ie, mg) or milligrams of antibody per kilogram of patient body weight (ie, mg / kg). In certain embodiments, a typical dosage of PCSK9 antigen binding protein can range from about 0.1 μg / kg of antigen binding protein to PCSK9 up to about 100 mg / kg or more. In certain embodiments, the dosage is from 0.1 μg / kg to up to about 100 mg / kg of the antigen binding protein for PCSK9; or 1 μg / kg to up to about 100 mg / kg; or 5 μg / kg to up to about 100 mg / kg. Or 1 mg / kg to about 50 mg / kg of the antigen binding protein for PCSK9; or 2 mg / kg to about 20 mg / kg of the antigen binding protein for PCSK9; or 2 mg / kg to about 10 mg / kg of the antigen binding protein for PCSK9. Can be

  In certain embodiments, the amount (or dosage) of the antigen binding protein for PCSK9 is at least about 120 mg to about 3000 mg, about 140 mg to about 2800 mg, about 140 mg to about 2500 mg, about 140 mg to about 2000 mg, about 140 mg to about 140 mg. 1800 mg, about 140 mg to about 1400 mg, about 120 mg to about 1200 mg, about 120 mg to about 1000 mg, about 120 mg to about 700 mg, about 140 mg to about 700 mg, about 140 mg to about 600 mg, about 140 mg to about 450 mg, about 120 mg to about 450 mg, About 120 mg to about 450 mg, about 140 mg to about 450 mg, about 210 mg to about 450 mg, or about 280 mg to about 450 mg, about 210 mg to about 420 mg, about 280 mg to about 420 mg, about 420 mg 3000 mg, about 700 mg to about 3000 mg, about 1000 mg to about 3000 mg, about 1200 to about 3000 mg, about 1400 mg to about 3000 mg, about 1800 mg to about 3000 mg, about 2000 mg to about 3000 mg, about 2400 mg to about 3000 mg, or about 2800 mg to about 3000 mg Range. In some embodiments of this aspect, the anti-PCSK9 antibody is administered to the patient in about 35 mg, about 45 mg, about 70 mg, about 105 mg, about 120 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg. About 200 mg, about 210 mg, about 280 mg, about 360 mg, about 420 mg, about 450 mg, about 600 mg, about 700 mg, about 1200 mg, about 1400 mg, about 1800 mg, about 2000 mg, about 2500 mg, about 2800 mg, or about 3000 mg. Administer.

  In certain embodiments, the frequency of dosing takes into account the pharmacokinetic parameters of the antigen binding protein for PCSK9 and / or any additional therapeutic agents in the formulation used. In certain embodiments, the clinician administers the formulation until a dosage is reached that achieves the desired effect. In certain embodiments, the formulation is thus in a single dose or over time as two or more doses (which may or may not contain the same dose of the desired molecule). Or as a continuous infusion via an implanted device or catheter. The formulation can also be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen-type delivery devices, as well as auto-injector delivery devices, apply to the delivery of the pharmaceutical formulations of the present invention. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the ambit of tasks routinely performed by those skilled in the art. In certain embodiments, appropriate dosages may be ascertained through use of appropriate dose response data. In some embodiments, the amount and frequency of dosing takes into account the desired cholesterol level (serum and / or total) to be obtained and the subject's current cholesterol, LDL, and / or LDLR levels. All of which can be obtained by methods well known to those skilled in the art.

  In certain embodiments, 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 of an antigen binding protein for PCSK9 in a patient in need thereof. Or a weekly (QW) dose of up to about 450 mg.

  In some other embodiments, the antigen binding protein to PCSK9 is administered to a patient in need thereof for 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 280 mg. Administer once every other week (or every two weeks) (Q2W) at a dosage of about 350 mg, or up to about 420 mg, or up to about 450 mg.

  In certain other embodiments, the antigen binding protein for PCSK9 is administered to a patient in need thereof for 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 400 mg. Once every four weeks (or once a month) at a dosage of 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 ( Q4W) dosing.

  In certain other embodiments, the antigen binding protein to PCSK9 is administered to a patient in need thereof for 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 700 mg. A dosage of about 1000 mg, or up to about 2000 mg, or up to about 3000 mg is administered once every eight weeks (or once every other month).

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 10% compared to the serum LDL cholesterol level before administration. 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 level is reduced by at least about 65%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 70%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 80%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 85%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 90%.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 10% relative to the pre-dose serum LDL cholesterol level, and the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 15% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. For at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 20% relative to the pre-dose serum LDL cholesterol level, and the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 25% relative to the pre-dose serum LDL cholesterol level, and the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 30% relative to the pre-administration serum LDL cholesterol level, and the reduction is at least about 3 days, at least about 5 days, relative to the pre-administration level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 35% relative to the pre-dose serum LDL cholesterol level, and the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 40% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 45% relative to the pre-administration serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-administration level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 50% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 55% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 60% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 65% relative to the pre-administration serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-administration level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 70% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 75% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 80% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 85% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

  In some embodiments, the serum LDL cholesterol level is reduced by at least about 90% relative to the pre-dose serum LDL cholesterol level, wherein the reduction is at least about 3 days, at least about 5 days, relative to the pre-dose level. Maintain for a period of at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days.

Certain Therapeutic Uses As will be appreciated by those skilled in the art, the methods provided herein provide for the binding of antigen binding proteins (eg, antibodies) to the proprotein convertase subtilisin / keksin type 9 (PCSK9). This is a method of treating a patient diagnosed with homozygous familial hypercholesterolemia used.

  In one embodiment, an antigen binding protein for PCSK9 is used to modulate serum LDL cholesterol levels in patients diagnosed with homozygous familial hypercholesterolemia. In some embodiments, an antigen binding protein for PCSK9 is used to reduce the amount of serum LDL cholesterol from abnormally high levels or even normal levels. In certain embodiments, the serum LDL cholesterol level is reduced by at least about 10% compared to the level before administration. In certain embodiments, the serum LDL cholesterol level is reduced by at least about 15%. In certain embodiments, the serum LDL cholesterol level is reduced by at least about 20%. In certain embodiments, the serum LDL cholesterol level is reduced by at least about 25%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 30%. In certain embodiments, the serum LDL cholesterol level is reduced by at least about 35%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 40%. In certain embodiments, the serum LDL cholesterol level is reduced by at least about 45%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 50%. In certain embodiments, the serum LDL cholesterol level is reduced by at least about 55%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 60%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 65%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 70%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 75%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 80%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 85%. In some embodiments, the serum LDL cholesterol level is reduced by at least about 90%.

  In one embodiment, an antigen binding protein for PCSK9 is used to modulate serum PCSK9 levels in patients diagnosed with homozygous familial hypercholesterolemia. In certain embodiments, the antigen binding protein to PCSK9 is neutralizing. In some embodiments, an antigen binding protein for PCSK9 is used to reduce PCSK9 levels, even from abnormally high levels or even normal levels. In some embodiments, the serum PCSK9 value is reduced by at least about 20% compared to the level before administration. In some embodiments, the serum PCSK9 value is reduced by at least about 25%. In some embodiments, the serum PCSK9 value is reduced by at least about 30%. In some embodiments, the serum PCSK9 value is reduced by at least about 35%. In some embodiments, the serum PCSK9 value is reduced by at least about 40%. In some embodiments, the serum PCSK9 value is reduced by at least about 45%. In some embodiments, the serum PCSK9 value is reduced by at least about 50%. In some embodiments, the serum PCSK9 value is reduced by at least about 55%. In some embodiments, the serum PCSK9 value is reduced by at least about 60%. In some embodiments, the serum PCSK9 value is reduced by at least about 65%. In some embodiments, the serum PCSK9 value is reduced by at least about 70%. In some embodiments, the serum PCSK9 value is reduced by at least about 75%. In some embodiments, the serum PCSK9 value is reduced by at least about 80%. In some embodiments, the serum PCSK9 value is reduced by at least about 85%. In some embodiments, the serum PCSK9 value is reduced by at least about 90%.

  In one aspect, an antigen binding protein for PCSK9 is used to modulate total cholesterol levels in patients diagnosed with homozygous familial hypercholesterolemia. In certain embodiments, the antigen binding protein to PCSK9 is neutralizing. In some embodiments, an antigen binding protein for PCSK9 is used to reduce the amount of total cholesterol from abnormally high levels or even normal levels. In some embodiments, the total cholesterol level is reduced by at least about 20% compared to the level before administration. In some embodiments, total cholesterol levels are reduced by at least about 25%. In some embodiments, total cholesterol levels are reduced by at least about 30%. In some embodiments, total cholesterol levels are reduced by at least about 35%. In some embodiments, total cholesterol levels are reduced by at least about 40%. In some embodiments, total cholesterol levels are reduced by at least about 45%. In some embodiments, total cholesterol levels are reduced by at least about 50%. In some embodiments, total cholesterol levels are reduced by at least about 55%. In some embodiments, total cholesterol levels are reduced by at least about 60%.

  In one aspect, an antigen binding protein for PCSK9 is used to modulate non-HDL cholesterol levels in patients diagnosed with homozygous familial hypercholesterolemia. In certain embodiments, the antigen binding protein to PCSK9 is neutralizing. In some embodiments, an antigen binding protein for PCSK9 is used to reduce non-HDL cholesterol levels, from abnormally high levels or even normal levels. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 30%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 35%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 40%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 50%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 55%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 60%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 65%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 70%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 75%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 80%. In some embodiments, the level of non-HDL cholesterol is reduced by at least about 85%.

  In one aspect, an antigen binding protein to PCSK9 is used to modulate ApoB levels in patients diagnosed with homozygous familial hypercholesterolemia. In certain embodiments, the antigen binding protein to PCSK9 is neutralizing. In some embodiments, an antigen binding protein for PCSK9 is used to reduce the amount of ApoB, from abnormally high levels or even normal levels. In some embodiments, the ApoB level is reduced by at least about 10% compared to the level before administration. In some embodiments, ApoB levels are reduced by at least about 15%. In some embodiments, the ApoB level is reduced by at least about 20%. In some embodiments, ApoB levels are reduced by at least about 25%. In some embodiments, the ApoB level is reduced by at least about 30%. In some embodiments, ApoB levels are reduced by at least about 35%. In some embodiments, the ApoB level is reduced by at least about 40%. In some embodiments, the ApoB level is reduced by at least about 45%. In some embodiments, the ApoB level is reduced by at least about 50%. In some embodiments, the ApoB level is reduced by at least about 55%. In some embodiments, ApoB levels are reduced by at least about 60%. In some embodiments, ApoB levels are reduced by at least about 65%. In some embodiments, the ApoB level is reduced by at least about 70%. In some embodiments, the ApoB level is reduced by at least about 75%.

  In one embodiment, an antigen binding protein for PCSK9 is used to modulate Lp (a) levels in patients diagnosed with homozygous familial hypercholesterolemia. In certain embodiments, the antigen binding protein to PCSK9 is neutralizing. In some embodiments, an antigen binding protein for PCSK9 is used to reduce the amount of Lp (a) from abnormally high levels or even normal levels. In some embodiments, Lp (a) levels are reduced by at least about 10% compared to pre-administration levels. In some embodiments, Lp (a) levels are reduced by at least about 15%. In some embodiments, Lp (a) levels are reduced by at least about 20%. In some embodiments, Lp (a) levels are reduced by at least about 25%. In some embodiments, Lp (a) levels are reduced by at least about 30%. In some embodiments, Lp (a) levels are reduced by at least about 35%. In some embodiments, Lp (a) levels are reduced by at least about 40%. In some embodiments, Lp (a) levels are reduced by at least about 45%. In some embodiments, Lp (a) levels are reduced by at least about 50%. In some embodiments, Lp (a) levels are reduced by at least about 55%. In some embodiments, Lp (a) levels are reduced by at least about 60%. In some embodiments, Lp (a) levels are reduced by at least about 65%.

Combination Therapy In certain embodiments, a method of treating homozygous familial hypercholesterolemia comprising administering a therapeutically effective amount of one or more antigen binding proteins to PCSK9 and another therapeutic agent. Is provided. In certain embodiments, the antigen binding protein to PCSK9 is administered prior to administration of at least one other therapeutic agent. In certain embodiments, the antigen binding protein to PCSK9 is administered concurrently with the administration of at least one other therapeutic agent. In certain embodiments, the antigen binding protein to PCSK9 is administered after administration of at least one other therapeutic agent.

  Therapeutic agents (apart from antigen binding proteins) include, but are not limited to, at least one other cholesterol lowering (serum and / or systemic cholesterol) agent. In some embodiments, the agent has been observed to increase LDLR expression, increase serum HDL levels, reduce LDL levels, or reduce triglyceride levels. Typical drugs include statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), nicotinic acid (niacin) (NIACOR, NIASPAN (slow release niacin), SLO-NIACIN ( Sustained release niacin), CORDAPTIVE (laropiprant)), fibric acid (LOPID (gemfibrozil), TRICOR (fenofibrate), bile acid sequestrant (QUESTRAN (cholestyramine), colesevelam (WELCHOL), COLESTID (cholestipol)), cholesterol absorption Inhibitors (ZETIA (ezetimibe)), a combination of statins and nicotinic acid (ADVICOR (LOVASTATIN and N ASPN), combinations of statins with absorption inhibitors (VYTORIN (ZOCOR and ZETIA)) and / or lipid modulators.In some embodiments, ABPs are PPARγ agonists, PPARα / γ agonists, squalene synthase inhibitors, CETP inhibitors, antihypertensives, antidiabetic drugs (sulfonylureas, insulin, GLP-1 analogs, DDPIV inhibitors and the like, for example, metaformin), ApoB modulators (Mipomersen and the like), In combination with MTP inhibitors and / or treatment of arteriosclerosis obliterans In some embodiments, ABPs are statins, certain cytokines such as oncostatin M, estrogen, and / or certain specifics such as berberine Like plant ingredients In some embodiments, the ABP is combined with an agent that increases the level of LDLR protein in the subject (e.g., certain antipsychotics, certain HIV protease inhibitors, high fructose, sucrose, cholesterol or Combined with dietary factors, such as certain fatty acids, and certain nuclear receptor agonists and antagonists for RXR, RAR, LXR, FXR, etc.), and agents that increase serum cholesterol levels in the subject. , ABP is combined with an agent that increases the level of PCSK9 in a subject, such as statin and / or insulin, and the combination of the two may allow ABP to reduce the undesirable side effects of other agents. .

  The following examples, including experiments performed and results achieved, are provided for illustrative purposes only and should not be construed as limiting the invention. It should be noted that ABP names are commonly used herein, unless explicitly stated otherwise (eg, “21B12” is as defined in Example 7). It can be used as in Table 2 or as in FIGS. 2 and / or 3 and / or FIGS. 13A, 13C, 13F-13J).

Example 1
Immunization and titration Preparation of anti-PCKS9 antibody and hybridoma

Antibodies to the mature form of PCSK9 (shown as the sequence in FIG. 1A and the prodomain is underlined) were conjugated to a XenoMouse® mouse, a mouse containing the human immunoglobulin gene (Abgenix, Fremont) , CA). To generate antibodies to PCSK9, XenoMouse® two groups of mice (Groups 1 and 2) were used. Group 1, producing fully human IgG2 _kappa and IgG2λ antibody XenoMouse (TM) containing the mouse strains XMG2-KL. Group 1 mice were immunized with human PCSK9. PCSK9 was prepared using standard recombination techniques using the GenBank sequence as reference (NM — 174936). Group 2 included mice of the XenoMouse® strain XMG4-KL producing fully human IgG4κ and IgG4λ antibodies. Group 2 mice were also immunized with human PCSK9.

  According to the schedule in Table 3, both groups of mice were injected 11 times with antigen. In the first immunization, each mouse was injected with a total of 10 μg of antigen delivered intraperitoneally into the abdomen. Subsequent boost immunizations were at a dose of 5 μg and the injection method was alternating intraperitoneal injection into the abdomen and subcutaneous injection at the base of the tail. For intraperitoneal injection, the antigen is prepared as an emulsion with TiterMax® Gold (Sigma, Catalog No. T2684), and for subcutaneous injection, the antigen is mixed with Alum (aluminum phosphate) and CpG oligo. . At injections 2-8 and 10, a total of 5 μg of antigen in adjuvant alum gel was injected into each mouse. A final injection of 5 μg of antigen per mouse is delivered in phosphate buffered saline and delivered to two sites (50% intraperitoneally intraperitoneally and 50% subcutaneously at the base of the tail). The immunization program is summarized in Table 1.1, shown below.

  The protocol used to titrate XenoMouse animals was as follows. Costar3368 medium binding plates were coated with 8 μg / mL (50 μL / well) neutravidin and incubated in 1 × PBS / 0.05% azide at 4 ° C. overnight. The plates were washed using a three cycle TiterTek wash with RO water. Plates were blocked with 250 μL of 1 × PBS / 1% milk and incubated at room temperature for at least 30 minutes. The blocks were washed off using a three cycle TiterTek wash with RO water. Then, b-human PCSK9 was captured at 2 μg / mL in 50 μL / well of 1 × PBS / 1% milk / 10 mM Ca 2+ (assay diluent) and incubated for 1 hour at room temperature. It was then washed using RO water and using a TiterTek 3 cycle wash. For primary antibody, serum was titrated 1: 3 from 1: 100 in duplicate. This was done in 50 μL / well of assay diluent and incubated for 1 hour at room temperature. It was then washed using RO water and using a TiterTek 3 cycle wash. Secondary antibody was 400 ng / mL goat anti-human IgG FcHRP in 50 μL / well of assay diluent. This was incubated at room temperature for 1 hour. It was then washed using RO water, using a three cycle TiterTek wash, and patted dry on a paper towel. For the substrate, a one-step TMB solution (Neogen, Lexington, Kentucky) was used (50 μL / well) and developed for 30 minutes at room temperature.

The protocol followed in the ELISA assay was as follows. The following protocol was used for samples containing b-PCSK9 without the V5His tag. Costar 3368 medium binding plates (Corning Life Sciences) were used. Plates were coated with 8 μg / mL neutravidin in 1 × PBS / 0.05% azide (50 μL / well). Plates were incubated overnight at 4 ° C. The plates were then washed using a Titertek M384 plate washer (Titertek, Huntsville, AL). Three cycles of washing were performed. Plates were blocked with 250 μL of 1 × PBS / 1% milk and incubated at room temperature for about 30 minutes. Next, the plate was washed using an M384 plate washing device. Three cycles of washing were performed. Capture was b-huPCSK9 without the V5 tag and added at 2 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ (40 μL / well). The plate was then incubated for 1 hour at room temperature. Three cycles of washing were performed. Serum was titrated 1: 3 in duplicate from 1: 100, and row H was blank for serum. Titration was performed in assay diluent at a volume of 50 μL / well. Plates were incubated for 1 hour at room temperature. Next, three cycles of washing were performed. 100 ng / mL (1: 4000) goat anti-human IgG FcHRP in 1 × PBS / 1% milk / 10 mM Ca ²⁺ (50 μl / well) was added to the plate and incubated for 1 hour at room temperature. The plate was washed once more using a 3-cycle wash. The plate was then dried by tapping with a paper towel. Finally, 1-step TMB (Neogen, Lexington, Kentucky) (50 μL / well) was added to the plate, and after 30 minutes at room temperature, the reaction was stopped with 1N hydrochloric acid (50 μL / well). The OD was read immediately at 450 nm using a Titertek plate reader.

  Positive controls for detecting PCSK9-bound plates were soluble LDL receptor (R & D Systems, Cat. No. 2148LD / CF) and polyclonal, titrated 1: 3 in duplicate from 3 μg / mL in assay diluent. Rabbit anti-PCSK9 antibody (Caymen Chemical # 10007185). LDLR was detected using goat anti-LDLR (R & D Systems, catalog number AF2148) and rabbit anti-goat IgG FcHRP at a concentration of 400 ng / mL. Rabbit polyclonal was detected using goat anti-rabbit IgGFc at a concentration of 400 ng / mL in the assay diluent. Negative controls were na ナ イ ve XMG2-KL and XMG4-KL sera titrated 1: 3 from 1: 100 in assay diluent in duplicate.

For samples containing b-PCSK9 with a V5His tag, the following protocol was used. Costar 3368 medium binding plates (Corning Life Sciences) were used. Plates were coated with 8 μg / mL neutravidin in 1 × PBS / 0.05% azide (50 μL / well). Plates were incubated overnight at 4 ° C. The plates were then washed using a Titertek M384 plate washer (Titertek, Huntsville, AL). Three cycles of washing were performed. Plates were blocked with 250 μL of 1 × PBS / 1% milk and incubated at room temperature for about 30 minutes. Next, the plate was washed using an M384 plate washing device. Three cycles of washing were performed. Capture was b-huPCSK9 with V5 tag and added at 2 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ (40 μL / well). The plate was then incubated for 1 hour at room temperature. Three cycles of washing were performed. Serum was titrated 1: 3 in duplicate from 1: 100, and row H was blank for serum. Titration was performed in assay diluent at a volume of 50 μL / well. Plates were incubated for 1 hour at room temperature. The plates were then washed using an M384 plate washer that was operated using a three cycle wash. 400 ng / mL goat anti-human IgG FcHRP in 1 × PBS / 1% milk / 10 mM Ca ²⁺ was added to the plate at 50 μL / well and the plate was incubated for 1 hour at room temperature. The plate was washed once more using a 3-cycle wash. The plate was then dried by tapping with a paper towel. Finally, 1-step TMB (Neogen, Lexington, Kentucky) (50 μL / well) was added to the plate, and after 30 minutes at room temperature, the plate was quenched with 1N hydrochloric acid (50 μL / well). The OD was read immediately at 450 nm using a Titertek plate reader.

  The positive control was LDLR, rabbit anti-PCSK9 titrated 1: 3 in duplicate from 3 μg / mL in assay diluent. LDLR is detected using goat anti-LDLR (R & D Systems, Cat # AF2148) and rabbit anti-goat IgG FcHRP at a concentration of 400 ng / mL. Rabbit poly was detected using goat anti-rabbit IgG Fc at a concentration of 400 ng / mL in the assay diluent. 1: 3 titrations of human anti-His 1.2.3 and anti-V51.7.1 in duplicate from 1 μg / mL in assay diluent. All were detected using goat anti-IgGFcHRP at a concentration of 400 ng / mL in the assay diluent. Negative controls were na ナ イ ve XMG2-KL and XMG4-KL sera titrated 1: 3 from 1: 100 in assay diluent in duplicate.

  Antibody titers against human PCSK9 were tested by ELISA assay on mice immunized with the soluble antigens described. Table 4 summarizes the ELISA data and shows that there were some mice that appeared to be specific for PCSK9. See, for example, Table 4. Therefore, at the end of the immunization program, 10 mice (bold in Table 1.2) were selected for collection and spleen and spleen from lymph nodes, respectively, as described herein. Cells and lymphocytes were isolated.

Example 2
Lymphocyte Harvesting, B Cell Isolation, Fusion and Hybridoma Production This example outlines how immune cells were harvested and hybridomas were produced. Selected immunized mice were sacrificed by cervical dislocation and draining lymph nodes from each cohort were collected and pooled. B cells were dissociated from lymphoid tissue by trituration in DMEM to release cells from the tissue, and the cells were suspended in DMEM. Cells were counted and 0.9 mL of DMEM per 100 million lymphocytes was added to the cell pellet to gently, but completely resuspend the cells.

Lymphocytes were mixed with non-secretory myeloma P3X63Ag8.653 cells purchased from ATCC, catalog number CRL1580 (Kearney et al., (1979) J. Immunol. 123, 1548-1550) at a ratio of 1: 4. The cell mixture was gently settled by centrifugation at 400 × g for 4 minutes. After tilting the container and removing the supernatant, the cells were mixed gently using a 1 mL pipette. Pre-heated PEG / DMSO solution (1 mL / million B cells) from Sigma (Cat. # P7306) was added slowly with gentle stirring over 1 minute followed by mixing for 1 minute. Then, pre-heated IDMEM (2 mL per million B cells) (glutamine, L-glutamine, penicillin / streptomycin, DMEM without MEM non-essential amino acids) for 2 minutes with gentle stirring (all from Invitrogen) Was obtained. Finally, over 3 minutes, it was added a pre-heated IDMEM (10 ⁶ cells of B cells per 8 mL).

  The fused cells were centrifuged at 400 × g for 6 minutes, and 20 mL of selection medium per 1 million B cells (L-glutamine, penicillin / streptomycin, MEM non-essential amino acids, sodium pyruvate, 2-mercaptoethanol (all , DMEM (supplied from Invitrogen), HA-azaserine hypoxanthine, and OPI (oxaloacetate, pyruvate, bovine insulin) (all from Sigma), and IL-6 (Boeringer Mannheim). (Invitrogen), resuspended in 15% FBS (Hyclone) Incubate cells at 37C for 20-30 minutes, then resuspend in 200 mL of selective medium and T175 flask before seeding into 96 wells And cultured for 3-4 days. As described above, a hybridoma producing an antigen-binding protein for PCSK9 was prepared.

Example 3
Selection of PCSK9 Antibodies This example outlines how various PCSK9 antigen binding proteins were characterized and selected. The binding of secreted antibody (produced from the hybridomas produced in Examples 1 and 2) to PCSK9 was evaluated. Antibody selection was based on binding data and inhibition and affinity of PCSK9 binding to LDLR. Binding to soluble PCSK9 was analyzed by ELISA as described below. BIAcore® (Surface Plasmon Resonance) was used to quantify binding affinity.

Primary screening Primary screening was performed for antibodies that bind to wild-type PCSK9. Primary screening was performed on the two collections. Primary screening, including an ELISA assay, was performed using the following protocol.

Costar 3702 medium-coupled 384-well plates (Corning Life Sciences) were used. Plates were coated with neutravidin at a concentration of 4 μg / mL in 1 × PBS / 0.05% azide in a volume of 40 μL / well. Plates were incubated overnight at 4 ° C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three cycles of washing were performed. Plates were blocked with 90 μL of 1 × PBS / 1% milk and incubated at room temperature for about 30 minutes. The plate was then washed. Again, three cycles of washing were performed. The capture sample was biotinylated PCSK9 without the V5 tag and was added at 0.9 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ in a volume of 40 μL / well. The plate was then incubated for 1 hour at room temperature. The plates were then washed using a Titertek plate washer operated using a three cycle wash. 10 μL of the supernatant was transferred into 40 μL of 1 × PBS / 1% milk / 10 mM Ca ^{2+ and} incubated at room temperature for 1.5 hours. Again, the plates were washed using a Titertek plate washer operated using a three cycle wash. 40 μL / well of goat anti-human IgG FcPOD at a concentration of 100 ng / mL (1: 4000) in 1 × PBS / 1% milk / 10 mM Ca ²⁺ was added to the plate and incubated at room temperature for 1 hour. The plate was washed once more using a 3-cycle wash. Finally, 1-step TMB (Neogen, Lexington, Kentucky) (40 μL / well) was added to the plate, and after 30 minutes at room temperature, the reaction was stopped with 1N hydrochloric acid (40 μL / well). The OD was read immediately at 450 nm using a Titertek plate reader.

  Primary screening yielded a total of 3104 antigen-specific hybridomas identified from the two collections. Based on the highest ELISA OD, 1500 hybridomas per collection were used for further manipulation for a total of 3000 positives.

Confirmatory Screen Next, 3000 positives were rescreened for binding to wild-type PCSK9 to confirm that stable hybridomas were established. Screening was performed as follows. Costar 3702 medium-coupled 384-well plates (Corning Life Sciences) were used. Plates were coated with 3 μg / mL neutravidin in 1 × PBS / 0.05% azide in a volume of 40 μL / well. Plates were incubated overnight at 4 ° C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three cycles of washing were performed. Plates were blocked with 90 μL of 1 × PBS / 1% milk and incubated at room temperature for about 30 minutes. Next, the plate was washed using an M384 plate washing device. Three cycles of washing were performed. The capture sample was b-PCSK9 without the V5 tag and was added at 0.9 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ in a volume of 40 μL / well. The plate was then incubated for 1 hour at room temperature. The plate was then washed using a three cycle wash. 10 μL of the supernatant was transferred into 40 μL of 1 × PBS / 1% milk / 10 mM Ca ^{2+ and} incubated at room temperature for 1.5 hours. Again, the plates were washed using a Titertek plate washer operated using a three cycle wash. 40 μL / well of goat anti-human IgG FcPOD at a concentration of 100 ng / mL (1: 4000) in 1 × PBS / 1% milk / 10 mM Ca ²⁺ was added to the plate and the plate was incubated for 1 hour at room temperature. The plate was washed once more using a Titertek plate washer that was operated using a three cycle wash. Finally, 40 μL / well of 1-step TMB (Neogen, Lexington, Kentucky) was added to the plate, and after 30 minutes at room temperature, the reaction was stopped with 1N hydrochloric acid (40 μL / well). The OD was read immediately at 450 nm using a Titertek plate reader. A total of 2441 positives were repeated in the second screen. These antibodies were then used in subsequent screening.

Mouse Cross-Reaction Screen A panel of hybridomas was then screened for cross-reactivity to mouse PCSK9 to confirm that the antibody could bind to both human and mouse PCSK9. The following protocol was used for cross-reactivity screening. Costar 3702 medium-coupled 384-well plates (Corning Life Sciences) were used. Plates were coated with 3 μg / mL neutravidin in 1 × PBS / 0.05% azide in a volume of 40 μL / well. Plates were incubated overnight at 4 ° C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three cycles of washing were performed. Plates were blocked with 90 μL of 1 × PBS / 1% milk and incubated at room temperature for about 30 minutes. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. The capture sample was biotinylated mouse PCSK9 and added at 1 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ in a volume of 40 μL / well. The plate was then incubated for 1 hour at room temperature. The plates were then washed using a Titertek plate washer operated using a three cycle wash. 50 μL of the supernatant was transferred to a plate and incubated at room temperature for 1 hour. Again, the plate was washed using a 3-cycle wash. 40 μL / well of goat anti-human IgG FcPOD at a concentration of 100 ng / mL (1: 4000) in 1 × PBS / 1% milk / 10 mM Ca ²⁺ was added to the plate and the plate was incubated for 1 hour at room temperature. The plate was washed once more using a 3-cycle wash. Finally, 40 μL / well of 1-step TMB (Neogen, Lexington, Kentucky) was added to the plate, and after 30 minutes at room temperature, the reaction was stopped with 1N hydrochloric acid (40 μL / well). The OD was read immediately at 450 nm using a Titertek plate reader. It was observed that the 579 antibody cross-reacted with mouse PCSK9. These antibodies were then used in subsequent screening.

D374Y Mutant Binding Screening The D374Y mutation in PCSK9 has been described in the literature in a human population (eg, Timms KM et al, "A mutation in PCSK9 due to autosomal-dominant enemy age. 114: 349-353, 2004). To determine whether the antibody was specific for wild type or bound to the D374Y form of PCSK9, the samples were then screened for binding to a mutant PCSK9 sequence containing the mutant D374Y. The protocol for screening was as follows. For screening, Costar3702 medium-coupled 384-well plates (Corning Life Sciences) were used. Plates were coated with 4 μg / mL neutravidin in 1 × PBS / 0.05% azide in a volume of 40 μL / well. Plates were incubated overnight at 4 ° C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three cycles of washing were performed. Plates were blocked with 90 μL of 1 × PBS / 1% milk and incubated at room temperature for about 30 minutes. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. Plates were coated with biotinylated human PCSK9D374Y at a concentration of 1 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ^{2+ and} incubated for 1 hour at room temperature. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. The late-depleted hybridoma culture supernatant was diluted 1: 5 in PBS / milk / Ca ²⁺ (10 mL + 40 mL) and incubated for 1 hour at room temperature. Next, 40 μL / well of rabbit anti-human PCSK9 (Cayman Chemical) and human anti-His 1.2.3 (1: 2) at 1 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ were plated on the plate. Titrated and then incubated for 1 hour at room temperature. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. 40 μL / well of goat anti-human IgG FcHRP at a concentration of 100 ng / mL (1: 4000) in 1 × PBS / 1% milk / 10 mM Ca ²⁺ was added to the plate and the plate was incubated for 1 hour at room temperature. 40 μL / well of goat anti-rabbit IgG FcHRP at a concentration of 100 ng / mL (1: 4000) in 1 × PBS / 1% milk / 10 mM Ca ²⁺ was added to the plate and the plate was incubated for 1 hour at room temperature. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. Finally, 40 μL / well of 1-step TMB (Neogen, Lexington, Kentucky) was added to the plate, and after 30 minutes at room temperature, the reaction was stopped with 1N hydrochloric acid (40 μL / well). The OD was read immediately at 450 nm using a Titertek plate reader. Over 96% of the positive hits against wild-type PCSK9 also bound mutant PCSK9.

Large Scale Receptor Ligand Blocking Screen To screen for antibodies that block PCSK9 binding to LDLR, an assay using the D374Y PCSK9 mutant was developed. Having a higher binding affinity for LDLR, the variants were used for this assay, allowing the development of a more sensitive receptor ligand blocking assay. The following protocol was used in the receptor ligand blocking screen. For screening, Costar3702 medium-coupled 384-well plates (Corning Life Sciences) were used. Plates were coated with 2 μg / mL goat anti-LDLR (R & D, catalog number AF2148) in 1 × PBS / 0.05% azide in a volume of 40 μL / well. Plates were incubated overnight at 4 ° C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three cycles of washing were performed. Plates were blocked with 90 μL of 1 × PBS / 1% milk and incubated at room temperature for about 30 minutes. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. The capture sample was LDLR (R & D, Catalog No. 2148LD / CF) and added at 0.4 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ in a volume of 40 μL / well. The plate was then incubated for 1 hour 10 minutes at room temperature. At the same time, 20 ng / mL of biotinylated human D374Y PCSK9 was incubated with 15 μL of hybridoma depleted supernatant in Nunc polypropylene plates to dilute the depleted supernatant concentration 1: 5. The plate was then pre-incubated for about 1 hour 30 minutes at room temperature. The plates were then washed using a Titertek plate washer operated using a three cycle wash. 50 μL / well of the pre-incubated mixture was transferred onto an LDLR-coated ELISA plate and incubated for 1 hour at room temperature. To detect b-PCSK9 bound to LDLR, 40 μL / well of 500 ng / mL streptavidin HRP in assay diluent was added to the plate. Plates were incubated for 1 hour at room temperature. Again, the plates were washed using a Titertek plate washer. Three cycles of washing were performed. Finally, 40 μL / well of 1-step TMB (Neogen, Lexington, Kentucky) was added to the plate, and after 30 minutes at room temperature, the reaction was stopped with 1N hydrochloric acid (40 μL / well). The OD was read immediately at 450 nm using a Titertek plate reader. Screening identified 384 antibodies that blocked the interaction between PCSK9 and LDLR wells, with 100 antibodies strongly blocking the interaction (OD <0.3). These antibodies inhibited the binding interaction between PCSK9 and LDLR by more than 90% (more than 90% inhibition).

Receptor Ligand Binding Assay for a Subset of Blockers A receptor ligand assay was then repeated using the mutant enzyme on a subset of 384 neutralizing agents identified in the first large scale receptor ligand inhibition assay. . The screening of the 384 blocker subset assay used the same protocol as that performed in the large receptor ligand blocking screen. This repeated screening confirmed the initial screening data.

  Screening of this subset of 384 members identified over 85% of 85 antibodies that block the interaction between the PCSK9 mutant enzyme and LDLR.

Receptor Ligand Binding Assay of Blockers that Bind Wild-Type PCSK9 but Do Not Bind the D374Y Mutant In an initial panel of 3000 supernatants, specific binding to wild-type PCSK9 but huPCSK9 (D374Y) There were 86 antibodies that were shown not to bind to. These 86 supernatants were tested for their ability to block binding of wild-type PCSK9 to the LDLR receptor. The following protocol was used. For screening, Costar3702 medium-coupled 384-well plates (Corning Life Sciences) were used. Plates were coated with 10 μg / mL anti-His 1.2.3 in 1 × PBS / 0.05% azide in a volume of 40 μL / well. Plates were incubated overnight at 4 ° C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three cycles of washing were performed. Plates were blocked with 90 μL of 1 × PBS / 1% milk and incubated at room temperature for about 30 minutes. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. LDLR (R & D Systems, # 2148LD / CF or R & D Systems, # 2148LD) was added at 5 μg / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ in a volume of 40 μL / well. The plate was then incubated for 1 hour at room temperature. The plates were then washed using a Titertek plate washer operated using a three cycle wash. At the same time, biotinylated human wild-type PCSK9 was pre-incubated with hybridoma depleted supernatant in Nunc polypropylene plates. Transfer 22 μL of hybridoma supernatant to 33 μL of b-PCSK9 at a concentration of 583 ng / mL in 1 × PBS / 1% milk / 10 mM Ca ²⁺ , final b-PCSK9 concentration = 350 ng / mL and 1: 2.5 The final dilution gave the depleted supernatant. Plates were preincubated for about 1 hour 30 minutes at room temperature. 50 μL / well of the pre-incubated mixture was transferred onto an ELISA plate with LDLR captured and incubated for 1 hour at room temperature. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. 40 μL / well of 500 ng / mL streptavidin HRP in assay diluent was added to the plate. Plates were incubated for 1 hour at room temperature. The plate was then washed using a Titertek plate washer. Three cycles of washing were performed. Finally, 40 μL / well of 1-step TMB (Neogen, Lexington, Kentucky) was added to the plate, and after 30 minutes at room temperature, the reaction was stopped with 1N hydrochloric acid (40 μL / well). The OD was read immediately at 450 nm using a Titertek plate reader.

Screening Results Based on the results of the described assays, several hybridoma strains were identified as producing antibodies with the desired interaction with PCSK9. Ultradilution was used to isolate a manageable number of clones from each strain. Clones were designated by the hybridoma strain number (eg, 21B12) and clone number (eg, 21B12.1). In general, no differences between the different clones of any particular strain were detected by the functional assays described herein. In a few cases, clones were identified from specific strains that behaved differently in the functional assay. For example, 25A7.1 does not block PCSK9 / LDLR, while 25A7.3 (referred to herein as 25A7) was found to be neutralizing. The isolated clones were each grown in 50-100 mL of hybridoma medium and grown to depletion (ie, less than about 10% cell viability). The concentration and potency of the antibody to PCSK9 in the supernatants of these cultures was determined by ELISA as described herein and by in vitro functional tests. As a result of the screening described herein, the hybridoma with the highest titer of antibodies to PCSK9 was identified. Selected hybridomas are shown in FIGS. 2A-3D and Table 2.

Example 4.1
Production of Human 31H4 IgG4 Antibodies from Hybridomas This example generally describes how to produce one of the antigen binding proteins from a hybridoma strain. In the preparation operation, protein A purification was performed after using 50 mL of depleted supernatant. The Integra production was scaled up and then performed. Hybridoma strain 31H4 was grown in T75 flasks in 20 mL of media (Integra Media, Table 5). When the hybridomas were nearly confluent in T75 flasks, they were transferred to Integra flasks (Integra Biosciences, Integra CL1000, Cat # 90005).

Integra flasks are cell culture flasks separated by a membrane into two chambers (small chamber and large chamber). A volume of 20-30 mL of hybridoma cells with a minimum cell density of 1 × 10 ⁶ cells / mL from the 31H4 hybridoma strain was placed in a small chamber of an Integra flask in Integra medium (for the components of Integra medium, see Table 4). See .1). Integra medium alone (1 L) was placed in the large chamber of the Integra flask. The membrane separating the two chambers is permeable to small molecular weight nutrients but impermeable to hybridoma cells and antibodies produced by the hybridoma cells. Therefore, the hybridoma cells and the antibodies produced by the hybridoma cells were kept in a small chamber.

  One week later, the media was removed from both chambers of the Integra flask and replaced with fresh Integra media. The medium collected from the small chamber was kept separately. After the second week of growth, the medium from the small chamber was collected again. The medium collected at week 1 from the hybridoma strain was combined with the medium collected at week 2 from the hybridoma strain. The collected media samples obtained from the hybridoma strain were centrifuged to remove cells and debris (3000 rpm for 15 minutes) and the resulting supernatant was filtered (0.22 μm). The clarified conditioned medium was loaded on a protein A-Sepharose column. Optionally, the medium can be first concentrated and then loaded on a Protein A Sepharose column. Non-specific binding was removed by extensive PBS washing. The bound antibody protein on the Protein A column was recovered by standard acidic antibody elution from the Protein A column (such as 50 mM citrate, pH 3.0). Aggregated antibody proteins in the Protein A Sepharose spool were removed by size exclusion chromatography or bound ion exchange chromatography on an anion exchange resin such as Q Sepharose resin. Specific IEX conditions for 31H4 protein are Q-Sepharose HP at pH 7.8-8.0. Antibodies were eluted using 25 column volumes of a 10 mM to 500 mM NaCl gradient.

Example 4.2
Production of Recombinant 31H4 Human IgG2 Antibody from Transfected Cells This example outlines how 31H4 IgG2 antibody is produced from transfected cells. 293 cells for transient expression and CHO cells for stable expression were transfected with plasmids encoding 31H4 heavy and light chains. Conditioned media from transfected cells was recovered by removing cells and cell debris. The clarified conditioned medium was loaded on a protein A-Sepharose column. Optionally, the medium can be first concentrated and then loaded onto a Protein A Sepharose column. Non-specific binding was removed by extensive PBS washing. The bound antibody protein on the Protein A column was recovered by standard acidic antibody elution from the Protein A column (50 mM citrate, pH 3.0, etc.). Aggregated antibody proteins in the Protein A Sepharose spool were removed by size exclusion chromatography or bound ion exchange chromatography on an anion exchange resin such as Q Sepharose resin. Specific IEX conditions for 31H4 protein are Q-Sepharose HP at pH 7.8-8.0. Antibodies were eluted using 25 column volumes of a 10 mM to 500 mM NaCl gradient.

Example 5
Production of Human 21B12 IgG4 Antibody from Hybridoma This example outlines how antibody 21B12 IgG4 was produced from hybridoma. Hybridoma strain 21B12 was grown in T75 flasks in media (Integra Media, Table 5). When the hybridomas were nearly confluent in T75 flasks, they were transferred to Integra flasks (Integra Biosciences, Integra CL1000, Cat # 90005).

Integra flasks are cell culture flasks separated by a membrane into two chambers (small chamber and large chamber). A volume of 20-30 mL of hybridoma cells with a minimum cell density of 1 × 10 ⁶ cells / mL from the 31H4 hybridoma strain was placed in a small chamber of an Integra flask in Integra media (for the components of Integra media, see Table 1). 5). Integra medium alone (1 L) was placed in the large chamber of the Integra flask. The membrane separating the two chambers is permeable to small molecular weight nutrients but impermeable to hybridoma cells and antibodies produced by the hybridoma cells. Therefore, the hybridoma cells and the antibodies produced by the hybridoma cells were kept in a small chamber.

  One week later, the media was removed from both chambers of the Integra flask and replaced with fresh Integra media. The medium collected from the small chamber was kept separately. After the second week of growth, the medium from the small chamber was collected again. The medium collected at week 1 from the hybridoma strain was combined with the medium collected at week 2 from the hybridoma strain. The collected media samples obtained from the hybridoma strain were centrifuged to remove cells and debris (3000 rpm for 15 minutes) and the resulting supernatant was filtered (0.22 μm). The clarified conditioned medium was loaded on a protein A-Sepharose column. Optionally, the medium is first concentrated and then loaded onto a Protein A Sepharose column. Non-specific binding was removed by extensive PBS washing. The bound antibody protein on the Protein A column was recovered by standard acidic antibody elution from the Protein A column (such as 50 mM citrate, pH 3.0). Aggregated antibody proteins in the Protein A Sepharose spool were removed by size exclusion chromatography or bound ion exchange chromatography on an anion exchange resin such as Q Sepharose resin. Specific IEX conditions for 21B12 protein are Q-Sepharose HP at pH 7.8-8.0. Antibodies were eluted using 25 column volumes of a 10 mM to 500 mM NaCl gradient.

Example 6
Production of Human 21B12 IgG2 Antibody from Transfected Cells This example outlines how 21B12 IgG2 antibody is produced from transfected cells. Cells (293 cells for transient expression and CHO cells for stable expression) were transfected with plasmids encoding 21B12 heavy and light chains. Conditioned media from hybridoma cells was recovered by removing cells and cell debris. The clarified conditioned medium was loaded on a protein A-Sepharose column. Optionally, the medium can be first concentrated and then loaded onto a Protein A Sepharose column. Non-specific binding was removed by extensive PBS washing. The bound antibody protein on the Protein A column was recovered by standard acidic antibody elution from the Protein A column (50 mM citrate, pH 3.0, etc.). Aggregated antibody proteins in the Protein A Sepharose spool were removed by size exclusion chromatography or bound ion exchange chromatography on a cation exchange resin such as SP-Sepharose resin. Specific IEX conditions for the 21B12 protein were SP-Sepharose HP at pH 5.2. Antibodies were eluted with 25 column volumes of buffer containing a 10 mM to 500 mM NaCl gradient 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 antibody light and heavy chains were then determined by Sanger (dideoxy) nucleotide sequencing. The amino acid sequence was then deduced for the nucleic acid sequence. The nucleic acid sequences for the variable domains are illustrated in FIGS. 3E-3JJ and 3LL-BBB.

  The cDNA sequences for the λ light chain variable regions of 31H4, 212B12, and 16F12 have been determined and are disclosed as SEQ ID NOs: 153, 95, and 105, respectively.

  The cDNA sequences for the heavy chain variable regions of 31H4, 212B12, and 16F12 have been determined and are disclosed as SEQ ID NOs: 152, 94, and 104, respectively.

  The λ light chain constant region (SEQ ID NO: 156) and the IgG2 and IgG4 heavy chain constant regions (SEQ ID NOs: 154 and 155) are shown in FIG. 3KK.

  The predicted polypeptide sequence from each of the cDNA sequences was determined. The predicted polypeptide sequences for the λ light chain variable region of 31H4, 21B12, and 16F12 are predicted and are disclosed as SEQ ID NOs: 12, 23, and 35, respectively, and the λ light chain constant region (SEQ ID NO: 156), 31H4, The heavy chain variable regions of 21B12 and 16F12 are disclosed as SEQ ID NOs: 67, 49, and 79, respectively. IgG2 and IgG4 heavy chain constant regions (SEQ ID NOs: 154 and 155).

  The FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 classification is shown in FIGS. 2A-3D and FIGS. 3CCC-3JJJ.

  Based on the sequence data, the germline gene from which each heavy or light chain variable region was obtained was determined. In FIGS. 2A-3D and 3CCC-3JJJ, germline gene identification is shown next to the corresponding hybridoma strain, each represented by a unique SEQ ID NO. 2A-3D and 3CCC-3JJJ also illustrate the determined amino acid sequences for additional antibodies that have been characterized.

Example 8
Production of Antibodies in E. coli Some of the antibodies are also produced in E. coli and have some slight amino acid differences from, for example, antibodies produced as in Examples 4-6. The first residue in the variable region was glutamic acid instead of glutamine for the heavy and light chains of 21B12 and the light chain of 31H4. In addition to differences in the sequence of the variable regions, there were also some differences in the constant regions of the antibodies described by coordinates (also due to the fact that the antibodies were produced in E. coli). FIG. 3 LLL highlights (by underlined shadow or bold) differences between the constant regions of 21B12, 31H4, and 31A4 Fab (produced 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 λ (SEQ ID NO: 156). The underlined glycine residue is an insertion, during which the 21B12 and 31H4 variable regions terminate and the λ sequence begins.

  For both 21B12 and 31H4, heavy chain constants are similar to human IgG4 (SEQ ID NO: 155). The highlighted differences in FIG. 3 LLL are shown in Table 8.1.

  With respect to 31A4, 31A4 has the same differences described above, but there are three further differences. As shown in FIG. 3 LLL, there are two additional amino acids at the beginning, due to incomplete processing of the signal peptide during E. coli expression. In addition, there is one additional substitution in the 31A4 heavy chain constant region when compared to SEQ ID NO: 155, which is an adjustment of L to H (in SEQ ID NO: 155). Finally, 31A4 has glutamine as the first amino acid of the Fab, rather than the adjustment to glutamic acid described above for 21B12 and 31H4.

  For all three antibodies, the ends of the heavy chains (circled in dark gray) are also different, but do not appear in the coordinates because the amino acids are not organized in the structure. As will be appreciated by those skilled in the art, the His tag is a necessary part of the ABP unless a reference to a particular SEQ ID NO including the histidine tag and the ABP sequence is explicitly referred to by the description “including the histidine tag”. It should not be considered part of the sequence of ABP, but rather the part.

Example 9
Characterization of Antibody Binding to PCSK9 Once several antigens that bind to PCSK9 were identified, several approaches were used to quantify and further characterize the binding. In one embodiment of the test, Biacore affinity analysis was performed. In another embodiment of the study, KinExA® affinity analysis was performed. The samples and buffers used in these tests are shown in Table 9.1 below.

BIAcore® Affinity Measurement BIAcore® (Surface Plasmon Resonance Instrument, Biacore, Inc., Piscataway, NJ) affinity analysis of the 21B12 antibody to PCSK9 described in this example was performed according to the manufacturer's instructions. I went according to the book.

  Briefly, surface plasmon resonance experiments were performed using a Biacore 2000 optical biosensor (Biacore, GE Healthcare, Piscataway, NJ). Each individual anti-PCSK9 antibody was immobilized on a research grade CM5 biosensor chip by amine conjugation at a level that gave a maximum analyte binding response (Rmax) not exceeding 200 resonance units (RU). The concentration of PCSK9 protein was varied at two-fold intervals (analyte) and injected onto the immobilized antibody surface (1.5 min, at a flow rate of 100 μL / min). Use 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 as binding buffer did. In a separate experiment for each of the human, mouse and cynomolgus PCSK9 proteins at pH 7.4, the binding affinity of each anti-PCSK9 antibody was measured (concentrations used were 100, 50, 25, 12.5, 6.25). , 3.125, and 0 nM).

Furthermore, the binding affinity of the antibody to human PCSK9 was also determined using a pH 6.0 HBS-P buffer supplemented with 0.01% BSA (pH 6.0, 0.01 M Hepes, 0.15 M NaCl, 0.005% surfactant). P-20, Biacore) at pH 6.0. The binding signal obtained was proportional to the free PCSK9 in solution. The dissociation equilibrium constant (K _D ) was determined using a double-curve one-site homogeneous binding model (KinExA® software, Sapidyne Instruments Inc., Boise, ID) (n = 1 for a 6.0 pH run). Obtained from non-linear regression analysis of competition curves. Interestingly, the antibody appeared to show a stronger binding affinity at lower pH ( _Kd was 12.5, 7.3, 31H4, 21B12, and 16F12, respectively). And 29 pM).

k _a (association rate constant), _{k d} (dissociation rate constant) and _{K D} antibody binding kinetics parameters including (dissociation equilibrium constant) using the BIA evaluation 3.1 computer program (BIAcore, Inc.Piscataway, NJ) Measured. Lower dissociation equilibrium constants indicate a greater affinity of the antibody for PCSK9. _{K D} values as determined by BIAcore (TM) affinity analysis are presented in Table 9.2 shown below.

Table 9.3 _shows the _{k on} and _{k off} rate.

KinExA® Affinity Measurement KinExA® (Sapidyne Instruments, Inc., Boise, ID) affinity analysis of 16F12 and 31H4 was performed according to the manufacturer's instructions. Briefly, Reacti-Gel ™ (6 ×) (Pierce) was pre-coated with one of the human V5-tagged cynomolgus monkey or His-tagged mouse PCSK9 protein and blocked with BSA. Then, 10 or 100 pM of either antibody 31H4 or one of the PCSK9 proteins was incubated with various concentrations of PCSK9 protein (0.1 pM to 25 nM) for 8 hours at room temperature before passing through the PCSK9-coated beads. . The amount of beads bound 31H4 was quantified by a fluorescently (Cy5) labeled goat anti-human IgG (H + L) antibody (Jackson Immuno Research). The binding signal is proportional to the concentration of free 31H4 at binding equilibrium. Equilibrium dissociation constants (K _D ) were obtained from two sets of non-linear regressions of the competition curve using a one-site homogeneous binding model. KinExA® Pro software was used in the analysis. The binding curves generated in this analysis are represented as FIGS.

Both the 16F12 and 31H4 antibodies showed similar affinities for human and cynomolgus PCSK9, but about 10-250-fold less affinity for mouse PCSK9. KinExA (R) of the two antibodies were tested using the system, antibodies 31H4, respectively, with a _{K D} of 3 and 2 pM, it showed a higher affinity for both human and cynomolgus PCSK9. 16F12 is a _{K D} of 15pM against human PCSK9, with a _{K D} of 16pM against Kainikuizaru PCSK9, it showed weak affinity than slightly.

  The results of the KinExA® affinity analysis are summarized in Table 9.4, shown below.

In addition, SDS PAGE was performed to check the quality and quantity of the sample and is shown in FIG. 5A. cPCSK9 was shown to be approximately 50% less on the gel, as indicated by the active binding concentration calculated from the KinExA® assay. Thus, _{K D} of mAb to CPCSK9 was adjusted as 50% of the active CPCSK9.

To determine the _Kd value for ABP21B12, a BIAcore solution equilibrium binding assay was used. 21B12.1 showed little signal using the KinExA assay, so a biacore solution equilibrium assay was applied. Since no significant binding was observed for the binding of the antibody to the immobilized PCSK9 surface, the 21B12 antibody was immobilized on the flow cell 4 of a CM5 chip using amine binding at a density of about 7000 RU. . Flow cell 3 was used as a background control. Serial dilutions of human PCSK9 or cynomolgus PCSK9 0.3, 1, and 3 nM in a 21B12.1 antibody sample (ranging from 0.001 to 25 nM) in PBS supplemented with 0.1 mg / mL BSA, 0.005% P20. And mixed. Free PCSK9 binding in the mixed solution was measured by injection onto the 21B12.1 antibody surface. 100% PCSK9 binding signal on the 21B12.1 surface was measured in the absence of the mAb in solution. A decreased binding response of PCSK9 with increasing concentrations of the mAb indicated binding of PCSK9 to the mAb in solution, thereby blocking PCSK9 binding to the surface of the immobilized peptibody. The PCSK9 binding signal was plotted against mAb concentration and triplicate sets of curves (immobilized PCSK9 concentrations of 0.3, 1, and 3 nM) using a one-site homogeneous binding model in KinExAPro ™ software. The _KD was calculated from cPCSK9 has the lower protein concentrations than observed from KinExA assay and SDS- gels, the concentration of cPCSK9 Since were not used for the calculation of _{K D,} where its concentration was not adjusted. The results are shown in Table 9.5 below and in FIGS. 5B-5D. FIG. 5B illustrates the results obtained from a solution equilibrium assay at three different hPCSK9 concentrations for hPCSK9. FIG. 5C illustrates a set of similar results for mPCSK9. FIG. 5D shows the results obtained from the biacore capture assay described above.

Example 10
Efficacy of 31H4 and 21B12 blocking D374Y PCSK9 / LDLR binding This example provides IC50 values for two of the antibodies in blocking the ability of PCSK9 D374Y to bind to LDLR. A clear 384-well plate (Costar) was coated with 2 μg / mL of a goat anti-LDL receptor antibody (R & D Systems) diluted in buffer A (100 mM sodium cacodylate, pH 7.4). After thoroughly washing the plate with Buffer A, it was blocked for 2 hours with Buffer B (1% milk in Buffer A). After washing, plates were incubated for 1.5 hours with 0.4 μg / mL LDL receptor (R & D Systems) diluted in buffer C (buffer B supplemented with 10 mM CaCl 2). At the same time as this incubation, 20 ng / mL of biotinylated D374Y PCSK9 with various concentrations of 31H4 IgG2, 31H4 IgG4, 21B12 IgG2, or 21B12 IgG4 antibodies diluted in buffer A or buffer A alone (control). Was incubated. The plate containing the LDL receptor was washed, and the biotinylated D374Y PCSK9 / antibody mixture was transferred to the plate and incubated for 1 hour at room temperature. Binding of biotinylated D374Y to the LDL receptor was detected by incubating with 500 ng / mL streptavidin-HRP (Biosource) in buffer C, followed by TMB substrate (KPL). The signal was quenched with 1N HCl and the absorbance was read at 450 nm.

The results of this binding study are shown in FIGS. In summary, to measure _{IC 50} values for each antibody, 199PM respect 31H4 IgG2 (Figure 6A), 31H4 IgG4 against 156PM (Figure 6B), 170 pM (FIG. 6C) relative 21B12 IgG2, and 21B12 It was found to be 169 pM for IgG4 (FIG. 6D).

  The antibody also blocked binding of wild-type PCSK9 to LDLR in this assay.

Example 11
Cellular LDL Uptake Assay This example demonstrates that various antigen binding proteins can reduce LDL uptake by cells. Human HepG2 cells were seeded at a concentration of 5 × 10 ⁵ cells / well in DMEM medium (Mediatech, Inc) supplemented with 10% FBS in clear bottomed black 96-well plates (Costar) and 37 Incubated overnight at 5 ° C. (5% CO 2). D374Y with various concentrations of antibody diluted in uptake buffer (DMEM with 1% FBS) or uptake buffer alone (control) for 1 hour at room temperature for PCSK9 and antibody complex formation. 2 μg / mL of human PCSK9 was incubated. After washing the cells with PBS, the D374Y PCSK9 / antibody mixture was transferred to the cells, and then LDL-BODIPY (Invitrogen) was diluted in uptake buffer to a final concentration of 6 μg / mL. After incubation at 37 ° C. (5% CO 2) for 3 hours, the cells were washed thoroughly with PBS and the cell fluorescence signal was measured by Safire ™ (TECAN) at 480-520 nm (excitation) and 520-600 nm (emission). Detected.

The results of the cell uptake assay are shown in FIGS. In summary, to measure _{IC 50} values for each antibody, 16.7 nM (Fig. 7A) relative 31H4 IgG2, 13.3nM (Figure 7B) against 31H4 IgG4, 13.3nM respect 21B12 IgG2 ( 7C) and 18 nM for 21B12 IgG4 (FIG. 7D). These results demonstrate that the applied antigen binding protein can reduce the effect of PCSK9 (D374Y) and block LDL uptake by cells. The antibody also blocked the effect of wild-type PCSK9 in this assay.

Example 12
Serum Cholesterol-Reducing Effect of 31H4 Antibody in 6-Day Test To evaluate the reduction of total serum cholesterol (TC) in wild-type (WT) mice via antibody treatment against PCSK9 protein, the following procedure was performed.

  Male WT mice (C57BL / 6 strain, 9-10 weeks old, 17-27 g) obtained from Jackson Laboratory (Bar Harbor, ME) were fed a normal diet (Harland-Teklad, Diet 2918) throughout the duration of the experiment. Gave. At T = 0, mice were administered either anti-PCSK9 antibody 31H4 (2 mg / mL in PBS) or control IgG (2 mg / mL in PBS) through the tail vein of mice at a level of 10 mg / kg. Naive mice were also separated as a naive control group. Dosing groups and sacrifice times are shown in Table 12.1.

  Mice were sacrificed using CO2 asphyxiation at the indicated times shown in Table 9. Blood was collected via the vena cava into Eppendorf tubes and allowed to clot for 30 minutes at room temperature. Samples were then spun down in a tabletop centrifuge at 12,000 xg for 10 minutes to separate serum. Serum total cholesterol and HDL-C were measured using a Hitachi 912 clinical analyzer and a Roche / Hitachi TC and HDL-C kit.

The results of the experiment are shown in FIGS. In summary, mice receiving antibody 31H4 displayed reduced serum cholesterol levels throughout the experiment (FIGS. 8A and 8B). It is further noted that mice show reduced HDL levels (FIGS. 8C and 8D). 8A and 8C, the% change is relative to control IgG at the same time point ( ^* P <0.01, #P <0.05). 8B and 8D,% changes are relative to total serum cholesterol and HDL levels measured in naive animals at t = 0 hours ( ^* P <0.01, #P <0.05).

  With respect to reduced HDL levels, those skilled in the art will appreciate that a decrease in HDL in mice does not suggest that a decrease in HDL occurs in humans, but merely reflects a decrease in serum cholesterol in this organism. It is noted that this is understood. It is noted that mice transport most of the serum cholesterol in high density lipoprotein (HDL) particles, which is different from humans, which have the most serum cholesterol on LDL particles. In mice, the measurement of total serum cholesterol most closely approximates the level of serum HDL-C. Mouse HDL contains apolipoprotein E (apoE), a ligand for the LDL receptor (LDLR), allowing LDLR to clear HDL. Therefore, examining HDL is an appropriate indicator in the present example in mice (it is understood that a decrease in HDL is not expected for humans). In contrast, for example, human HDL does not contain apoE and is not a ligand for LDLR. Because the PCSK9 antibody increases LDLR expression in mice, the liver reduces serum HDL-C levels because the liver can eliminate more HDL.

Example 13
Effect of Antibody 31H4 on LDLR Levels in a 6 Day Study This example shows that, as expected, the antigen binding protein changes the level of LDLR in a subject over time. Western blot analysis was performed to confirm 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 cell debris. The protein concentration in the supernatant was measured using BioRad protein assay reagent (BioRad laboratories). 100 μg of protein was denatured at 70 ° C. for 10 minutes and separated on a 4-12% Bis-Tris SDS gradient gel (Invitrogen). Transfer proteins to 0.45 μm PVDF membrane (Invitrogen) and wash buffer containing 5% non-fat milk for 1 hour at room temperature (50 mM Tris PH7.5, 150 mM NaCl, 2 mM CaCl ₂ , and 0.05% Tween 20). Blocked inside. The blots were then probed with goat anti-mouse LDLR antibody (R & D system) 1: 2000 or anti-β-actin (sigma) 1: 2000 at room temperature for 1 hour. Blots were 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 1 hour incubation at room temperature, the blot was washed thoroughly and immunoreactive bands were detected using the ECL plus kit (Amersham biosciences). Western blots showed an increase in LDLR protein levels in the presence of antibody 31H4, as illustrated in FIG.

Example 14
Serum Cholesterol-Reducing Effect of Antibody 31H4 on 13-day Study In a 13-day study, the following procedure was used to evaluate total serum cholesterol (TC) reduction in wild-type (WT) mice via antibody treatment against PCSK9 protein. went.

  Male WT mice (C57BL / 6 strain, 9-10 weeks old, 17-27 g) obtained from Jackson Laboratory (Bar Harbor, ME) were given normal diet (Harland-Teklad, Diet 2918) throughout the duration of the experiment. Gave. At T = 0, mice were administered either anti-PCSK9 antibody 31H4 (2 mg / mL in PBS) or control IgG (2 mg / mL in PBS) through the tail vein of mice at a level of 10 mg / kg. Naive mice were also separated as a naive control group.

  Dosing groups and sacrifice times are shown in Table 14.1. The animals were sacrificed, the liver was excised and prepared as in Example 11.

  Extending the 6-day experiment to the 13-day test, the same serum cholesterol-lowering effect observed in the 6-day test was observed in the 13-day test. More specifically, animals dosed at 10 mg / kg showed a 31% decrease in serum cholesterol on day 3 and gradually returned to pre-dose levels by day 13. FIG. 10A illustrates the results of this experiment. FIG. 10C illustrates the results of repeating the above procedure using a 10 mg / kg dosage of 31H4, and also using another antibody 16F12 at 10 mg / kg. Dosing groups and sacrifice times are shown in Table 14.2.

As shown in FIG. 10C, both 16F12 and 31H4 resulted in a significant and significant reduction in total serum cholesterol after only a single dose, and were beneficial over a week (10 days or more). Was. The results of the repeated 13-day study were consistent with the results of the first 13-day study, and a 26% reduction in serum cholesterol levels was observed on the third day. For FIGS. 10A and 10B, the% change is relative to control IgG at the same time point ( ^* P <0.01). Referring to FIG. 10C, the% change is relative to control IgG at the same time point ( ^* P <0.05).

Example 15
Effect of Antibody 31H4 on HDL Levels in a 13-day Study The HDL levels on the animals in Example 14 were also determined. HDL levels were reduced in mice. More specifically, animals dosed at 10 mg / kg showed a 33% decrease in HDL levels on day 3 and gradually returned to pre-dose levels by day 13. FIG. 10B illustrates the results of this experiment. HDL levels decreased 34% on day three. FIG. 10B illustrates the results of a repeated 13-day experiment.

  As will be appreciated by those skilled in the art, antibodies reduce mouse HDL, but due to differences in HDL in humans and other organisms (such as mice), this is not expected to occur in humans. Thus, a decrease in mouse HDL does not indicate a decrease in human HDL.

Example 16
Example 14 To determine if repeated administration of antibodies could extend the results obtained in the above example with respect to the additional benefit of additional dosing resulting in continued benefit of the antigen binding peptide, see Example 14 The experiments in and were repeated (the dosing schedule is illustrated in FIG. 11A). The results are shown in FIG. 11B. As evident from the graph in FIG. 11B, both groups of mice showed a significant decrease in total serum cholesterol and received an additional injection of 31H4 ABP, as all mice received the first injection of 31H4 antigen binding protein. Mice showed a continuous decrease in total serum cholesterol, whereas mice that received only control injections eventually showed an increase in total serum cholesterol. Referring to FIG. 11, the% changes are for naive animals at t = 0 hours ( ^* P <0.01, ^** P <0.001).

  The results obtained from this example show that, unlike other cholesterol treatments where repeated applications result in reduced efficacy due to biological regulation in the subject, the approach of the present invention provides Shows that this problem seems to be non-existent. Furthermore, this indicates that the return of total serum cholesterol or HDL cholesterol levels to baseline observed in the previous example was not due to any resistance to the treatments developed by the subject, but was available in the subject. This suggests that this is due to antibody depletion.

Example 17
Use of a PCSK9 Antibody to Treat Hypercholesterolemia A human patient exhibiting symptoms of hypercholesterolemia is administered a therapeutically effective amount of a PCSK9 antibody, such as 31H4 (or, for example, 21B12). At regular time points during treatment, human patients are monitored to determine if serum cholesterol levels have decreased. After treatment, patients receiving treatment with the PCSK9 antibody are found to have reduced serum cholesterol levels compared to untreated arthritis patients.

Example 18
Use of PCSK9 antigen binding protein to prevent hypercholesterolemia Human patients at risk of developing hypercholesterolemia are identified via analysis of family history and / or lifestyle and / or current cholesterol levels You. Subjects are periodically (eg, weekly) administered a therapeutically effective amount of a PCSK9 antibody, 31H4 (or, eg, 21B12). At regular time points during treatment, patients are monitored to determine if their serum cholesterol levels have decreased. Following treatment, subjects receiving prophylactic treatment with PCSK9 antibodies are found to have reduced serum cholesterol levels as compared to untreated subjects.

Example 19
PCSK9 ABP further up-regulated LDLR in the presence of statins This example shows that when used in the presence of statins, ABP against PCSK9 resulted in a further increase in LDLR availability; We show that additional benefits can be achieved with the combination of the two.

  HepG2 cells were seeded in DMEM supplemented with 10% fetal bovine serum (FBS) and grown to approximately 90% confluency. Cells were treated with the indicated amounts of mevinolin (statin, Sigma) and PCSK9 ABP (FIGS. 12A-12C) in DMEM with 3% FBS for 48 hours. Whole cell lysates were prepared. 50 mg of the total protein was separated by gel electrophoresis and transferred to a PVDF membrane. Immunoblot was performed using rabbit anti-human LDL receptor antibody (Fitzgerlad) or rabbit anti-human b-actin antibody. The results of the enhanced chemiluminescence are shown in the top panels of FIGS. Band intensities were quantified by ImageJ software and normalized by b-actin. The relative levels of LDLR are shown in the lower panels of FIGS. ABP 21B12 and 31H4 are PCSK9 neutralizing antibodies, while 25A7.1 is a non-neutralizing antibody.

  HepG2-PCSK9 cells were also generated. These were stable HepG2 cell lines transfected with human PCSK9. Cells were seeded in DMEM supplemented with 10% fetal bovine serum (FBS) and grown to approximately 90% confluency. Cells were treated with the indicated amounts of mevinolin (Sigma) and PCSK9 ABP (FIGS. 12D-12F) in DMEM with 3% FBS for 48 hours. Whole cell lysates were prepared. 50 mg of the total protein was separated by gel electrophoresis and transferred to a PVDF membrane. Immunoblot was performed using rabbit anti-human LDL receptor antibody (Fitzgerlad) or rabbit anti-human b-actin antibody. The results of the enhanced chemiluminescence are shown in the top panel. Band intensities were quantified by ImageJ software and normalized by b-actin.

  As can be seen from the results illustrated in FIGS. 12A-12F, increasing amounts of neutralizing antibodies and increasing amounts of statins generally resulted in increased levels of LDLR. This increase in the effectiveness of increasing levels of ABP is particularly evident in FIGS. 12D-12F where cells have also been transfected with PCSK9 and ABP can show its efficacy to a greater extent.

  Interestingly, the effect of ABP concentration on LDLR levels was dramatically increased when PCSK9 was produced by the cells, as shown by the results of the comparison of FIGS. 12D-12F through 12A-12C. Furthermore, it is clear that neutralizing ABPs (21B12 and 31H4) resulted in a greater increase in LDLR levels even in the presence of statins than 25A7.1 ABPs (non-neutralizing substances), indicating that We show that additional benefits can be achieved by using both ABPs.

Example 20
Consensus Sequence The consensus sequence was determined using standard phylogenetic analysis of the CDRs corresponding to the anti-PCSK9 ABP _VH and _VL . The consensus sequence was determined by retaining adjacent CDRs within the same sequence corresponding to _VH or _VL . Briefly, amino acid sequences corresponding to the entire variable domain, either _VH or _VL , were converted to FASTA format to facilitate performing comparative alignments and estimating phylogeny. Second, due to accidental events, such as irrelevant antibodies that accidentally share a common germline framework inheritance, while still flanking the CDRs within the same sequence corresponding to the _VH or _VL. Replace the framework regions of these sequences with artificial linker sequences ("bbbbbbbbbbbb" surrogate sequences, non-specific nucleic acid constructs) so that only CDRs can be tested without introducing any amino acid position weighting bias. Was. Then, standard ClutalW like algorithms: using a program that uses the (Thompson et al, 1994, Nucleic Acids Res.22. 4673-4680 reference should be a), the sequence similarity juxtaposed inspection, _{V H} of this format Alternatively, the _VL sequence was provided. A gap creation penalty of 8.0 was used, with a gap extension penalty of 2.0. Similarly, the program builds and illustrates sequence group similarities and differences via branch length comparisons and groupings to identify unpaired pair group methods using arithmetic averagings (UPGMA) or adjacent weighting methods. A phyllogram (illustration of a phylogenetic tree) was created based on sequence similarity alignment using the Neighbor-Joining method (see Saitou and Nei, 1987, Molecular Biology and Evolution 4: 406-425). Although both methods yielded similar results, the phylogenetic tree obtained by UPGMA was ultimately used because the UPGMA method uses a simpler, more conservative set of hypotheses. A phylogenetic tree obtained by UPGMA (similar group of sequences (see notes in phylogenetic tree illustration for scale) was defined as having less than 15 substitutions per 100 residues among each sequence in the group) ) Was used to define the consensus sequence set. The results of the comparison are illustrated in FIGS. 13A-13J and FIGS. 31A and 31B. In FIG. 13E, the groups were selected such that the sequence in the light chain forming the clade was also a clade in the heavy chain and had less than 15 substitutions.

Example 21
11F1 Binding Specificity The results from this assay demonstrate that 11F1 binds to PCSK9 but does not bind to PCSK1, PCSK2, PCSK7, or furin, demonstrating the specificity of 11F1 for PCSK9.

  Biotinylated PCSK9 diluted in Buffer A (25 mM Tris, 150 mM NaCl, 0.1% BSA, 0.05% Tween, pH 7.5) was added to a 96-well plate coated with neutravidin in an amount of 0.2 μg. / ML and bound for 1 hour at room temperature. Separately, 11F1 (0.4 μg / mL) was added to any of PCSK1, PCSK2, PCSK7, PCSK9, or furin (R & D Systems, Minneapolis, MN) (diluted in buffer Aw / o Tween). Incubated for 1 hour at room temperature at concentrations (range 0-20 μg / mL). Furin inhibitor was included in all reactions containing furin at 4.5 μg / mL. The PCSK9 coated streptavidin plate was washed with buffer A, the antibody / proprotein convertase mixture was added to the plate and incubated for 1 hour at room temperature. After washing, bound antibody was detected by incubation with goat α-human Fc-HRP (160 ng / mL, diluted in buffer A) (Jackson Laboratories, Bar Harbor, ME), and then with TMB substrate. . The reaction was stopped with 1N HCl and the absorbance was read at a wavelength of 450 nm on a Spectramax Plus 384 spectrophotometer (Molecular Devices Inc., Sunnyvale, Calif.).

  This assay relied on the ability of the proprotein convertase in solution to compete with the binding of 11F1 to PCSK9 captured by the plate. By pre-incubation of 11F1 and PCSK9 in solution, the amount of 11F1 binding to PCSK9 captured by the plate was reduced in a dose-dependent and robust manner and detected as a decrease in OD450 (FIG. 14). All results were expressed as mean OD450 values ± standard deviation vs. concentration of proprotein convertase. Preincubation of 11F1 with PCSK1, PCSK2, PCSK7, or furin in solution did not significantly affect the binding of 11F1 to PCSK9 captured by the plate. Thus, at the protein concentrations tested, 11F1 bound only to PCSK9 and did not bind to the other proprotein convertase family members tested.

Example 22
Efficacy of 11F1 Inhibition of LDLR: PCSK9 Binding This example demonstrates that nanomolar 11F1 is capable of inhibiting both D374Y and wild-type PCSK9 binding to LDLR under the conditions of this assay. are doing.

  Briefly, clear 384-well plates were coated with 2 μg / mL of goat anti-LDL receptor antibody (R & D Systems, Minneapolis, MN) diluted in PBS and incubated overnight at 4 ° C. After thoroughly washing the plate with buffer A (100 mM sodium cacodylate, pH 7.5), buffer B (1% nonfat dry milk in buffer A [Bio-Rad Laboratories, Hercules, CA]) was used. Blocked for 2 hours at room temperature. After washing, plates were incubated for 1.5 hours at room temperature with 0.4 μg / mL LDL receptor (R & D Systems, Minneapolis, MN) diluted in buffer C (buffer B supplemented with 10 mM CaCl 2). . Simultaneously with this incubation, 20 ng / mL of biotinylated D374Y PCSK9 or 100 ng / mL of biotinylated wild-type PCSK9 was incubated with various concentrations of anti-PCSK9 antibody 11F1 diluted in buffer A (final). Concentrations ranged from 6.0 μg / mL to 200 μg / mL for the D374Y PCSK9 assay or from 3.1 μg / mL to 25 μg / mL for the wild-type PCSK9 assay). The plate coated with LDLR was washed and a biotinylated PCSK9 / antibody mixture was added. LDLR plates were incubated for 1 hour at room temperature. Binding of biotinylated PCSK9 to the LDL receptor was detected by incubating with streptavidin-HRP (500 ng / mL in buffer C) and then with TMB substrate. The reaction was stopped with 1N HCl and the absorbance was read at a wavelength of 450 nm on a Spectramax Plus 384 spectrophotometer (Molecular Devices Inc., Sunnyvale, CA). Using GraphPad Prism (v4.01) software, the log of antibody concentration versus OD450 was plotted and IC50 values were determined by non-linear regression.

  11F1 inhibited LDLR: PCSK9 binding. The IC50 values for 11F1 in the D374Y PCSK9 assay ranged from 7.3 nM to 10.1 nM, with a mean (± standard deviation) 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 a mean (± standard deviation) of 5.9 nM ± 1.9 nM (n = 3). Note that these IC50 values are dependent on the amount of recombinant D374Y PCSK9 or wild-type PCSK9 used in the binding assay. Representative dose response curves for both the D374Y and wild type assays are shown in FIGS. 15 and 16, respectively.

Example 23
Effect of 11F1 on blocking cellular LDL uptake 11F1 can block the interaction between PCSK9 and LDLR in vitro and prevent PCSK9-mediated reduction of LDL uptake in HepG2 cells.

  Briefly, 5x104 cells / well in DMEM medium (Mediatech Inc., Herndon, VA) supplemented with 10% FBS and 1% antimicrobial antifungal solution (Mediatech Inc., Herndon, VA). At a concentration, human HepG2 cells were seeded in clear bottom black 96-well plates (Fisher Scientific CO LLC, Santa Clara, CA). Cells were incubated overnight at 37 ° C. (5% CO 2). In the range of 666.7 nM to 0.7 nM (to block D374Y PCSK9) or (to block wild-type PCSK9) to form a complex of D374Y PCSK9 and antibody or antibody of wild-type PCSK9. Serial dilutions (1: 2) of 11F1 ranging from 3.3 μm to 3.3 nM) were prepared in formulation buffer (25 mM HEPES, pH 7.5, 0.15 M NaCL). Either D374Y PCSK9 (2 μg / mL) or wild-type PCSK9 (25 μg / mL) was diluted in uptake buffer (DMEM with 1% FBS) and various concentrations of 11F1 or uptake buffer alone (negative control) And incubated at room temperature with shaking for 1 hour. BODIPY-LDL (Invitrogen, Carlsbad, CA) was diluted to a concentration of 12 μg / mL in uptake buffer. After overnight incubation, HepG2 cells were washed twice with DPBS (Mediatech Inc., Herndon, VA). 25 μL of a complex of 11F1 and D374Y PCSK9 or wild-type PCSK9 and 25 μL of diluted BODIPY-LDL (Invitrogen, Carlsbad, Calif.) Were added to the cells and incubated at 37 ° C. (5% CO 2) for 3 hours. Cells were washed with DPBS for 5 hours and resuspended in 100 μL DPBS. Fluorescence signals were detected as relative fluorescence units (RFU) at 480-520 nm (excitation) and 520-600 nm (emission) using a Safire plate reader (Tecan Systems Inc., San Jose, CA).

  Using GraphPad Prism (version 4.02, GraphPad Software Inc., San Diego, Calif.) Software, plot the log of antibody concentration versus RFU and use a sigmoidal dose response (variable slope) curve fitting program to perform non-linear regression. To determine the EC50 value.

This example shows that 11F1 blocked D374Y PCSK9 or wild-type PCSK9-mediated reduction of LDL uptake in HepG2 cells in a dose-dependent manner. By adding recombinantly purified D374Y PCSK9 (2 μg / mL) or wild-type PCSK9 (25 μg / mL) to HepG2 cells, BODIPY-LDL uptake was approximately 50-60% of the level measured in untreated cells and Each was reduced to about 40%. The antibody returned LDL uptake in a dose-dependent manner to the levels observed in untreated cells. The mean (± standard deviation) EC50 value of 11F1 for blocking D374Y PCSK9-mediated reduction of LDL uptake was 35.3 ± 9.1 nM (n = 6, FIG. 17). The EC50 value for the ability of 11F1 to block wild-type PCSK9-mediated reduction of LDL uptake was 124.2 ± 28.5 nM (n = 3, FIG. 18). Note that these EC50 values are dependent on the amount of recombinant D374Y PCSK9 or wild-type PCSK9 used in the cell assay. Since the binding affinity of D374Y PCSK9 to LDLR is 5-30 fold over that of wild-type PCSK9, EC50 values are lower for D374Y PCSK9 than for wild-type PCSK9 because less D374Y PCSK9 was used in the assay. (Cunningham et al, 2007; Fisher et al, 2007; Kwon et al, 2008).
The EC50 values reported here are representative averages from 3-6 separate measurements for 11F1.

Example 24
Effect of 11F1 and 8A3 blocking human PCSK9 expressed via adeno-associated virus in a mouse model Serum non-HDL in mice expressing human PCSK9 by AAV by single intravenous bolus administration of anti-PCSK9 antibody 11F1 or 8A3 Leading to a significant reduction in C and TC. This example demonstrates both effects of anti-PCSK9 antibodies blocking the function of human PCSK9 in vivo.

  Briefly, 120 C57BL / 6 mice expressing human PCSK9 were generated by infection with a modified adeno-associated virus (AAV) encoding human PCSK9 to produce low density lipoprotein cholesterol (LDL-C). Increased circulation levels resulted. Serum cholesterol analysis was performed using a Cobas Integra 400 plus chemical analyzer (Roche Diagnostics, Indianapolis, Ind.). Animals were randomized to treatment groups with similar levels of non-HDL-C (LDL-C and VLDL-C), HDL-C, and TC. On day 0 of treatment (T = 0), some of the mice were euthanized and serum was collected to establish baseline levels for that day. The remaining mice were then administered 30 mg / kg of 11F1, 8A3, or anti-keyhole limpet hemocyanin (KLH) IgG2 control antibody via tail vein injection. One to five days after injection, some of the mice were euthanized and whole blood was collected from the vena cava and allowed to clot for 30 minutes at room temperature. After centrifugation at 12,000 rpm for 10 minutes in a tabletop centrifuge, serum was collected. Serum cholesterol analysis was performed using a Cobas Integra 400 plus chemical analyzer.

  The serum concentration of PCSK9 was determined using a sandwich ELISA assay. Clear 96-well plates were coated overnight with 2 μg / ml of monoclonal anti-PCSK9 antibody (31H4) diluted in 1 × PBS. 1 × PBS /. After thoroughly washing the plate with 05% Tween, the plate was blocked with 3% BSA / 1 × PBS for 2 hours. After washing, plates were incubated for 2 hours with serum diluted in common assay diluent (Immunochemistry Technologies, Bloomington, MN). Recombinant human PCSK9 (1 ng / ml to 500 ng / ml) was evaluated simultaneously and used to generate a standard curve on each ELISA plate. After addition of rabbit polyclonal biotinylated anti-PCSK9 antibody (D8773, Amgen Inc, CA) at 1 μg / ml (in 1% BSA / PBS), neutravidin-HRP was added at 200 ng / ml (in 1% BSA / PBS). Was added. Bound PCSK9 was detected by incubation with TMB substrate. The reaction was stopped with 1N HCl and the absorbance was read at 450 nm on a Spectramax Plus 384 spectrophotometer (Molecular Devices Inc., Sunnyvale, Calif.). The corresponding concentration of PCSK9 in serum samples was determined using a standard curve (4-parameter logistic fit) generated with recombinant human PCSK9.

  The serum concentration of PCSK9 was determined using a sandwich ELISA assay. Polyclonal goat anti-human Fc IgG and HRP-labeled goat anti-human IgG Fcγ polyclonal reagents (both obtained from Jackson ImmunoResearch Laboratories Inc, West Grove, PA) were used as capture and detection antibodies, respectively. A 3,3 ′, 5,5 ′ tetramethylbenzidine (TMB) substrate solution was reacted with peroxide and in the presence of horseradish peroxidase (HRP), proportional to the amount of each anti-PCSK9 antibody bound by the capture reagent. A colorimetric signal was generated. Color (optical density, OD) intensity was measured at 450-650 nm using a microplate reader (Spectra Max Plus 384). Data were analyzed using a Watson version 7.0.0.01 (Thermo Scientific, Waltham, Mass.) Data reduction package using a separately prepared standard curve logistic (automatic estimation) regression model. The lower limit of quantification (LLOQ) of the assay was 34.4 ng / mL.

Calculation of Pharmacokinetic Parameters in AAV Mice Using WinNonlin Enterprise version 5.1.1 (Pharsight, St. Louis, MO), non-compartmental analysis of serum concentration (NCA) using predetermined reference time points for each subject went. Data points for estimating the terminal phase elimination rate constant and half-life were selected by visual inspection of the concentration-time profile. The reported NCA parameters included the apparent half-life (t1 / 2), the area under the serum concentration-time curve from time 0 to the last measured concentration (AUC0-t), and the apparent serum clearance (CL0). -T) is included. AUC0-t was determined using the linear / log-linear trapezoidal method, and CLO-t was calculated by dosage / AUC0-t for 11F1, 8A3, and 31H4 antibodies. Post-test dosage solution analysis showed that the actual dosage was within 20% of the 30 mg / kg target. However, analysis showed that for the IgG2 control, the actual dosage was only 40% of the target of interest. Therefore, a corrected dosage of 12 mg / kg was used for calculating the CL0-t of the IgG2 control. The parameters were reported with three significant figures, except for the half-life reported with two significant figures.

Statistical analysis All cholesterol results were expressed as mean ± sem. All pharmacokinetic data were expressed as mean ± standard deviation. Using a p-value of 0.05 determined by one-way analysis of variance as a threshold, the statistical significance between animals injected with anti-KLH IgG2 control antibody and animals dosed with anti-PCSK9 antibody at the same time point was determined. .

Effect of anti-PCSK9 antibody on serum non-HDL-C, HDL-C, and TC To establish a baseline, a portion of mice expressing human PCSK9 were euthanized prior to antibody injection 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 (mean ± standard error), respectively. PCSK9 levels in na ナ イ ve animals were determined to be 4921 ng / mL ± 2044 ng / mL.

  Compared to mice injected with an anti-KLH IgG2 control antibody (control animals), injection of 11F1 resulted in a significant reduction in non-HDL-C on days 1, 2 and 4 post injection (up to 59%). ), But TC resulted in a significant reduction (22%) only on day 4 (FIGS. 19, 20). No significant reduction in HDL-C was observed at any time point (FIG. 21).

  Compared to control animals, injection of 8A3 resulted in a significant decrease (up to 65%) in non-HDL-C on days 1, 2, and 4 post-injection, while TC Day 2 resulted in a significant reduction (up to 24%) (FIGS. 19, 20). No significant decrease in HDL-C was observed at any time point (FIG. 21).

Pharmacokinetics At 30 mg / kg iv, 11F1 and 8A3 had very similar pharmacokinetic behavior (FIG. 22). For these two molecules, AUC0-t exposure, estimated CL0-t, and apparent half-life were comparable (table in FIG. 23). The anti-KLH IgG2 control antibody unexpectedly reduced AUC0-t exposure than 11F1 and 8A3, likely due to the administration of the antibody at a lower than intended dosage (30 mg / kg). Dose solution analysis showed that the antibody concentration was 12 mg / kg and the antibody concentration was 40% of the target in contrast to the anti-KLH IgG2 control antibody CL0-t, which was calculated using the corrected dose, was 11F1 And 8A3, the apparent half-life of the anti-KLH IgG2 control antibody was estimated to be more than 120 hours, since the CL0-t values of 11F1 and 8A3 were closer to the anti-KLH IgG2 control antibody, the AAV model The effect of PCSK9 ligand on the pharmacokinetics of the antibody is less pronounced for 11F1 and 8A3 when compared to other antibodies administered in Absence was shown by these data.

Overview Expression of human PCSK9 by AAV (about 5 μg / mL) in mice resulted in serum non-HDL-C levels of about 33 mg / dL. After 30 mg / kg injection of 11F1, a significant reduction in serum non-HDL-C was observed on days 1, 2, and 4 post injection (up to 59% compared to control animals). A significant reduction in TC was seen only on day 4. Injection of 8A3 resulted in a similar pattern of up to 65% non-HDL-C reduction compared to control animals. However, 8A3 administration resulted in significant TC reduction only on day 2 after injection, up to 24%. No significant reduction in HDL-C was observed in animals receiving either 11F1 or 8A3. Analysis of serum antibody levels of 11F1 and 8A3 demonstrated a similar profile as the anti-KLH IgG2 control antibody.

Example 25
Effect of a single subcutaneous administration of 11F1, 21B12, and 8A3 on cynomolgus monkey serum lipids A single subcutaneous administration of 11F1, 8A3, or 21B12 to cynomolgus monkeys results in a significant reduction in serum LDL-C and TC. This test demonstrates that anti-PCSK9 antibodies are capable of reducing serum cholesterol in non-human primates.

  Briefly, naive male cynomolgus monkeys were acclimated to the environment for at least two weeks before the experiment. Animals were randomized into treatment groups based on serum TC, HDL-C, LDL-C, and triglyceride levels, and pre-screening of body weight. One week later, the animals were fasted overnight, blood was collected from the peripheral vasculature (cephalic or saphenous vein), and baseline serum lipid levels were measured at designated time points (T = 0). The animals were then given 0.5 mg / kg of any of the anti-KLH IgG2 control antibodies, 11F1, 21B12, or 8A3 (all in 10 mM NaOAc, pH 5.2, 9% sucrose) at 0.5 mg / kg (all 0.4 mL / kg). (By weight). Thereafter, fasting blood samples were collected from the animals at designated times over a period of 45 days.

  At the specified time points, blood was collected from the peripheral vasculature (radiocutaneous or saphenous vein) of animals under overnight fasting conditions. Whole blood was allowed to clot for 30 minutes at room temperature. After centrifugation at 3,000 rpm for 20 minutes, the serum was collected. Serum cholesterol analysis was performed directly using a Cobas Integra 400 analyzer (Roche Diagnostics Inc, Indianapolis, Ind.). Apolipoprotein B serum levels at specific time points (Day 0, Day 3, Day 6, Day 15, Day 24, and Day 33) were determined by Analytics, MD in the following manner. A 17 μL aliquot of the sample (no prior preparation) was used for analysis on a Hitachi 717 analyzer using a 6-point standard curve. If the initial value of the sample was higher than the standard curve linearity, the sample was diluted and the result multiplied by the appropriate dilution factor and repeated. Reagents for the assay (APO-B reagent kit number 86071, antibody set number 86060, control set number 86103) were obtained from DiaSorin (Stillwater, MN).

  Antibody concentrations in serum were determined using an enzyme-linked immunosorbent assay (ELISA) in an assay range of 34.4-3000 ng / mL (34.4 ng / mL is the lower limit of quantification [LLOQ]).

  Non-compartmental analysis (NCA) of serum concentrations was performed by Watson® LIMS version 7.0.0.01 (Thermo Scientific, Waltham, Mass.) Using predetermined reference time points for each subject. Data points for estimating the terminal elimination rate constant and half-life were selected by visual inspection of the concentration-time profile and optimal linear fit (typically about 360 hours, when the antibody concentration was below the lower limit of quantification). Until it falls). Reported NCA parameters included terminal half-life (t1 / 2, z), maximum serum concentration (Cmax), area under the serum concentration-time curve from time 0 to infinity (AUC0-inf), and apparent Serum clearance (CL / F) is included. AUC0-inf was calculated using the linear / log-linear trapezoidal method. All parameters were all reported with three significant figures except for the half-life reported with two significant figures.

Statistical analysis Statistical models that considered the baseline as a covariate and the treatment group as a parametric effect were fitted to the log-transformed responses at each time point for LDL-C, HDL-C, TC, and triglycerides. Tukey's multiple comparison correction was applied to adjust the pairwise comparison at each time point. Statistical significance was evaluated at α = 0.05 using adjusted p-values.

Effect of 11F1, 21B12, and 8A3 on Serum LDL Cholesterol The maximal LDL-C reduction of 11F1 was observed on day 9 post-injection, and LDL-C was reduced by 57% 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. The maximum LDL-C reduction of 21B12 was observed on day 3 after injection, with a 64% reduction in LDL-C compared to control animals. LDL-C returned to levels similar to control animals by day 6. The maximum LDL-C reduction of 8A3 was observed 4 days after injection, with a 54% reduction in LDL-C 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 A maximal TC reduction of 11F1 was observed on day 9 post injection, with a 27% reduction in TC 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. The maximum TC reduction of 21B12 was observed on day 3 post injection, with a 20% reduction in TC compared to control animals. TC returned temporarily to levels similar to those observed in vehicle-treated monkeys by day 4, but dropped significantly between days 14-18 (including days 14 and 18). The maximum TC reduction of 8A3 was observed on day 9 after injection, with a 22% reduction in TC compared to control animals. TC returned to levels similar to those observed in control animals by day 30 (FIG. 25).

Effect of 11F1, 21B12, and 8A3 on Serum HDL Cholesterol and Triglycerides On average and at each time point, HDL-C or triglyceride levels in animals treated with 11F1 or 8A3 were similar to those observed in anti-KLH IgG2 control antibody-treated monkeys. Not significantly different (based on α = 0.05 significance level). However, 21B12 showed a statistically significant change in HDL-C at a single time point (18 days after injection) (FIGS. 26 and 27).

Effect of 11F1, 21B12, and 8A3 on Apolipoprotein B (ApoB) Serum ApoB levels were measured on days 3, 6, 15, 24, and 33 post-injection. 11F1 and 8A3 were associated with ApoB reduction on days 3-24 compared to anti-KLH IgG2 control antibody treated monkeys (FIG. 28). 21B12 was associated with statistically significantly lower ApoB levels only on day 3.

Pharmacokinetic Profiles of 11F1, 21B12, and 8A3 A summary plot of the mean concentration-time profiles with treatment is shown in FIG. The estimated mean pharmacokinetic parameters of animals receiving 11F1, 21B12, 8A3, and anti-KLH IgG2 control antibodies are shown in the table of FIG.

  Antibody absorption in all groups was consistent with the characteristics of subcutaneous antibody administration. The 21B12 pharmacokinetic behavior for CL / F, Cmax, and AUC0-inf was consistent with that observed in previous studies where 21B12 was administered at the same dose. The pharmacokinetics of 11F1 and 8A3 are significantly different from 21B12, with lower CL / F observed (約 15% 21B12 CL / F) and a longer half-life estimated (40 hours of 21B12). About 200 hours). In particular, the pharmacokinetics of 11F1 and 8A3 were indistinguishable from each other and from anti-KLH IgG2 control antibodies. Given that 11F1 and 8A3 have the same exposure profile as an anti-KLH IgG2 control antibody that has no affinity for PCSK9, the pharmacokinetics of 11F1 and 8A3 are more affected than 21B12 in association with the PCSK9 target. These data suggest that to a much lesser degree.

Summary of Results In a study over 45 days, a statistically significant reduction in TC and LDL-C was observed in animals that received 11F1, 21B12, or 8A3 compared to anti-KLH IgG2 control antibodies. 11F1 was associated with a statistically significant LDL-C reduction (compared to the anti-KLH IgG2 control antibody) on days 2-24 (including days 2 and 24). 21B12 showed a statistically significant LDL-C reduction (compared to the anti-KLH IgG2 control antibody) on days 1-4 (including days 1 and 4). 8A3 showed a statistically significant LDL-C reduction (compared to the anti-KLH IgG2 control antibody) on days 1-24 (including days 1 and 24). The changes in TC and ApoB were very similar to those observed with LDL-C in all groups. 11F1 achieved maximal reduction in LDL-C 9 days post-injection (compared to anti-KLH IgG2 control antibody at the same time point) (-57%). 21B12 achieved maximal reduction in LDL-C 3 days after injection (compared to anti-KLH IgG2 control antibody at the same time point) (-64%). 8A3 achieved maximal reduction of LDL-C 4 days after injection (compared to anti-KLH IgG2 control antibody at the same time point) (-54%). 21B12 reduced HDL-C at a single time point, 18 days after injection. No statistically significant change was observed in HDL-C levels after 11F1 or 8A3 administration. No statistically significant change in triglyceride levels after 11F1, 21B12, or 8A3 administration was observed.

Example 26
Two-part study to evaluate the safety, resistance, and efficacy of human anti-PCSK9 antibody to LDL-C in subjects with heterozygous familial hypercholesterolemia Study design: This is a two-part study is there. Part A is an open-label, single-arm, multicenter pilot study. Part B is the same design as Part A except that the entry has been extended, antibody, 21B12, double-blind (heavy chain SEQ ID NO: 592 and light chain SEQ ID NO: 591), randomized, placebo control, This is a multicenter trial. Both the inclusion / exclusion criteria and the evaluation schedule are the same for Part A and Part B.

The inclusion criteria include:
• Men and women between 12 and 65 years old • Diagnosis of homozygous familial hypercholesterolemia • Stable lipid reduction therapy for at least 4 weeks • LDL cholesterol> 130 mg / dl (3.4 mmol / L) • Triglycerides Is less than 400 mg / dL (4.5 mmol / L).
Exclusion criteria are:
• LDL or plasma apheresis within 8 weeks prior to randomization • New York Heart Association (NYHA) class III or IV or latest known left ventricular ejection fraction <30% • 3 months prior to randomization Within myocardial infarction, unstable angina, percutaneous coronary intervention (PCI), coronary artery bypass graft (CABG), or seizures, planned cardiac surgery or revascularization, uncontrolled cardiac arrhythmias, poor management Including hypertension.

  Evaluation schedules include, but are not limited to, adverse event collection (AE) and significant adverse event (SAE) data, vital signs, concomitant medications, and laboratory tests.

  Subjects who meet the inclusion / exclusion criteria are instructed to follow the NCEP Adult Treatment Panel TLC (or equivalent) diet and are required to maintain current lipid-lowering therapy throughout the study.

  The 21B12 formulation is presented as a sterile, clear, colorless frozen liquid. Each sterile vial fills a 1 mL deliverable volume of 70 mg / mL 21B12 formulation combined with 10 mM sodium acetate, 9% (w / v) sucrose, 0.004% (w / v) polysorbate 20, pH 5.2. . Each vial is for single use. Placebo is presented in the same container as a clear, colorless, sterile, protein-free frozen solution, 10 mM sodium acetate, 9% (w / v) sucrose, 0.004% (w / v) polysorbate 20, Formulated as pH 5.2.

  In part A, 8 subjects enrolled on stable lipid-lowering medication for more than 4 weeks (or 4 weeks), genetically confirmed to be homozygous familial hypercholesterolemia, were enrolled. Received the 21B12 formulation open-label. Table 26.1 shows the genotype of the patients in the study.

  The 21B12 formulation (420 mg) is administered subcutaneously every 4 weeks for 12 weeks, and treated at 4 week intervals for an additional 12 weeks, followed by AMG145 420 mg every 2 weeks for 12 weeks. Study visits are performed at least every 4 weeks. During these visits, adverse event (AE) and important adverse event (SAE) data, vital signs, concomitant medications, laboratory tests, etc. will be collected.

  Changes in lipid-related parameters and% changes are shown in Table 38.2. At week 12 of every 4 weeks of treatment, the average LDL cholesterol by ultracentrifugation decreased 16.5% (70.6 mg / dL; 1.8 mmol / L) from baseline, from + 5.2% to -43%. 0.6%. Four (50%) patients had a reduction of 15% or more, and three of the four (38%) achieved LDL cholesterol reduction of 30% or more. Patients with negative LDL receptor activity do not have reduced LDL cholesterol.

  After 12 weeks of biweekly treatment, the mean LDL cholesterol reduction from baseline was 13.9% (60.8 mg / dL; 1.6 mmol / L). LDL cholesterol lowering was not observed in LDL receptor negative patients, but a greater reduction occurs in patients with receptor dysfunction (Table 26.3). Three patients (38%) have reduced LDL cholesterol by more than 30%. Receptor deficient patients average 22.9% and 23.6%, respectively, over the 12 week treatment period when administered every 4 weeks and every 2 weeks (Table 26.3).

  The changes from baseline at week 12 in apolipoprotein B and related lipoproteins at 4 weekly and biweekly doses are shown in Table 26.2. The average change in Lp (a) was -11.7% and -18.6% at 4 weekly and 2 weekly doses, respectively, which does not appear to be related to LDL receptor activity. It is. Triglycerides are reduced by 5.7% and increased by 5.9%, respectively, when administered every 4 weeks and every 2 weeks. HDL-cholesterol and apolipoprotein A1 are substantially unchanged when administered every 4 weeks or every 2 weeks (Table 26.2). Treatment every 4 weeks with 420 mg of the 21B12 formulation reduces free PCSK9 by 22.7% and 87.6% at 12 weeks of dosing every 4 weeks and every 2 weeks, respectively (Table 26.2).

Values are means (standard error) unless otherwise specified.
^{* To} convert cholesterol values to millimoles per liter, multiply by 0.0259. To convert apolipoprotein A1 or apolipoprotein B values to grams per liter, multiply by 0.01. To convert triglyceride values to millimoles per liter, multiply by 0.0113. To convert the value of free PCSK9 to nanomoles per liter, divide by 72.
^† Median (interquartile range).
Q4W: every 4 weeks, Q2W: every 2 weeks, SE: standard error, LDL: low density lipoprotein, HDL: high density lipoprotein, PCSK9: proprotein convertase subtilisin / kexin type 9.

  About 51 new subjects are registered in Part B. Enrolled subjects will be randomized to two treatment groups of 420 mg 21B12Q4W sc or placebo Q4W sc in a 2: 1 distribution. Randomization is stratified by baseline LDL-C level. Investigational visits are made every four weeks and two random visits are made on the second and tenth weeks. Visits will involve the collection of AE and SAE data, vital signs, concomitant medications, laboratory tests, etc. Fasting lipid panels are collected at 6 weeks to assess nadir LDL-C levels in response to 21B12 treatment. The 21B12 formulation is administered on days 1, 4, and 8. End-of-trial (EOS) visits and final lipid estimates are made at week 12 for all subjects.

INCORPORATION BY REFERENCE All references cited herein, including patents, patent applications, papers, textbooks, and references cited therein, if not already incorporated, Which is incorporated herein by reference in its entirety. If any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the terms and definitions of the present invention will control.

Equivalents The foregoing 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 envisioned by the inventors. However, no matter how detailed the preceding description is in the text, the invention may be embodied in many forms and the invention shall be construed in accordance with the appended claims and any equivalents thereof. It is understood that it should.

Claims (11)

Light chain complementarity determining region (CDR) 1 of the CDRL1 sequence of SEQ ID NO: 23, CDRL2 of the CDRL2 sequence of SEQ ID NO: 23, CDRL3 of the CDRL3 sequence of SEQ ID NO: 23, and heavy chain complementarity of the CDRH1 sequence of SEQ ID NO: 49 1 and determining region (CDR), a CDRH2 of CDRH2 sequence of SEQ ID NO: 49, is administered an anti-PCSK9 antibody comprising a CDRH3 of CDRH3 sequence of SEQ ID NO: 49 420mg seen including, every 2 weeks to patients at a dose of 420mg a pharmaceutical formulation for decreasing serum LDL cholesterol in patients with dysfunction of LDL receptor activity. Light chain complementarity determining region (CDR) 1 of the CDRL1 sequence of SEQ ID NO: 23, CDRL2 of the CDRL2 sequence of SEQ ID NO: 23, CDRL3 of the CDRL3 sequence of SEQ ID NO: 23, and heavy chain complementarity of the CDRH1 sequence of SEQ ID NO: 49 and determining region (CDR) 1, a CDRH2 of CDRH2 sequence of SEQ ID NO: 49, is administered an anti-PCSK9 antibody comprising a CDRH3 of CDRH3 sequence of SEQ ID NO: 49 420mg seen including, to a patient at a dose of one 420mg in 1 month that the pharmaceutical preparation for reducing serum LDL cholesterol in patients with dysfunction of LDL receptor activity.   CDRL1 is SEQ ID NO: 158, CDRL2 is SEQ ID NO: 162, CDRL3 is SEQ ID NO: 395, CDRH1 is SEQ ID NO: 308 or 368, CDRH2 is SEQ ID NO: 175, CDRH3 is SEQ ID NO: 180, The pharmaceutical preparation according to claim 1.   The pharmaceutical preparation according to any one of claims 1 to 3, wherein the anti-PCSK9 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49. Anti-PCSK9 antibody
(A) the light chain constant sequence of SEQ ID NO: 156, or
(B) the light chain constant sequence of SEQ ID NO: 157, or
(C) the heavy chain constant sequence of SEQ ID NO: 154, or
(D) the heavy chain constant sequence of SEQ ID NO: 155, or
(E) the light chain constant sequence of SEQ ID NO: 156 and the heavy chain constant sequence of SEQ ID NO: 154, or
(F) the light chain constant sequence of SEQ ID NO: 157 and the heavy chain constant sequence of SEQ ID NO: 154, or
(G) the light chain constant sequence of SEQ ID NO: 156 and the heavy chain constant sequence of SEQ ID NO: 155, or
(H) the light chain constant sequence of SEQ ID NO: 157 and the heavy chain constant sequence of SEQ ID NO: 155,
The pharmaceutical preparation according to any one of claims 1 to 4, further comprising:   The pharmaceutical formulation according to any one of claims 1 to 5, wherein the anti-PCSK9 antibody further comprises a glycine residue at the C-terminal of the light chain variable domain.   The pharmaceutical preparation according to any one of claims 1 to 3, wherein the anti-PCSK9 antibody has a light chain including the amino acid sequence of SEQ ID NO: 593 and a heavy chain including the amino acid sequence of SEQ ID NO: 592.   The pharmaceutical preparation according to any one of claims 1 to 3, wherein the anti-PCSK9 antibody comprises a light chain variable region including the amino acid sequence of SEQ ID NO: 591 and a heavy chain variable region including the amino acid sequence of SEQ ID NO: 590. .   The pharmaceutical formulation according to any one of claims 1 to 8, which is formulated for parenteral administration.   9. A pharmaceutical formulation according to any one of the preceding claims, formulated for intravenous administration.   The pharmaceutical formulation according to any one of claims 1 to 8, which is formulated for subcutaneous administration.

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