Antigen-binding proteins for proprotein converterases subtilisin keksin type 9 (PCSK9)

Abstract

This invention provides an antigen-binding protein that binds to proprotein converterase subtilisin kexin type 9 (PCSK9), as well as a method for using and producing the antigen-binding protein. [Solution] The present invention provides an isolated neutralizing antigen-binding protein that interacts with proprotein converterase subtilisin kexin type 9 (PCSK9), and which contains a specific amino acid sequence that binds to the PCSK9 protein and reduces the LDLR-reducing effect of PCSK9 on LDLR.

Description

[Technical Field]

[0001] Related applications This application is based on U.S. Provisional Application No. 60 / 957,668, filed on August 23, 2007. Application No. 61 / 008,965 filed on December 21, 2007, and application filed on January 9, 2008. Issues 61 / 010,630 (these in their entirety are incorporated herein by reference) They claim priority.

[0002] Field of Invention This invention relates to proprotein converterase subtilisin keksin type 9 (PCSK9). This relates to antigen-binding proteins and methods for using and producing said antigen-binding proteins. ru. [Background technology]

[0003] Proprotein converterase subtilisin kexin type 9 (PCSK9) is a low-density protein serine proteas involved in regulating the levels of defibrillator receptor (LDLR) proteins (Horton et al., 2007; Seidah and Prat (2007). In vitro experiments showed that the addition of PCSK9 to HepG2 cells increased the cell surface L This has been shown to reduce DLR levels (Benjannet et al., 2004;Lagace et al., 2006;Maxwell et al., 2005; Park et al., 2004). Experiments using mice were conducted using PCSK. 9. Increasing protein levels reduces LDLR protein levels in the liver. (Benjannet et al., 2004; Lagace et al., 2006; Maxwell et al., 2005; Park et al. (2004) PCSK9 knockout mice showed increased levels of LDLR in the liver. It was shown that it possesses (Rashid et al., 2005). Furthermore, plasma LD Various human PCSK9 mutations resulting in either increased or decreased levels of L have been identified. (Kotowski et al., 2006;Zhao et al., 2 006). PCSK9 directly interacts with the LDLR protein and, together with LDLR, interacts with the cell. It is taken up internally and emits immunofluorescence simultaneously with LDLR throughout the endosomal pathway (L (agace et al., 2006) has shown that PCSK9 affects LDL No degradation of R was observed, and the mechanism by which it reduces extracellular LDLR protein levels remains unknown. That is the case.

[0004] PCSK9 is a proforma in the subtilisin (S8) family of serine proteases. It is an om-proprotein converterase (Seidah et al., 2003). Humans have nine prohormones that can be divided into the S8A and S8B subfamilies. It possesses a protein converterase (Rawlings et al, 2006). Furylin, PC1 / PC3, PC2, PACE4, PC4, PC5 / PC6 and PC7 / PC8 / LPC / SPC7 are classified under the subfamily S8B. Mouse furin The crystal and NMR structures of different domains derived from PC1 are subtilisin-like prodomains. And the catalytic domain and the P domain immediately at the C-terminus of the catalytic domain are revealed (Hen (och et al, 2003; Tangrea et al, 2002). This Based on the similarity of amino acid sequences within the family, all seven members have similar structures. It is expected to have (Henrich et al., 2005). SKI-1 / S1 P and PCSK9 belong to the subfamily S8A. The sequences of these proteins are shown. The comparison also suggests the presence of subtilisin-like prodomains and catalytic domains (Sakai et al., 1998; Seidah et al., 2003; (h et al, 1999). In these proteins, the catalytic domain is C-terminus The amino acid sequence is more variable and does not suggest the presence of a P-domain.

[0005] Prohormone-proprotein converterase is expressed as zymogen and undergoes multi-step processing. It matures over time. Prodomain has two functions in this process. First, Prodomain The cytoplasm acts as a chaperone and is required for the proper folding of the catalytic domain (I Kemura et al, 1987). Once the catalytic domain is folded, Prodomei Autocatalysis occurs between the prodomain and the catalytic domain. After this initial cleavage reaction, the prodomain It remains bound to the catalytic domain and then acts as an inhibitor of catalytic activity (Fu et al, 2000). If the condition is correct, maturation occurs in the region within the prodomain. This process proceeds along with a second autocatalytic phenomenon (Anderson et al, 1997). ). After this second cleavage phenomenon occurs, the prodomain and catalytic domain dissociate, and the active Administer protease.

[0006] The autocatalytic reaction of PCSK9 Zimogen occurs between Gln152 and Ser153 (VFA Q / SIP) (Naureckiene et al, 2003), the portion from cells It has been shown to be necessary for secretion (Seidah et al, 2003). PC No secondary autocatalytic phenomenon was observed at the site within the prodomain of SK9. PCSK9 has two species (17Kd) that can be separated by non-reducing SDS-PAGE. It consists of a rod domain and a catalyst + C-terminal domain at 65Kd. PCSK9 is Since it has not been isolated without the inhibitory prodomain, the measurement of the catalytic activity of PCSK9 is variable. Naureckiene et al, 2003; Seidah et al, 2 003). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Horton et al., 2007 [Non-Patent Document 2] Seidah and Prat, 2007 [Non-Patent Document 3] Benjannet et al., 2004 [Non-Patent Document 4] Lagace et al., 2006 [Non-Patent Document 5] Maxwell et al., 2005 [Non-Patent Document 6] Park et al., 2004 [Non-Patent Document 7] Rashid et al., 2005 [Non-Patent Document 8] Kotowski et al., 2006 [Non-Patent Document 9] Zhao et al., 2006 [Non-Patent Document 10] Seidah et al., 2003 [Non-Patent Document 11] Rawlings et al, 2006 [Non-Patent Document 12] Henoch et al, 2003 [Non-Patent Document 13] Tangrea et al, 2002 [Non-Patent Document 14] Henrich et al., 2005 [Non-Patent Document 15] Sakai et al., 1998 [Non-Patent Document 16] Seidah et al, 1999 [Non-Patent Document 17] Ikemura et al, 1987 [Non-Patent Document 18] Tu et al, 2000 [Non-Patent Document 19] Anderson et al, 1997 [Non-Patent Document 20] Naureckiene et al, 2003 [Overview of the Initiative]

[0008] In some embodiments, the present invention includes an antigen-binding protein for PCSK9. .

[0009] In some embodiments, the present invention relates to A)(i) Sequence IDs 74, 85, 71, 72, 67 , 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and 60 CDRH1 obtained from CDRH1 in a sequence selected from the following group; (ii) Sequence ID 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 5 6, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78 Obtained from CDRH2 in sequences selected from the group consisting of 83, 69, 81, and 60. CDRH2;(iii) Sequence IDs 74, 85, 71, 72, 67, 87, 58, 52, 5 1, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65 Selected from the group consisting of 79, 80, 76, 77, 78, 83, 69, 81, and 60. CDRH3 obtained from CDRH3 in the sequence; and (iv) one of the amino acids below 4 (i), (ii) and ( iii) One or more heavy chain complementarity determination regions selected from the group consisting of CDRH (CDRH), B) (i) Sequence numbers 5, 7, 9, 10, 12, 13, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, A selection from the group consisting of 35, 36, 37, 38, 39, 40, 42, 44 and 46 CDRL1 obtained from CDRL1 in the column; (ii) Sequence numbers 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 3 From 0, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44 and 46 CDRL2 obtained from CDRL2 in sequences selected from the group; (iii) Sequence ID 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 2 3, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40 CDR obtained from CDRL3 in sequences selected from the group consisting of 42, 44, and 46 L3; and (iv) substitution or deletion of one or more amino acids, including 4 or fewer amino acids. Alternatively, select from the group consisting of (i), (ii), and (iii) CDRLs containing insertions. One or more light chain complementarity determining regions (CDRLs); or one of C)A) or containing one or more heavy chain CDRH and B) light chain CDRL, It contains an isolated antigen-binding protein that binds to PCSK9. In some embodiments, The isolated antigen-binding protein is at least one CDRH of A) and at least one of B) Each contains one CDRL. In some embodiments, isolated antigen-binding protein This includes at least two CDRHs of A) and at least two CDRLs of B). In that embodiment, the isolated antigen-binding proteins are CDRH1, CDRH2 , including CDRH3, CDRL1, CDRL2 and CDRL3. In some embodiments Therefore, CDRH of A) is selected from the group consisting of (i) Sequence IDs 67, 78, 89 and 49. CDRH1 amino acid sequence selected from CDRH1 in the sequence; (ii) Sequence ID 67 CDRH2 selected from the sequence selected from the group consisting of 79, 89, and 49 RH2 amino acid sequence; (iii) Select from the group consisting of SEQ ID NOs. 67, 79, 89 and 49. CDRH3 amino acid sequences selected from CDRH3 in the sequence; and (iv) 2 or (i), (i i) and (iii) are selected from the group consisting of CDRH. Furthermore, B The CDRL of (i) is selected from the group consisting of sequence numbers 12, 35, 32 and 23. CDRL1 amino acid sequence selected from CDRL1 in the column; (ii) Sequence IDs 12, 35 CDRL2 selected from the sequence selected from the group consisting of 32 and 23 (iii) Selected from the group consisting of SEQ ID NOs: 12, 35, 32, and 23. CDRL3 amino acid sequences selected from CDRL3 in the sequence; and (iv) the following A (i), (ii) and (iii) CDRL or one or more heavy chain CDRHs of (iii)A) and one of B Selected from at least one of the group consisting of one or more light chain CDRLs. In the embodiment of A), CDRH is (i) the CDRH1 amino acid compound in SEQ ID NO: 67 (ii) CDRH1 amino acid sequence in sequence number 67 RH2 amino acid sequence; (iii) CDRH3 amino acid sequence in SEQ ID NO: 67 Amino acid sequence; and (iv) one or more amino acid sequences of 2 or fewer amino acids A group consisting of (i), (ii), and (iii) CDRH containing substitution, deletion, or insertion. (i) The CDRL in (i) Sequence ID 12 (ii) CDRL1 amino acid sequence in L1 amino acid sequence; (ii) CDRL2 amino acid sequence in Sequence ID No. 12 (iii) CDRL2 amino acid sequence in sequence number 12; (iii) CDRL3 amino acid sequence in sequence number 12 The CDRL3 amino acid sequence of the column; and (iv) one or more amino acids less than or equal to 2. CDs (i), (ii), and (iii) containing the above amino acid substitutions, deletions, or insertions. One or more heavy chains of RL or C)A) and one or more of B) Selected from at least one of the group consisting of the above light chain CDRL. In some embodiments Therefore, the antigen-binding protein is A) CDRH1 of the CDRH1 sequence in Sequence ID No. 67, sequence number CDRH2 of the CDRH2 sequence in sequence 67 and CDRH3 of the CDRH3 sequence in sequence number 67 3, and B) CDRL1 of the CDRL1 sequence in sequence number 12, CDR in sequence number 12 Includes CDRL2 in the L2 sequence and CDRL3 in the CDRL3 sequence in sequence number 12. In this embodiment, the antigen-binding protein is sequence numbers 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 6 From 4, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and 60 A heavy chain variable region having at least 80% sequence identity with an amino acid sequence selected from the group. Domain (VH), and / or sequence numbers 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 3 A group selected from the group consisting of 5, 36, 37, 38, 39, 40, 42, 44, and 46 It contains a light chain variable region (VL) that has at least 80% sequence identity with the no-acid sequence. In this embodiment, VH is sequence numbers 74, 85, 71, 72, 67, 87, 58, 5 2, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89 Select from the group consisting of 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. The amino acid sequence has at least 90% sequence identity with the sequence, and / or VL is sequence Numbers 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, An amino acid sequence selected from the group consisting of 40, 42, 44, and 46 and at least 90% It has sequence identity. In some embodiments, VH is sequence numbers 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 5 0, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 Selected from the group consisting of and 60, and / or VL is sequence numbers 5, 7, 9, 10, 1 2, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28 , 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44 and 46 It is selected from the group consisting of the following.

[0010] In some embodiments, the present invention is made possible by any of the ABPs disclosed herein. It contains isolated antigen-binding proteins that specifically bind to the epitope to which they are bound.

[0011] In some embodiments, the present invention relates to A)(i) Sequence IDs 74, 85, 71, 72, 67 , 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and 60 One of the sequences selected from the group has at least 80% sequence identity with CDRH1. CDRH1; (ii) Sequence IDs 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 6 Selected from the group consisting of 5, 79, 80, 76, 77, 78, 83, 69, 81 and 60 CDRH2 having at least 80% sequence identity with one of the sequences; and (iii) Sequence numbers 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 8 One of the sequences selected from the group consisting of 0, 76, 77, 78, 83, 69, 81, and 60. A small number of the group consisting of CDRH3 that has at least 80% sequence identity with the CDRH3 inside One or more heavy-chain CDR(CDRH);B)(i) sequence numbers selected from one or more. No. 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 4 CDRL1 and at least one of the sequences selected from the group consisting of 0, 42, 44, and 46 CDRL1 also has 80% sequence identity; (ii) Sequence IDs 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 3 From 0, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44 and 46 One of the sequences selected from the group has at least 80% sequence identity with CDRL2. CDRL2; and (iii) Sequence numbers 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 3 Selected from the group consisting of 3, 35, 36, 37, 38, 39, 40, 42, 44 and 46 From a CDRL3 that has at least 80% sequence identity with one of the sequences One or more light chain CDRs (CDRLs) selected from at least one of the groups; Or C) One or more heavy chain CDRHs from A) and B) One or more Contains isolated antigen-binding proteins that bind PCSK9, including light chain CDRL. In that embodiment, the antigen-binding protein is A)(i)Sequence IDs 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 5 0, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and CDRH1 and at least 90% of the sequence selected from the group of 60 CDRH1 with identity; (ii) Sequence IDs 74, 85, 71, 72, 67, 87, 5 8, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62 A group consisting of 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and 60. A CDR having at least 90% sequence identity with CDRH2 among the selected sequences. H2; and (iii) Sequence IDs 74, 85, 71, 72, 67, 87, 58, 52, 5 1, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65 Selected from the group consisting of 79, 80, 76, 77, 78, 83, 69, 81, and 60. It consists of CDRH3 having at least 90% sequence identity with one of the CDRH3 sequences. One or more CDRH, B)(i) sequence numbers selected from at least one of the groups No. 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 4 CDRL1 and at least one of the sequences selected from the group consisting of 0, 42, 44, and 46 CDRL1 also has 90% sequence identity; (ii) Sequence numbers 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 3 From 0, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44 and 46 One of the sequences selected from the group has at least 90% sequence identity with CDRL2. CDRL2; and (iii) Sequence numbers 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 3 Selected from the group consisting of 3, 35, 36, 37, 38, 39, 40, 42, 44 and 46 CDRL3 in one of the sequences has at least 90% sequence identity with CDRL3. One or more CDRLs selected from at least one of the groups; or C)A) One or more heavy chain CDRH and one or more light chain CDRL of B) include.

[0012] In some embodiments, the present invention relates to an isolated antigen-binding protein that binds PCSK9. The substance contains the antigen-binding protein A)(i)Sequence numbers 67, 79 and 49 CDRH3 selected from the sequence selected from the group, CDRH3 of (ii)(i) In the amino acid sequence obtained from RH3, the addition, deletion, or removal of two or fewer amino acids may occur. CDRH3 with different substitutions, and (iii)X1X2X3X4X5X6X7X8X9X1 0X 11 X 12 X 13 X 14 (Sequence ID 404) (X1 is D, A, R and no amino acids) X2 is selected from the group consisting of Y, I, G, and no amino acids, X 3 is selected from the group consisting of D, A, G and no amino acids, and X4 is F, A, L and amino acids. The group consisting of no acid is selected, and X5 is selected from the group consisting of W, L, A and no amino acids. X6 is selected from the group consisting of S, Y, A and no amino acids, and X7 is selected from the group consisting of A, Y, R and Selected from the group consisting of Y, P, and the group consisting of no amino acids, X8 is selected from the group consisting of Y, P, and no amino acids. Selected, X9 is selected from the group consisting of Y, G, and no amino acid, X 10 is selected from the group consisting of D, G, and no amino acid, X 11 is selected from the group consisting of A, M, and no amino acid selected, X 12 is selected from the group consisting of F, D, and no amino acid, X 13 is D, V, and selected from the group consisting of no amino acid, X 14 is selected from the group consisting of V and no amino acid selected. ) consisting of at least one selected from the group of heavy chain complementarity determining regions (CDRH ), B) (i) CDRL selected within the sequence selected from the group consisting of SEQ ID NO: 12, 35, and 23 3 selected CDRL3; (ii) in the amino acid sequence obtained from the CDRL3 of (i), CDRL3 with different amino acid addition, deletion, or substitution of two or fewer amino acids, and ( iii) X1X2X3X4X5X6X7X8X9X 10 X 11 (SEQ ID NO: 405) (X1 is selected from the group consisting of Q and G, X2 is selected from the group consisting of S, T, A, and no amino acid selected, X3 is selected from the group consisting of Y, no amino acid, and W, X4 is D and amino selected from the group consisting of acid and no amino acid, X5 is selected from the group consisting of S and no amino acid, X 6 is selected from the group consisting of S and no amino acid, X7 is selected from the group consisting of L, T, and no amino acid selected, X8 is selected from the group consisting of no amino acid, A, and S, X9 is amino selected from the group consisting of no acid, G, A, and V, X 10 is selected from the group consisting of no amino acid, S, Y, and V selected, X 11 is selected from the group consisting of no amino acid and V. ) or selected from at least one of the group consisting of CDRL3 amino acid sequences selected from the group consisting of It includes the light chain complementarity determination region (CDRL).

[0013] In some embodiments, the present invention relates to 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 and several combinations thereof Isolated antigen-binding protein containing a light chain having an amino acid sequence selected from the group consisting of Contains chlorine.

[0014] In some embodiments, the antigen-binding protein is the antigen disclosed herein. It specifically binds to an epitope that is bound by at least one binding protein. In several embodiments, the isolated antigen-binding proteins are 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 9 1, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, 60 and heavy chains having amino acid sequences selected from a group consisting of several combinations thereof Furthermore, it includes. In some embodiments, the amino acid sequence of ABP is SEQ ID NOs: 12, 35 Selected from the group consisting of 23 and several combinations thereof. In some embodiments In this context, the heavy chain of ABP consists of CDRH3 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 67, and the It includes CDRH1 in sequence number 67, and the light chain is CDRL3 in sequence number 12, sequence number Includes 12 CDRL2s and CDRL1 of sequence number 12. In some embodiments, single The detached antigen-binding proteins include monoclonal antibodies, polyclonal antibodies, and recombinant antibodies. These are human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, or fragments of these antibodies. In several embodiments, the isolated antigen-binding protein consists of a Fab fragment and a Fab' fragment. These are F(ab')2 fragments, Fv fragments, diabodies, or single-chain antibody molecules. In the embodiment, the isolated antigen-binding protein is a human antibody. Several embodiments In this study, the isolated antigen-binding protein is a monoclonal antibody. Several implementations Morphologically, isolated antigen-binding proteins are IgG1, IgG2, IgG3, or I It is of type gG4. In some embodiments, the isolated antigen-binding protein is IgG It is type 4 or IgG2. In some embodiments, the isolated antigen-binding protein is , it is bound to the labeling group. In some embodiments, the isolated antigen-binding protein is Regarding binding to PCSK9, it competes with the antigen-binding proteins described herein. In some embodiments, the isolated antigen-binding protein is a monoclonal antibody. Polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, multiple specific antibodies The body or fragments of these antibodies. In some embodiments, isolated antigen-binding proteins The components are Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies or It is a single-chain antibody molecule. In some embodiments, the isolated antigen-binding protein is It is bound to a labeling group. In some embodiments, the isolated antigen-binding protein is L Reduces the binding of PCSK9 to DLR. In some embodiments, isolated antigen The binding protein, when administered to a target, reduces the amount of LDL present in the target. In some embodiments, when the isolated antigen-binding protein is administered to a subject... In some embodiments, this reduces the amount of serum cholesterol present in the subject. The isolated antigen-binding protein, when administered to the subject, is released to the LDL present in the subject. Increase the amount of R.

[0015] In some embodiments, the present invention relates to a vector comprising nucleic acid molecules described herein. —Includes. In some embodiments, the present invention includes nucleic acid molecules described herein. Includes host cells containing [the specified component].

[0016] In some embodiments, the present invention relates to bonding to PCSK9 as described herein. It contains isolated antigen-binding proteins that compete with the antigen-binding proteins already present.

[0017] In some embodiments, the present invention relates to antigen-binding proteins disclosed herein. Contains the coding nucleic acid molecule.

[0018] In some embodiments, the present invention relates to at least one antigen described herein. The pharmaceutical composition includes a binding protein.

[0019] In some embodiments, the present invention relates to the treatment of symptoms associated with elevated serum cholesterol levels. For patients in need of treatment or prevention, at least one isolated as disclosed herein This involves administering an effective amount of the antigen-binding protein to the patient, which is associated with elevated serum cholesterol levels. This includes methods for treating or preventing symptoms accompanied by high levels of tetraol.

[0020] In some embodiments, the present invention relates to at least one antigen disclosed herein. The binding of PCSK9 to LDLR in the subject, including administering an effective amount of the binding protein. This includes methods for inhibition.

[0021] In some embodiments, the present invention relates to an antigen-binding protein that selectively binds to PCSK9. It contains, and the antigen-binding protein has a K content less than 100 pM. D Then it connects to PCSK9.

[0022] In some embodiments, the present invention relates to the treatment of symptoms associated with elevated serum cholesterol levels. Drugs that increase the availability of LDLR protein in subjects requiring treatment or prevention. Simultaneously or sequentially, at least one isolated antigen binding as disclosed herein Elevated serum cholesterol levels in subjects, including administration of an effective amount of protein. This includes methods for treating or preventing symptoms accompanied by these symptoms.

[0023] In some embodiments, the present invention relates to at least one isolated as disclosed herein. This involves administering an effective amount of the antigen-binding protein to the target, including the serum cholesterol of the target. This includes methods to lower the role level.

[0024] In some embodiments, the present invention relates to a drug that increases the availability of LDLR protein. Simultaneously or sequentially, at least one isolated antigen binding as disclosed herein This includes administering an effective amount of protein to the target, thereby increasing serum cholesterol levels in the target. This includes methods to reduce it.

[0025] In some embodiments, the present invention relates to at least one isolated as disclosed herein. This includes administering an effective amount of the antigen-binding protein to the target of the LDLR-tank. This includes methods for increasing protein content.

[0026] In some embodiments, the present invention relates to a drug that increases the availability of LDLR protein. Simultaneously or sequentially, at least one isolated antigen binding as disclosed herein This includes administering an effective amount of protein to the target, thereby increasing the LDLR protein level in the target. Includes methods for increasing it.

[0027] In some embodiments, the present invention relates to the ABP and LDLR technologies disclosed herein. The pharmaceutical composition includes an agent that increases the availability of protein levels. Several implementations In this context, drugs that increase the availability of LDLR protein include statins. In that embodiment, the statin is atrovastatin, cerivastatin, fluvastatin Lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, The selection is made from a group consisting of simvastatin and several combinations thereof.

[0028] In some embodiments, the present invention relates to antigen-binding proteins described herein. The process described herein includes a step of preparing the antigen-binding protein from a secreting host cell. This includes a method for producing antigen-binding proteins.

[0029] In some embodiments, the present invention relates to at least one antigen described herein. The pharmaceutical composition includes a binding protein and a pharmaceutically acceptable excipient. Several implementations In form, the pharmaceutical composition further comprises additional active factors. In some embodiments... The further activating factors include radioisotopes, radionuclides, toxins, or therapeutic groups and chemotherapy Selected from a group consisting of elements.

[0030] In some embodiments, the present invention relates to elevated serum cholesterol levels in patients. This includes methods for treating or preventing symptoms of elevated serum cholesterol in patients. For patients who require treatment or prevention of symptoms associated with a blood pressure level, the information disclosed herein is available. This involves administering an effective amount of at least one isolated antigen-binding protein. In some embodiments, the symptom is hypercholesterolemia.

[0031] In some embodiments, the present invention relates to at least one antigenic compound described herein. The binding of PCSK9 to LDLR in patients, including administering an effective amount of the combined protein. This includes methods of inhibition.

[0032] In some embodiments, the present invention relates to K less than 100 pM. d Connect to PCSK9 It contains an antigen-binding protein. In some embodiments, the antigen-binding protein is 10p K is smaller than M. d It binds via 5p. In some embodiments, the antigen-binding protein is 5p K is smaller than M. a They are joined together.

[0033] In some embodiments, the present invention relates to the treatment of symptoms associated with elevated serum cholesterol levels. Drugs that increase the availability of LDLR protein in subjects requiring treatment or prevention. Simultaneously or sequentially, at least one isolated antigen binding as described herein Elevated serum cholesterol levels in subjects, including administration of an effective amount of protein. The method includes treating or preventing symptoms accompanied by [unclear]. In some embodiments, LDLR protein Drugs that increase the availability of nitrates include statins. In some embodiments, statins Chin is atrovastatin, cerivastatin, fluvastatin, lovastatin, mevasta Tin, pitavastatin, pravastatin, rosuvastatin, simvastatin and these It is selected from a group consisting of several combinations.

[0034] In some embodiments, the present invention relates to a method for lowering serum cholesterol levels in a subject. This method includes the method of at least one isolated antigenic compound described herein. This includes administering an effective amount of the combined protein to the target population.

[0035] In some embodiments, the present invention relates to a drug that increases the availability of LDLR protein. Simultaneously or sequentially, at least one isolated antigen binding as described herein This includes administering an effective amount of protein to the target, thereby increasing serum cholesterol levels in the target. This includes methods for reducing the availability of LDLR protein. In some embodiments, the availability of LDLR protein Drugs that increase include statins. In some embodiments, statins are used in Atrobas. Tachin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin Pravastatin, rosuvastatin, simvastatin, and several combinations thereof It is selected from the group consisting of the following.

[0036] In some embodiments, the present invention relates to at least one isolated as described herein. By administering an effective amount of the antigen-binding protein to the target, the LDLR in the target This includes methods for increasing protein levels.

[0037] In some embodiments, the present invention relates to a drug that increases the availability of LDLR protein. Simultaneously or sequentially, at least one isolated antigen binding as described herein By administering an effective amount of protein to the target, the LDLR protein level in the target can be reduced. This includes methods for increasing LDLR protein levels. In some embodiments, the utilization of LDLR protein levels Drugs that increase the likelihood include statins. In some embodiments, statins are Trovastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pita Vastatin, pravastatin, rosuvastatin, simvastatin, and several combinations thereof. Selected from a group consisting of combinations.

[0038] In some embodiments, the present invention is coupled to PCSK9 and PCSK9 to LDLR It contains a neutralizing antibody that reduces the low-density lipoprotein receptor (LDLR) depressant effect. In one embodiment, the antibody specifically binds to PCSK9. In some embodiments, Then, the antibody binds to the catalytic domain of PCSK9. In some embodiments, the antibody It binds to epitopes within residues 31 to 447 of SEQ ID NO: 3. In some embodiments, The antibody has an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, which is PCSK9 Combine.

[0039] In some embodiments, the present invention comprises a neutralizing antigen-binding protein that binds to PCSK9. Furthermore, the antigen-binding protein is located at the position between residues 31 and 447 of SEQ ID NO: 3, and is PCSK It binds to 9. In some embodiments, the antigen-binding protein binds to PCSK9. If so, the antibody will be 8 angstroms from at least one of the following residues of PCSK9. Located at or below the base level: S153, I154, P155, R194, D238 , A239, I369, S372, D374, C375, T377, C378, F379 , V380, S381, W156, N157, L158, E159, H193, E195 , H229, R237, G240, K243, D367, I368, G370, A371 , S373, S376, Q382, W72, F150, A151, Q152, T214, R215, F216, H217, A220, S221, K222, S225, H226, C255, Q256, G257, K258, N317, F318, T347, L348, G349, T350, L351, E366, D367, D374, V380, S381, Q382, S383, G384, K69, D70, P71, S148, V149, D18 6, T187, E211, D212, G213, R218, Q219, C223, D22 4, G227, H229, L253, N254, G259, P288, A290, G29 1, G316, R319, Y325, V346, G352, T353, G365, I36 8, I369, S372, S373, C378, F379, T385, S386, Q38 7, S153, S188, I189, Q190, S191, D192, R194, E19 7, G198, R199, V200, D224, R237, D238, K243, S37 3, D374, S376, T377, F379, I154, T187, H193, E19 5, I196, M201, V202, C223, T228, S235, G236, A23 9, G244, M247, I369, S372, C375 or C378. Several implementations In this state, the antibody is 8 angstroms from at least one of the following residues of PCSK9. Or located below: S153, I154, P155, R194, D238, A 239, 1369, S372, D374, C375, T377, C378, F379, V 380, S381, W156, N157, L158, E159, H193, E195, H 229, R237, G240, K243, D367, I368, G370, A371, S 373, S376, or Q382. In some embodiments, the antibody is the following of PCSK9 It is located at least 5 angstroms or less from one of the residues. S153 , I154, P155, R194, D238, A239, I369, S372, D374 , C375, T377, C378, F379, V380 or S381. Several embodiments In this case, the antibody is 5 angstroms from at least two of the following residues of PCSK9 or These are located below: S153, I154, P155, R194, D238, A2 39, I369, S372, D374, C375, T377, C378, F379, V3 80 or S381. In some embodiments, the antibody has fewer of the following residues of PCSK9. At most four to five angstroms or less. (S153, I154, P155) , R194, D238, A239, I369, S372, D374, C375, T377 , C378, F379, V380 or S381. In some embodiments, the antibody is P At least one of the following residues of CSK9 located 8 angstroms or less Yes. W72, F150, A151, Q152, T214, R215, F216, H21 7, A220, S221, K222, S225, H226, C255, Q256, G25 7, K258, N317, F318, T347, L348, G349, T350, L35 1, E366, D367, D374, V380, S381, Q382, S383, G38 4, K69, D70, P71, S148, V149, D186, T187, E211, D 212, G213, R218, Q219, C223, D224, G227, H229, L 253, N254, G259, P288, A290, G291, G316, R319, Y 325, V346, G352, T353, G365, I368, I369, S372, S 373, C378, F379, T385, S386, or Q387. In some embodiments... The antibody is derived from at least one of the following residues of PCSK9, ranging from 5 angstroms or more. It is located below: W72, F150, A151, Q152, T214, R215. F216, H217, A220, S221, K222, S225, H226, C255, Q256, G257, K258, N317, F318, T347, L348, G349, T350, L351, E366, D367, D374, V380, S381, Q382, S383 or G384. In some embodiments, the antibody is the following residue of PCSK9 It is located at least 2 to 5 angstroms or less. W72, F150 , A151, Q152, T214, R215, F216, H217, A220, S221 , K222, S225, H226, C255, Q256, G257, K258, N317 , F318, T347, L348, G349, T350, L351, E366, D367 , D374, V380, S381, Q382, S383 or G384. Several embodiments. In this case, the antibody is found in at least four to five angstroms of the following residues of PCSK9 or These are located below: W72, F150, A151, Q152, T214, R21 5, F216, H217, A220, S221, K222, S225, H226, C25 5, Q256, G257, K258, N317, F318, T347, L348, G34 9, T350, L351, E366, D367, D374, V380, S381, Q38 2. S383 or G384. In some embodiments, the antibody is the following residue of PCSK9. It is located at least 8 angstroms or less from the base. S153, S 188, I189, Q190, S191, D192, R194, E197, G198, R 199, V200, D224, R237, D238, K243, S373, D374, S 376, T377, F379, I154, T187, H193, E195, I196, M 201, V202, C223, T228, S235, G236, A239, G244, M 247, I369, S372, C375, or C378. In some embodiments, antibodies This is located at least one of the following residues of PCSK9 at a position of 5 angstroms or less. They are located at: S153, S188, I189, Q190, S191, D192, R194 , E197, G198, R199, V200, D224, R237, D238, K243 , S373, D374, S376, T377 or F379. In some embodiments, The antibody is 5 angstroms or less from at least two of the following residues of PCSK9. It is located at S153, S188, I189, Q190, S191, D192, R1 94, E197, G198, R199, V200, D224, R237, D238, K2 43, S373, D374, S376, T377, or F379. In some embodiments... The antibody is found in at least four to five angstroms of the following residues of PCSK9 or It is located as follows: S153, S188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, or F379.

[0040] In some embodiments, the present invention comprises a neutralizing antibody that binds to PCSK9, wherein the antibody is P It binds to CSK9, reducing the likelihood of PCSK9 binding to LDLR.

[0041] In some embodiments, an antibody or antigen-binding molecule that binds to PCSK9 is envisioned. The antibody binds to PCSK9 at a position within residues 31 to 447 of SEQ ID NO: 3. In some embodiments, when an antibody or antigen-binding molecule is bound to PCSK9, At least one of the following residues of PCSK9 is located 8 angstroms or less It is. S153, I154, P155, R194, D238, A239, I369, S 372, D374, C375, T377, C378, F379, V380, S381, W 156, N157, L158, E159, H193, E195, H229, R237, G 240, K243, D367, I368, G370, A371, S373, S376, Q 382, W72, F150, A151, Q152, T214, R215, F216, H2 17, A220, S221, K222, S225, H226, C255, Q256, G2 57, K258, N317, F318, T347, L348, G349, T350, L3 51, E366, D367, D374, V380, S381, Q382, S383, G3 84, K69, D70, P71, S148, V149, D186, T187, E211, D212, G213, R218, Q219, C223, D224, G227, H229, L253, N254, G259, P288, A290, G291, G316, R319, Y325, V346, G352, T353, G365, I368, I369, S372, S373, C378, F379, T385, S386, Q387, S153, S188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T187, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375 or C378.

[0042] In some embodiments, provided is an isolated antibody or antigen-binding molecule that blocks the binding of an antibody to PCSK9 within 8 angstroms of a residue of PCSK9. In some embodiments, the residue of PCSK9 is selected from at least one of the following PCSK9 residues. S153, I154, P 155, R194, D238, A239, I369, S3 72, D374, C375, T377, C378, F379, V380, S381, W1 56, N157, L158, E159, H193, E195, H229, R237, G2 40, K243, D367, I368, G370, A371, S373, S376, Q3 82, W72, F150, A151, Q152, T214, R215, F216, H21 7, A220, S221, K222, S225, H226, C255, Q256, G25 7, A220, S221, K222, S225, H226, C255, Q256, G25 7, K258, N317, F318, T347, L348, G349, T350, L35 1, E366, D367, D374, V380, S381, Q382, S383, G38 4, K69, D70, P71, S148, V149, D186, T187, E211, D 212, G213, R218, Q219, C223, D224, G227, H229, L 253, N254, G259, P288, A290, G291, G316, R319, Y 325, V346, G352, T353, G365, I368, I369, S372, S 373, C378, F379, T385, S386, Q387, S153, S188, I 189, Q190, S191, D192, R194, E197, G198, R199, V 200, D224, R237, D238, K243, S373, D374, S376, T 377, F379, I154, T187, H193, E195, I196, M201, V 202, C223, T228, S235, G236, A239, G244, M247, I 369, S372, C375 or C378.

[0043] In some embodiments, an isolated antibody or antigen-binding molecule that binds to PCSK9 is provided at a position that overlaps with the position where LDLR binds to PCSK9. In some embodiments, at the position where LDLR binds to PCSK9, at least one amino acid residue selected from the group consisting of S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380 and S381 is included. いて、PCSK9に結合する単離された抗体又は抗原結合分子が提供される。幾つかの実 施形態において、LDLRがPCSK9に結合する位置には、S153、I154、P1 55、R194、D238、A239、I369、S372、D374、C375、T3 77、C378、F379、V380及びS381からなる群から選択される少なくとも 1つのアミノ酸残基が含まれる。

[0044] In some embodiments, isolated antibodies or antigen-binding molecules that bind to PCSK9 are Provided. In some embodiments, the antibody or antigen-binding molecule is placed on PCSK9 below EGFa binds to PCSK9 within 8 angstroms of at least one residue. It reduces the probability. S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380, S381, W156, N157, L158, E159, H193, E195, H229, R237, G240, K243, D367, I368, G370, A371, S373, S376 or Q382.

[0045] In some embodiments, EGFa is bound, Ab21B12 is bound, and / or Antibodies and antigen-binding proteins that bind to the surface of PCSK9 that overlaps with the surface to which 31H4 binds. A substance or antigen-binding molecule is provided. In some embodiments, as illustrated in the drawings... Antibodies, antigen-binding proteins, or antigen-binding molecules that bind to PCSK9 in a similar manner are provided. To be served.

[0046] In some embodiments, the above embodiment is a neutralizing antibody or an antigen-binding protein. In some embodiments, the antigen-binding protein is LDLR or a fragment thereof (EGFa (and so on)

[0047] In some embodiments, the present invention comprises an isolated neutralizing antibody that reacts with PCSK9. When bound, the antibody is 8 angus from at least one of the following residues of PCKS9. Located in or below Rohm. Sequence ID 3: T468, R469, M470, A471 , T472, R496, R499, E501, A502, Q503, R510, H512 , F515, P540, P541, A542, E543, H565, W566, E567 , V568, E569, R592, E593, S465, G466, P467, A473 , I474, R476, G497, E498, M500, G504, K506, L507 , V508, A511, N513, A514, G516, V536, T538, A539 , A544, T548, D570, L571, H591, A594, S595 and H59 7. In some embodiments, the antibody is from at least one of the following residues of PCSK9 located at 5 angstroms or less. T468, R469, M47 0, A471, T472, R496, R499, E501, A502, Q503, R51 0, H512, F515, P540, P541, A542, E543, H565, W56 6, E567, V568, E569, R592 and E593.

[0048] In some aspects, the present invention includes an isolated antigen-binding protein. The antigen-binding ta n protein includes A) CDRH1 of the CDRH1 sequence in SEQ ID NO: 89, CD RH2 of the CDRH2 sequence in SEQ ID NO: 89 and CDRH3 of the CDRH3 sequence in SEQ ID NO: 89, and B) CDRL1 of the CDRL1 sequence in SEQ ID NO: 32, CD RL2 of the CDRL2 sequence in SEQ ID NO: 32 and CDRL3 of the CDRL3 sequence in SEQ ID NO: 32.

[0049] In some aspects, the present invention includes an isolated antigen-binding protein that binds to the PCSK9 protein of SEQ ID NO: 1, and the isolated antigen-binding protein and variant PC SK9 have a binding affinity difference of less than or equal to 10-fold compared to the binding affinity of the isolated antigen-binding protein and the PCSK9 protein of SEQ ID NO: 1. The binding between the SK9 protein and the isolated antigen-binding protein and SEQ ID NO: 1 and / or The binding rate between the PCSK9 protein and the sequence number 303 is less than 50%. In the application form, the variant PCSK9 protein is 207 as shown in SEQ ID NO: 1. ,208,185,181,439,513,538,539,132,351,390 , 413, 582, 162, 164, 167, 123, 129, 311, 313, 337 At positions consisting of or selected from the group including 519, 521, and 554, the residue It includes at least one mutation. In some embodiments, at least one mutation is R 207E, D208R, E181R, R185E, R439E, E513R, V538R This includes E539R, T132R, S351R, A390R, A413R and E582R. Or selected from the group consisting of these. In some embodiments, at least one variant The differences are D162R, R164E, E167R, S123R, E129R, A311R, D Selected from the group consisting of 313R, D337R, R519E, H521R, and Q554R ru.

[0050] In some embodiments, the present invention provides the PCSK-9 protein of SEQ ID NO: 303 with a first Antigen-binding proteins that bind in one manner and to PCSK9 variants in a second manner. Includes. PCSK9 variants include SEQ ID NOs. 303 and / or SEQ ID NOs. 1, 207, 20 8, 185, 181, 439, 513, 538, 539, 132, 351, 390, 41 3, 582, 162, 164, 167, 123, 129, 311, 313, 337, 51 Positions including or selected from the group consisting of 9, 521, and 554, with fewer residues Both have one point variation. In some embodiments, the first form is the first EC 50 , First B max or the first EC 50 and the first B max This includes, in some embodiments, The second form is the second EC 50 , second B max or the second EC 50 and the second B max of Includes. The values ​​for the first form are different from the values ​​for the second form. In some embodiments... In this context, the first form is the first EC 50 The second form includes the second EC 50 including, and The point mutations are R207E, D208R, E181R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R and E58 Selected from the group consisting of or including 2R. In some embodiments, the first E C 50 is the second EC 50 And it differs by at least 20%. In some embodiments, the first EC 50 is the second EC 50 And it differs by at least 50%. In some embodiments, the second EC 50 is the first EC 50 It is a larger number. In some embodiments, the first EC50 is measured by a multiple bead binding assay. In some embodiments, Second EC 50 It is larger than 1 μm. In some embodiments, the antigen-binding protein is medium It is a neutralizing antigen-binding protein. In some embodiments, the neutralizing antigen-binding protein competes It is a neutralizing antigen-binding protein. In some embodiments, the neutralizing antigen-binding protein The protein is a non-competitive neutralizing antigen-binding protein. In some embodiments, the first form is First B max This includes, and the second form is the first B max A different second B max Includes Hmm. PCSK9 variants are D162R, R164E, E167R, S123R, E1 29R, A311R, D313R, D337R, R519E, H521R and Q554R It has at least one point mutation selected from or consisting of the following groups. In this embodiment, the second B max is the first B max It is about 10% of the total. Several implementation forms In the state, the first B max is the second B max And it differs by at least 20%. Several implementations In terms of form, the first B max is the second B max It differs by at least 50%.

[0051] In some embodiments, the present invention relates to isolation of the PCSK9 protein of SEQ ID NO: 3. It contains an antigen-binding protein, and the epitope of the antigen-binding protein is the same as in SEQ ID NO: 1. Contains at least one of the following amino acids: 207, 208, 181, 185, 439, 51 3, 538, 539, 132, 351, 390, 413, 582, 162, 164, 16 7, 123, 129, 311, 313, 337, 519, 521, and 554.

[0052] In some embodiments, the present invention relates to the PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1 It comprises an isolated neutralizing antigen-binding protein that binds to a substance, and the neutralizing antigen-binding protein This reduces the LDLR-reducing effect of PCSK9 on LDLR. In some embodiments, Furthermore, antigen-binding proteins are LDLR non-competitive neutralizing antigen-binding proteins. In this embodiment, the antigen-binding protein is an LDLR competitively neutralizing antigen-binding protein. .

[0053] In some embodiments, the present invention comprises an isolated antigen-binding protein, and the antigen-binding The protein is A) CDRH1 of the CDRH1 sequence in SEQ ID NO: 49, CD in SEQ ID NO: 49 CDRH2 of the RH2 sequence and CDRH3 of the CDRH3 sequence in sequence number 49, and B) CDRL1 in the CDRL1 sequence in sequence number 23, CD in the CDRL2 sequence in sequence number 23 Includes RL2 and CDRL3 of the CDRL3 sequence in sequence number 23.

[0054] In some embodiments, the present invention relates to crystallized PCSK9 protein and PCSK9 The composition contains an antigen-binding protein that binds to the target. The composition contains approximately 2.2 ang straws The three-dimensional structure of the PCSK9 protein can be determined to a resolution of 10 or higher. It contains crystallized PCSK9 protein, such as in some embodiments. Proteins are antibodies or fragments thereof.

[0055] In some embodiments, the present invention relates to crystallized PCSK9 protein and LDLR The protein contains at least the EGFa portion, and the EGFa portion of the LDLR protein is PCS The crystallized PCSK9 protein, bound by the K9 protein, is approximately 2.2 To achieve angstrom or higher resolution, the three-dimensional structure of the PCSK9 protein This is a crystallized PCSK9 protein that can determine the structure. In several embodiments, Furthermore, the molecular models are located on a computer-readable medium.

[0056] In some embodiments, the present invention relates to the preparation of pharmaceuticals for lowering serum cholesterol. This includes the use of antigen-binding proteins as described herein.

[0057] In some embodiments, the present invention relates to elevated serum cholesterol levels in a subject. In the preparation of a pharmaceutical product for treating or preventing the symptoms described herein, Includes the use of the original binding protein.

[0058] In some embodiments, the present invention relates to an isolated antigen-binding protein that binds PCSK9. The substance contains the antigen-binding protein A)(i) from Sequence IDs 67, 79, 89 and 49 CDRH1 selected from the sequence selected from the group, (ii) 2 or less Only amino acid addition, deletion, or substitution of amino acids results in a different amino acid sequence from (i) CDRH1. CDRH1, and (iii)X1X2X3X4X5X6X7X8X9X 10 (Sequence 406)(X1 is selected from the group consisting of G, and X2 is selected from the group consisting of Y, F, and G) X3 is selected from the group consisting of T and S, and X4 is selected from the group consisting of L and F. X5 is selected from the group consisting of T, S, and N, and X6 is selected from the group consisting of S and A. X7 is selected from the group consisting of Y and F, and X8 is selected from the group consisting of G, S and Y. X9 is selected from the group consisting of I, M, and W, X 10 Select from the group consisting of S, N, and H. At least one of the groups consisting of CDRH1 amino acid sequences selected from the group consisting of ) B)(i) Sequence IDs 12, 3 CDRL1 selected from the sequence selected from the group consisting of 5 and 23, ( ii) Only amino acid addition, deletion, or substitution of 2 or fewer amino acids, (i) CDRL3 and ami CDRL1 and (iii)X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 (Sequence ID 407) (X1 consists of no T and no amino acids) X2 is selected from the group consisting of G and S, and X3 consists of S, T and G. Selected from the group, X4 is selected from the group consisting of S, X5 is selected from the group consisting of S, X6 is selected from the group consisting of N, D, and S, and X7 is selected from the group consisting of I, V, and N. X8 is selected from the group consisting of G and I, and X9 is selected from the group consisting of A and G. X 10 X is selected from the group consisting of G, Y, S, and N. 11 Select from the group consisting of Y and N Selected, X 12 X is selected from the group consisting of D, S, T, and F. 13 From the group consisting of V Selected, X 14 The group is selected from the group consisting of S, N, and H. Selected from at least one of the group consisting of CDRL1 amino acid sequences, one of which is selected from one. It contains a light chain complementarity determining region (CDRL). Those skilled in the art will know that a single ABP or antibody is one of the above options. It can satisfy one or more of the following conditions, and further the invention described for this embodiment To understand what it means to belong.

[0059] In some embodiments, the present invention relates to an isolated antigen-binding protein that binds PCSK9. The antigen-binding protein contains the following: A) (i) SEQ ID NOs: 67, 79, 89 and CDRH2 selected from the sequence selected from the group consisting of 49, (i i) Only amino acid addition, deletion, or substitution of 2 or fewer amino acids, (i) CDRH2 and amino CDRH2 with a different acid sequence, and (iii)X1X2X3X4X5X6X7X8X9X1 0X 11 X 12 X 13 X 14 X 15 X 16 X 17 (Sequence code 408) (X1 is W, S, L) X2 is selected from the group consisting of V, I, and E. X3 is selected from the group consisting of S, W, and I, and X4 is selected from the group consisting of F, S, and N. X5 is selected from the group consisting of Y, S, D, and H, and X6 is selected from the group consisting of N, S, and G. X7 is selected from the group consisting of S and G, and X8 is selected from N, Y, D and R. X9 is selected from the group consisting of T, I and E, X 10 is N, S, Y and Selected from the group consisting of and D, X 11 X is selected from the group consisting of Y, 12 Are A and N? Selected from the following group, X 13 X is selected from the group consisting of Q, D, and P. 14 is K and Selected from the group consisting of S, X 15 X is selected from the group consisting of L and V. 16 is Q and Selected from the group consisting of K, X 17 The group consisting of G and S is selected. A heavy chain selected from at least one of the group consisting of CDRH2 amino acid sequences selected from Complementarity Determination Region (CDRH), B) The following: (i) Sequence IDs 12, 32, 35 and 2 (ii)2 selected from CDRL3 within the sequence selected from the group consisting of 3 Only the addition, deletion, or substitution of the following amino acids, (i) CDRL3 and amino acid sequence (iii)X1X2X3X4X5X6X7 (Sequence ID 409) (X1 is selected from the group consisting of G, E, S, and D, and X2 is selected from the group consisting of N, V, and Y) X3 is selected from the group consisting of S and N, and X4 is selected from the group consisting of N, Q and K. X5 is selected from the group consisting of R, X6 is selected from the group consisting of P, and X7 is Selected from the group consisting of S. ) From the CDRL2 amino acid sequence selected from the group consisting of It includes a light chain complementarity determination region (CDRL) selected from at least one of the following groups.

[0060] In some embodiments, the present invention relates to an isolated antigen-binding protein that binds PCSK9. The substance contains the antigen-binding protein A) the following: (i) SEQ ID NOs: 67, 79, 8 CDRH3 selected from the sequence selected from the group consisting of 9 and 49, ( ii) Only amino acid addition, deletion, or substitution of 2 or fewer amino acids, (i) CDRH3 and ami CDRH3 with a different anoic acid sequence, and (iii)X1X2X3X4X5X6X7X8X9 X 10 X 11 X 12 X 13 X 14 (Sequence ID 410) (X1 is D and no amino acids) X2 is selected from the group consisting of Y, A and no amino acids, and X3 is D, Selected from the group consisting of I and no amino acids, X4 is the group consisting of F, A and no amino acids. X5 is selected from the group consisting of W, A and no amino acids, and X6 is selected from the group consisting of S, L and Selected from the group consisting of A, Y, G, and the group consisting of no amino acids, X7 is the group consisting of A, Y, G, and no amino acids. X8 is selected from the group consisting of Y, Q and no amino acids, and X9 is selected from the group consisting of G, Y and Selected from the group consisting of and L, X 10 X is selected from the group consisting of Y, D, and V. 11 teeth Selected from the group consisting of G, A, and P, X 12 X1 is selected from the group consisting of M and F. 3 is selected from the group consisting of D, X 14 (is selected from the group consisting of V and Y.) Selected from the group consisting of CDRH3 amino acid sequences, at least one of the group selected from the group. Heavy chain complementarity determination region (CDRH), B) The following: (i) Sequence IDs 12, 32, 35 and CDRL3 selected from the sequence selected from the group consisting of b and 23; (ii ) Only amino acid addition, deletion, or substitution of 2 or fewer amino acids, (i) CDRL3 and amino acids CDRL3 with a different sequence, and (iii)X1X2X3X4X5X6X7X8X9X1 0X 11 (Sequence ID 411) (X1 is selected from the group consisting of Q, A, G, and no amino acid) X2 is selected from the group consisting of S, V, T, and no amino acids, and X3 is selected from the group consisting of Y, N, and W. X4 is selected from the group consisting of S and D, and X5 is selected from the group consisting of S, Y and D. X6 is selected from the group consisting of S and T, and X7 is selected from the group consisting of L and S. X8 is selected from the group consisting of S, T, and N, and X9 is selected from the group consisting of G, S, and A. Selected from the following group, X 10 is selected from the group consisting of S, M, W, and Y, and X 11 The group consisting of V is selected. ) CDRL3 amino acid combination selected from the group consisting of It includes a light chain complementarity determination region (CDRL) selected from at least one of a group consisting of columns. In some embodiments, any of the above amino acids are substituted by a conservative amino acid substitution. It is possible.

[0061] In some embodiments, the present invention relates to an isolated antigen-binding protein that binds PCSK9. The substance contains the antigen-binding protein A)(i)Sequence codes 47, 48, 49, 50, 5 C in a sequence selected from the group consisting of 1, 52, 53, 54, 55, 56, 57, and 58 CDRH1 selected from DRH1, (ii) amino acid addition or deletion of 2 or fewer amino acids or By substitution, (i) CDRH1 has a different amino acid sequence from CDRH1, and (iii) )X1X2X3X4X5X6X7X8X9X 10 (Sequence No. 412) (X1 is G, P and X2 is selected from the group consisting of A, X3 is selected from the group consisting of Y, W, F, T and S, X3 X4 is selected from the group consisting of T, P, S and A, C, V, L, and I, and X4 is L, F, I, V X5 is selected from the group consisting of M, A, and Y, and X5 is selected from the group consisting of T, P, S, and A. X6 is selected from the group consisting of S, T, A, and C, and X7 is selected from Y, W, F, T, and S. X8 is selected from the group consisting of G, P and A, and X9 is selected from the group consisting of I, L and V. Selected from the group consisting of M, A, and F, X 10 The group is selected from S, T, A, and C. At least one of the groups consisting of CDRH1 amino acid sequences selected from the group consisting of ) B)(i) Sequence IDs 14, 15, 16 Within the sequence selected from the group consisting of 17, 18, 19, 20, 21, 22, 23, and 24 CDRL1 selected from CDRL1; (ii) CDRL1 with an amino acid sequence different from that of CDRL3 in (i) due to addition, deletion or substitution of 2 or fewer amino acids, and (i i) X1X2X3X4X5X6X7X8X9X X 10 X 11 X 12 X 13 X 14 (SEQ NO: 413) (X1 is selected from the group consisting of T and S, X2 is selected from the group consisting of G, P and A, X3 is selected from the group consisting of T and S, X4 is selected from the group consisting of S, N, T, A, C and Q , X5 is selected from the group consisting of S, T, A and C, X6 is selected from the group consisting of D and E, X7 is selected from the group consisting of V, I, M, L, F and A, X8 is selected from the group consisting of G, P and A, X9 is selected from the group consisting of G, A, R, P, V, L, K, K , Q and N, X 10 is selected from the group consisting of Y, W, F, T and S , X 11 is selected from the group consisting of N and Q, X 12 is selected from the group consisting of Y, S, W, F, T, A and C, X 13 is selected from the group consisting of V, I, M, L, F and A , X 14 is selected from the group consisting of S, T, A and C. ) and at least one selected from the group consisting of CDRL1 amino acid sequences selected from at least one of the groups selected from the group consisting of CDRL1 amino acid sequences selected from at least one of the groups, and contains a light chain complementary determining region (CDRL).

[0062] In some embodiments, the invention includes an isolated antigen-binding protein that binds to PCSK9, where the antigen-binding protein is A) (i) C within a sequence selected from the group consisting of SEQ ID NOs: 47, 48, 49, 50, 5 1, 52, 53, 54, 55, 56, 57 and 58 CDRH2 selected from DRH2, (ii) amino acid addition, deletion or only substitution of 2 or fewer amino acids, a CDRH2 with an amino acid sequence different from the CDRH2 of (i), and (iii) X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO: 414) (X1 is selected from the group consisting of W, Y, and F, X2 is selected from the group consisting of V, I, M, L, F, and A, X3 is selected from the group consisting of S, T, A, and C selected, X4 is selected from the group consisting of A, F, V, L, I, Y, and M, X5 is selected from the group consisting of Y, W, F , T, and S, X6 is selected from the group consisting of N and Q, X7 is selected from the group consisting of G , P, and A, X8 is selected from the group consisting of N and Q, X9 is selected from the group consisting of T and S, X 10 is selected from the group consisting of N and Q, X 11 is Y , W, F, T, and S, X 12 is selected from the group consisting of A, V, L, and I selected, X 13 is selected from the group consisting of Q, E, N, and D, X 14 is selected from the group consisting of K, R, Q, and N, X 15 is selected from the group consisting of L, F, V, I, M, A, and Y selected, X 16 is selected from the group consisting of Q and N, X 17 [[ID=5�]]is selected from the group consisting of G, P, and A selected. ) selected from the group consisting of CDRH2 amino acid sequences, at least one selected from the group of heavy chain complementarity determining regions (CDRH), B) (i) SEQ ID NOs: 14, 15 Selected from the group consisting of 16, 17, 18, 19, 20, 21, 22, 23, and 24. CDRL2 selected from CDRL3 in the sequence; (ii) amino acid assignment of 2 or fewer amino acids Addition, deletion or substitution only, CDRL2 with an amino acid sequence different from (i) CDRL3, and (iii) X1X2X3X4X5X6X7 (Sequence number 415) (X1 consists of E and D) X2 is selected from the group consisting of V, I, M, L, F and A, and X3 is S, X4 is selected from the group consisting of T, A and C, X4 is selected from the group consisting of N and Q, and X5 is X6 is selected from the group consisting of R, K, Q, and N, and X is selected from the group consisting of P and A. 7 is selected from the group consisting of S, T, A, and C. Light chain complementation selected from at least one of the group consisting of selected CDRL2 amino acid sequences. Includes the decision region (CDRL).

[0063] In some embodiments, the present invention relates to an isolated antigen-binding protein that binds PCSK9. The substance contains the antigen-binding protein A)(i)Sequence codes 47, 48, 49, 50, 5 C in a sequence selected from the group consisting of 1, 52, 53, 54, 55, 56, 57, and 58 (ii) Addition or deletion of amino acids of 2 or fewer amino acids from DRH3, or (i) CDRH3 has a different amino acid sequence from (iii) X1X2X3X4X5X6 (Sequence ID 416) (X1 is without G, P, A and amino acids) X2 is selected from the group consisting of Y, W, F, T and S, and X3 is G, Selected from the group consisting of V, P, A, I, M, L, and F, and X4 is from M, L, F, and I. X5 is selected from the group consisting of D and E, and X6 is selected from the group consisting of V, I, M, L, F CDRH3 amino acid sequences selected from the group consisting of A and A. A heavy chain complementarity determination region (CDRH) selected from at least one of the group consisting of (B)(i ) From sequence numbers 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 CDRL3 selected from the sequence selected from the group; (ii) 2 or less Due to the addition, deletion, or substitution of amino acids, the amino acid sequence of (i) CDRL3 is different. CDRL3, and (iii)X1X2X3X4X5X6X7X8X9 (Sequence number 4 17) (X1 is selected from the group consisting of S, N, T, A, C and Q, and X2 is selected from the group consisting of S, T, A and X3 is selected from the group consisting of C, and X is selected from the group consisting of Y, W, F, T and S. 4 is selected from the group consisting of T and S, and X5 is selected from the group consisting of S, T, A and C. X6 is selected from the group consisting of S, T, A, and C, and X7 is selected from the group consisting of N, S, Q, T, A, and C. X8 is selected from the group consisting of M, V, L, F, I and A, and X9 C is selected from the group consisting of V, I, M, L, F, and A. Light chain complementarity determination region selected from at least one of the group consisting of DRL3 amino acid sequences. Includes CDRL. [Brief explanation of the drawing]

[0064] [Figure 1A] Figure 1A illustrates the amino acid sequence of the mature form of PCSK9, with the prodomain underlined. [Figure 1B1] Figure 1B1 illustrates the amino acid and nucleic acid sequences of PCSK9, with the prodomain underlined and the signal sequence shown in bold. [Figure 1B2]Figure 1B2 illustrates the amino acid and nucleic acid sequences of PCSK9, with the prodomain underlined and the signal sequence shown in bold. [Figure 1B3] Figure 1B3 illustrates the amino acid and nucleic acid sequences of PCSK9, with the prodomain underlined and the signal sequence shown in bold. [Figure 1B4] Figure 1B4 illustrates the amino acid and nucleic acid sequences of PCSK9, with the prodomain underlined and the signal sequence shown in bold. [Figure 2A] Figure 2A is a comparative table of light chain sequences of various antigen-binding proteins. [Figure 2B] Figure 2B is a comparative table of light chain sequences of various antigen-binding proteins. [Figure 2C] Figure 2C is a comparative table of various light chain sequences of various antigen-binding proteins. Figure 2C is a continuation of the sequences that began in Figure 2A. [Figure 2D] Figure 2D is a comparative table of light chain sequences of various antigen-binding proteins. Figure 2D is a continuation of the sequences that began in Figure 2B. [Figure 3A] Figure 3A is a comparative table of the heavy chain sequences of various antigen-binding proteins. [Figure 3B] Figure 3B is a comparative table of the heavy chain sequences of various antigen-binding proteins. [Figure 3C] Figure 3C is a comparative table of the sequences of various heavy chains of various antigen-binding proteins. Figure 3C is a continuation of the sequences that began in Figure 3A. [Figure 3D] Figure 3D is a comparative table of various heavy chain sequences of various antigen-binding proteins. Figure 3D is a continuation of the sequences that began in Figure 3B. [Figure 3E] Figure 3E illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3F] Figure 3F illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3G]Figure 3G illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3H] Figure 3H illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3I] Figure 3I illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3J] Figure 3J illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3K] Figure 3K illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3L] Figure 3L illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3M] Figure 3M illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3N] Figure 3N illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3O] Figure 3O illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3P] Figure 3P illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3Q] Figure 3Q illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3R] Figure 3R illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3S] Figure 3S illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3T]Figure 3T illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3U] Figure 3U illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3V] Figure 3V illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3W] Figure 3W illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3X] Figure 3X illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3Y] Figure 3Y illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3Z] Figure 3Z illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3AA] Figure 3AA illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3BB] Figure 3BB illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3CC] Figure 3CC illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3DD] Figure 3DD illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3EE] Figure 3EE illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3FF] Figure 3FF illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3GG]Figure 3GG illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3HH] Figure 3HH illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3II] Figure 3II illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3JJ] Figure 3JJ illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3KK] Figure 3KK illustrates the amino acid sequences for various constant domains. [Figure 3LL] Figure 3LL illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3MM] Figure 3MM illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3NN] Figure 3NN illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of the antigen-binding protein. [Figure 300] Figure 300 illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3PP] Figure 3PP illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3QQ] Figure 3QQ illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3RR] Figure 3RR illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3SS] Figure 3SS illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3TT]Figure 3TT illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3UU] Figure 3UU illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3VV] Figure 3VV illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3WW] Figure 3WW illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3XX] Figure 3XX illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3YY] Figure 3YY illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3ZZ] Figure 3ZZ illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3AAA] Figure 3AAA illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3BBB] Figure 3BBB illustrates the amino acid and nucleic acid sequences for the variable domains of several embodiments of antigen-binding proteins. [Figure 3CCC] Figure 3CCC is a comparative table of various heavy and light chain sequences for several embodiments of antigen-binding proteins. [Figure 3DDD] Figure 3DDD is a comparative table of various heavy and light chain sequences for several embodiments of antigen-binding proteins. [Figure 3EEE] Figure 3EEE is a comparative table of various heavy and light chain sequences for several embodiments of antigen-binding proteins. [Figure 3FFF] Figure 3FFF is a comparative table of various heavy and light chain sequences for several embodiments of antigen-binding proteins. [Figure 3GGG]Figure 3GGG is a comparative table of various heavy and light chain sequences for several embodiments of antigen-binding proteins. [Figure 3HHH] Figure 3HHH is a comparative table of various heavy and light chain sequences for several embodiments of antigen-binding proteins. [Figure 3III] Figure 3III is a comparative table of various heavy and light chain sequences for several embodiments of antigen-binding proteins. [Figure 3JJJ] Figure 3JJJ is a comparative table of various heavy and light chain sequences for several embodiments of antigen-binding proteins. [Figure 4A] Figure 4A shows the binding curve of antigen-binding proteins to human PCSK9. [Figure 4B] Figure 4B shows the binding curve of antigen-binding proteins to human PCSK9. [Figure 4C] Figure 4C shows the binding curve of antigen-binding proteins to PCSK9 in cynomolgus monkeys. [Figure 4D] Figure 4D shows the binding curve of antigen-binding proteins to PCSK9 in cynomolgus monkeys. [Figure 4E] Figure 4E shows the binding curve of antigen-binding proteins to mouse PCSK9. Figure 4F shows the binding curve of antigen-binding proteins to mouse PCSK9. [Figure 4F] Figure 4F shows the binding curve of antigen-binding proteins to mouse PCSK9. [Figure 5A] Figure 5A illustrates the results of SDSPAGE experiments using PCSK9 and various antigen-binding proteins, showing the relative purity and concentration of the proteins. [Figure 5B] Figure 5B shows a graph obtained from the biacore solution equilibrium assay for 21B12. [Figure 5C] Figure 5C shows a graph obtained from the biacore solution equilibrium assay for 21B12. [Figure 5D] Figure 5D shows a kinetic graph obtained from the biacore capture assay. [Figure 5E]Figure 5E shows a bar graph illustrating the binning results for the three ABPs. [Figure 6A] Figure 6A shows the inhibition curve of the antigen-binding protein 31H4IgG2 to PCSK9 in an in vitro PCSK9:LDLR binding assay. [Figure 6B] Figure 6B shows the inhibition curve of the antigen-binding protein 31H4IgG4 to PCSK9 in an in vitro PCSK9:LDLR binding assay. [Figure 6C] Figure 6C shows the inhibition curve of the antigen-binding protein 21B12IgG2 to PCSK9 in an in vitro PCSK9:LDLR binding assay. [Figure 6D] Figure 6D shows the inhibition curve of the antigen-binding protein 21B12IgG4 to PCSK9 in an in vitro PCSK9:LDLR binding assay. [Figure 7A] Figure 7A shows the inhibition curve of the antigen-binding protein 31H4IgG2 in a cellular LDL uptake assay, demonstrating the effect of ABP on reducing the LDL uptake blocking effect of PCSK9. [Figure 7B] Figure 7B shows the inhibition curve of the antigen-binding protein 31H4IgG4 in a cellular LDL uptake assay, demonstrating the effect of ABP on reducing the LDL uptake blocking effect of PCSK9. [Figure 7C] Figure 7C shows the inhibition curve of the antigen-binding protein 21B12IgG2 in a cellular LDL uptake assay, demonstrating the effect of ABP on reducing the LDL uptake blocking effect of PCSK9. [Figure 7D] Figure 7D shows the inhibition curve of the antigen-binding protein 21B12IgG4 in a cellular LDL uptake assay, demonstrating the effect of ABP on reducing the LDL uptake blocking effect of PCSK9. [Figure 8A] Figure 8A is a graph illustrating the serum cholesterol-lowering ability of ABP31H4 in mice (change compared to IgG control-treated mice) (*p<0.01). [Figure 8B]Figure 8B is a graph illustrating the serum cholesterol-lowering ability of ABP31H4 in mice (change compared to time = zero) (*p<0.05). [Figure 8C] Figure 8C is a graph illustrating the effect of ABP31H4 on HDL cholesterol levels in C57B1 / 6 mice (*p<0.01). [Figure 8D] Figure 8D is a graph illustrating the effect of ABP31H4 on HDL cholesterol levels in C57B1 / 6 mice (#p<0.05). [Figure 9] Figure 9 illustrates the Western blot analysis of the ability of ABP31H4 to increase the amount of liver LDLR protein present at various time points. [Figure 10A] Figure 10A is a graph illustrating the ability of the antigen-binding protein 31H4 to lower total serum cholesterol in wild-type mice. [Figure 10B] Figure 10B is a graph illustrating the ability of the antigen-binding protein 31H4 to lower HDL in wild-type mice. [Figure 10C] Figure 10C is a graph illustrating the serum cholesterol-lowering ability of various antigen-binding proteins 31H4 and 16F12. [Figure 11A] Figure 11A illustrates an injection protocol for investigating the duration and ability of antigen-binding proteins to lower serum cholesterol. [Figure 11B] Figure 11B is a graph illustrating the results of the protocol shown in Figure 11A. [Figure 12A] Figure 12A illustrates LDLR levels in HepG2 cells in response to a combination of statin and ABP21B12. [Figure 12B] Figure 12B illustrates LDLR levels in HepG2 cells in response to statin and ABP31H4 combinations. [Figure 12C] Figure 12C illustrates LDLR levels in HepG2 cells in response to a combination of statins and the non-neutralizing antibody ABP25A7.1 (unlike the neutralizing antibody "25A7"). [Figure 12D] Figure 12D illustrates LDLR levels in PCSK9-overexpressing HepG2 cells in response to a combination of statin and ABP21B12. [Figure 12E] Figure 12E illustrates LDLR levels in PCSK9-overexpressing HepG2 cells in response to a combination of statin and ABP31H4. [Figure 12F] Figure 12F illustrates LDLR levels in PCSK9-overexpressing HepG2 cells in response to a combination of statins and the non-neutralizing antibody ABP25A7.1 (unlike the neutralizing antibody "25A7"). [Figure 13A] Figure 13A illustrates various light chain amino acid sequences of various ABPs relative to PCSK9. A dot (.) indicates the absence of an amino acid. [Figure 13B] Figure 13B illustrates the light chain bifurcation diagrams for various ABPs relative to PCSK9. [Figure 13C] Figure 13C illustrates various heavy-chain amino acid sequences of various ABPs relative to PCSK9. A dot (.) indicates the absence of an amino acid. [Figure 13D] Figure 13D illustrates the heavy chain dendrograms for various ABPs relative to PCSK9. [Figure 13E] Figure 13E shows a comparison of light-chain and heavy-chain CDRs and the notation for the group that gives the consensus. [Figure 13F] Figure 13F illustrates the consensus sequences for groups 1 and 2. [Figure 13G] Figure 13G illustrates the consensus sequences for groups 3 and 4. [Figure 13H] Figure 13H illustrates the consensus sequences for groups 1 and 2. Dots (.) indicate identical residues. [Figure 13I] Figure 13I illustrates the consensus sequence for group 2. Dots (.) indicate identical residues. [Figure 13J] Figure 13J illustrates the consensus sequences for groups 3 and 4. Dots (.) indicate identical residues. [Figure 14A] Figure 14A is a graph illustrating the in vivo LDL-lowering ability of various ABPs (at 10 mg / kg). [Figure 14B] Figure 14B is a graph illustrating the in vivo LDL-lowering ability of various ABPs (at 30 mg / kg). [Figure 15A] Figure 15A is a comparative table of various light chain sequences for different embodiments of antigen-binding proteins. [Figure 15B] Figure 15B is a comparative table of various light chain sequences for different embodiments of antigen-binding proteins. Figure 15B is a continuation of the sequences that began in Figure 15A. [Figure 15C] Figure 15C is a comparative table of various light chain sequences for different embodiments of antigen-binding proteins. [Figure 15D] Figure 15D is a comparative table of various light chain sequences for different embodiments of antigen-binding proteins. Figure 15D is a continuation of the sequences starting from Figure 15C. [Figure 16A] Figure 16A is an illustration of the gel used to investigate the ability of Ab21B12 to bind to the ProCat or VD portion of PCSK9. [Figure 16B] Figure 16B is an illustration of the gel used to investigate the ability of Ab31H4 to bind to the ProCat or VD portion of PCSK9. [Figure 17] Figure 17 illustrates the structure of the EGFa portion of PCSK9 and LDLR. [Figure 18A] Figure 18A is an illustration of the structures of PCSK9 and 31H4Ab. [Figure 18B] Figure 18B is an illustration of the structures of PCSK9 and 31H4Ab. [Figure 19A] Figure 19A illustrates the structures of PCSK9, 31H4Ab, and 21B12Ab. [Figure 19B] Figure 19B is an illustration of the structures of PCSK9 and 21B12Ab. [Figure 20A] Figure 20A is a diagram illustrating the structures of PCSK9 and LDLR-derived EGFa superimposed on the structures of antibodies 31H4 and 21B12 bound to PCSK9. [Figure 20B] Figure 20B is an illustration of the structural models of PCSK9 and LDLR. [Figure 20C] Figure 20C is an illustration of the structural models of PCSK9 and LDLR obtained from a different angle. [Figure 20D] Figure 20D illustrates the structural models of PCSK9 and LDLR, along with their structural representations, including 31H4 and 21B12. [Figure 20E] Figure 20E is an illustration of the structural model of Figure 20D, rotated 90° around the indicated axis. [Figure 20F] Figure 20F is an illustration of the structural model of Figure 20D, rotated 180° around the indicated axis. [Figure 21A] Figure 21A illustrates the structures of PCSK9 and 31A4. [Figure 21B] Figure 21B illustrates the structures of PCSK9 and 31A4. Figure 21C illustrates the structures of PCSK9 and 31A4. [Figure 21C] Figure 21C illustrates the structures of PCSK9 and 31A4. [Figure 21D] Figure 21D is an illustration of the structural models of the full-length PCSK9 and 31A4. [Figure 22] Figure 22 shows a group of ABP sequences that identify various differences between human ABP sequences and ABP sequences produced in E. coli and used for crystal structure. [Figure 23] Figure 23 is a table showing the results of various binning operations. [Figure 23A] Figure 23A is the first part of a table illustrating the results of various binning methods. [Figure 23B] Figure 23B is the second part of a table illustrating the results of various binning methods. [Figure 23C] Figure 23C is the third part of a table illustrating the results of various binning methods. [Figure 23D] Figure 23D is the fourth part of a table illustrating the results of various binning operations. [Figure 24A]Figure 24A shows a Western blot under non-reducing conditions. [Figure 24B] Figure 24B shows a Western blot under reducing conditions. [Figure 25A] Figure 25A illustrates the surface coating of PCSK9. [Figure 25B] Figure 25B is an illustration of the surface coating of PCSK9. [Figure 25C] Figure 25C illustrates the surface coating of PCSK9. [Figure 25D] Figure 25D illustrates the surface coating of PCSK9. [Figure 25E] Figure 25E illustrates the surface coating of PCSK9. [Figure 25F] Figure 25F is an illustration of the surface coating of PCSK9. [Figure 26] Figure 26 shows a comparison of all sequences of the PCSK9 amino acid sequence and mutated residues in the PCSK9 variant to investigate the epitopes of various antibodies. [Figure 27A] Figure 27A illustrates the hit of the 21B12 epitope mapped onto the crystal structure of PCSK9 with 21B12. [Figure 27B] Figure 27B illustrates the hits of the 31H4 epitope mapped onto the crystal structure of PCSK9 with 31H4 and 21B1. [Figure 27C] Figure 27C illustrates the hits of the 31H4 epitope mapped onto the crystal structure of PCSK9 with 31H4 and 21B12. [Figure 27D] Figure 27D illustrates the hits of the 12H11 epitope mapped onto the crystal structure of PCSK9 with 31H4 and 21B12. [Figure 27E] Figure 27E illustrates the hits of the 3C4 epitope mapped onto the crystal structure of PCSK9 with 31H4 and 21B12. [Figure 28A] Figure 28A is a graph showing the ability of various ABPs to bind to different parts of PCSK9. [Figure 28B]Figure 28B is a graph showing the ability of various ABPs to bind to different parts of PCSK9. [Figure 28C] Figure 28C is a graph comparing the LDLR binding capabilities of two ABPs. [Figure 28D] Figure 28D is a graph comparing the cellular LDL uptake activity of two ABPs. [Modes for carrying out the invention]

[0065] Antigen-binding proteins (antibodies and their functional binding fragments, etc.) that bind to PCSK9 are Disclosed in the specification. In some embodiments, the antigen-binding protein is PCSK9 It binds to and prevents PCSK9 from functioning in various ways. In some embodiments Furthermore, antigen-binding proteins block PCSK9's ability to interact with other substances, or To reduce. For example, in some embodiments, the antigen-binding protein is PCSK9 It binds to PCSK9 in a manner that prevents or reduces the possibility of binding to LDLR. In this embodiment, the antigen-binding protein binds to PCSK9, but PCSO binds to LDLR. It does not block the ability to interact with it. In some embodiments, the antigen-binding protein is It is a human monoclonal antibody.

[0066] As will be understood by those skilled in the art, in light of the disclosure of the present invention, PCSK9 and LDLR Changing the interaction between them increases the amount of LDLR available for binding to LDL. Next, this reduces the amount of serum LDL in the subject and raises the serum cholesterol level of the subject. This leads to a decrease in serum. Therefore, the antigen-binding protein for PCSK9 is elevated in serum. Individuals with cholesterol levels, or those at risk of elevated serum cholesterol levels. Various treatments for patients or patients for whom a reduction in serum cholesterol levels may be beneficial. It can be used in the following methods and compositions. Therefore, the increase in serum cholesterol Various methods and techniques for reducing, maintaining, or preventing are also described herein. In some embodiments, the antigen-binding protein is involved in the binding between PCSK9 and LDLR. This makes it possible, but antigen-binding proteins inhibit the harmful activity of PCSK9 against LDLRs. or reduces. In some embodiments, the antigen-binding protein is P to LDLR. It inhibits or reduces CSK9 binding.

[0067] For convenience, the following sections provide a general overview of the various meanings of terms used in this specification. Observe. Following this discussion, a general description of antigen-binding proteins is given. Subsequently, the characteristics of various embodiments of antigen-binding proteins and how to use them are described. The text then provides specific examples demonstrating whether this is possible.

[0068] Definitions and Embodiments The above general description and the following detailed explanation are merely illustrative and descriptive, and in particular It must be understood that this does not limit the invention as described in the claims. No. In this application, the use of the singular form includes the plural form unless otherwise specified. In this context, the use of "or" means "and / or" unless otherwise specified. The use of the term "includes" as well as other forms such as "include" and "included" The use is not limited. Also, terms such as "element" or "component" are used unless otherwise noted. If not listed, elements and components containing one unit and elements containing two or more subunits It includes both elements and components. Also, the use of the term "part" implies a part of the constituent parts or It may include all of its constituent parts.

[0069] The headings used in this specification are for organizational purposes only and do not include the headings used in the text. It should not be interpreted as limiting the subject matter. Patents, patent applications, articles, books and papers, etc. (However, this does not apply) all documents or parts thereof cited in this application For all purposes, the entirety of it is expressly incorporated herein by reference. When used in this disclosure, the following terms have the meanings set forth below unless otherwise noted. We need to understand those around us.

[0070] "Proprotein converterases subtilisin keksin type 9" or "PCSK9" The term refers to the polypeptide or fragment thereof described in Sequence ID No. 1 and / or 3, and Allele variant, splice variant, derivative variant, substitution variant, deletion Lost variants and / or inserted variants (including the addition of N-terminal methionine), fusion poly Related polypeptides, including (but not limited to) peptides and interspecific homologs, are listed. In certain embodiments, the PCSK9 polypeptide is a leader sequence residue, targeted Residues, amino-terminal methionine residues, lysine residues, tag residues and / or fusion protein residues It includes terminal residues such as (but not limited to) these. "PCSK9" is FH3, NARC1, HCHOLA3, Proprotein Converterse Subtilisin / Keksin 9 It is also called type 1 and neuronal apoptosis regulatory converterase 1. The PCSK9 gene is A proprotein belonging to the proteinase K subfamily of the secretory subtylases family. It encodes a protein converterase protein. The term "PCSK9" refers to the protein... This refers to both the product generated after the autocatalysis of the protein and proprotein. (e.g., cleaved) Only autocatalyzed products (for antigen-binding proteins that selectively bind to PCSK9) When cited, the protein is described as "mature," "cleaved," or "processing." It may be referred to as "activated" or "active" PCSK9. In this case, the protein is either "inactive," "pro-type," or "processed" by PCSK9. This can be described as a form that is "not attached". The term PCSK9 as used herein refers to This also includes naturally occurring alleles such as mutations D374Y, S127R, and F216L. The term CSK9 refers to glycosylated, PEGylated PCSK9 sequences and their signaling The PCSK9 sequence is cleaved from the catalytic domain to its prodomain. However, PCSK9 sequences that have not been separated from the catalytic domain (e.g., Figures 1A and 1B), etc. This also includes PCSK9 molecules that incorporate post-translational modifications to the PCSK9 amino acid sequence.

[0071] The term "PCSK9 activity" encompasses all biological effects of PCSK9. In this embodiment, PCSK9 activity interacts with the substrate or receptor, or the substrate or the ability of PCSK9 to bind to the receptor. In some embodiments, PCSK PCSK9 activity is represented by the ability of PCSK9 to bind to the LDL receptor (LDLR). In several embodiments, PCSK9 binds to and catalyzes a reaction involving LDLR. In one embodiment, PCSK9 activity alters the availability of LDLR (for example). This includes the ability of PCSK9 to reduce activity. In some embodiments, PCSK9 activity is This includes the ability of PCSK9 to increase the amount of LDL in the target. In some embodiments, PCSK9 activity reduces the amount of LDLR available for binding to LDL. This includes the ability to perform PCSK9 signaling. In some embodiments, "PCSK9 activity" refers to the ability to perform PCSK9 signaling. This includes all biological activities resulting from the transmission. Typical activities include the transmission of PCSK9 to LDLR. PCSK9 enzyme activity that binds, cleaves LDLR or other proteins, and promotes PCSK9 action. PCSK9 binding to proteins other than LDLR, and altering APOB secretion. SK9(Sun XM et al, “Evidence for effect of mutant PCSK9 on apoliprotein B secret ion as the cause of unusually severe dom inant hypercholesterolemia”, Human Molec ular Genetics 14:1161-1169, 2005 and August uerram K et al, “Apolipoprotein B100 met abolism in autosomal-dominant hyperchole sterolemia related to mutations in PCSK9 , Arterioscler thromb Vase Biol.24:1448- 1453, 2004), the role of PCSK9 in liver regeneration and neuronal differentiation (S eidah NG et al, “The secretory profession n convertase neural apoptosis-regulated convertase 1(NARC-I):Liver regeneration and neuronal differentiation” PNAS 100:9 28-933, 2003) and the role of PCSK9 in hepatic glucose metabolism (Co stet et al., “Hepatic PCSK9 expression i s regulated by nutritional status via in sulin and sterol regulatory element-bind ing protein 1c” J.Biol.Chem.281(10):6211 This includes, but is not limited to, those mentioned in -18, 2006.

[0072] As used herein, the term "hypercholesterolemia" refers to cholesterol This describes a symptom where the level has risen above a desired level. In some embodiments, This indicates that serum cholesterol levels are elevated. In some embodiments, The desired level is known to those skilled in the art (and is described or referenced herein). Take into account various "risk factors."

[0073] The term "polynucleotide" or "nucleic acid" refers to single-stranded and double-stranded nucleotides. It contains both polynucleotides. Nucleotides containing polynucleotides are either ribonucleotides or deonucleotides. It may be a modified form of xylibonenucleotide or any type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, and 2',3'-dideoxy Ribose modifications such as siribose, as well as phosphorothioate, phosphorodioate, and phosphate. Holoselenoart, phosphoroserenoart, phosphoroanirotioart, phosphorani This includes internucleotide bond modifications such as ladders and phosphoramidates.

[0074] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotides. It means nucleotide. In some embodiments, an oligonucleotide is 10 to It is 60 bases long. In other embodiments, the oligonucleotides are 12, 13, 1 They are 4, 15, 16, 17, 18, 19, or 20 to 40 nucleotides long. Rheotides are used, for example, in the construction of mutant genes, in single-stranded or double-stranded form. It's possible. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides are radiolabeled, fluorescently labeled, or haptened for detection assays. It can include labels such as antigenic labels. Oligonucleotides can be used, for example, in PCR Can be used as a lymer, cloning primer, or hybrid formation probe. can.

[0075] "Isolated nucleic acid molecules" are isolated polynucleotides that contain the polynucleotides that are normally found within them. Polynucleotides that are not accompanied by all or part of a renucleotide, or that are not originally linked. The genomic DNA, RNA, mRNA, cDNA, or synthetic origin linked to the rheotide. Or means some combination thereof. In this disclosure, a certain nucleotide sequence It should be understood that "nucleic acid molecules containing" do not include a complete chromosome. Isolated nucleic acid molecules "containing" the specified nucleic acid sequence may contain up to 10 additional nucleic acids in addition to the specified sequence. Alternatively, it may contain coding sequences for up to 20 other proteins or parts thereof. A moduloable linkage that modulates the expression of the coding region of a cited nucleic acid sequence. It may include a control array and / or a vector array.

[0076] Unless otherwise stated, all single-stranded polynucleotides discussed herein The left end of the sequence is the 5' end, and the direction of the left hand of the double-stranded polynucleotide sequence is the 5' direction. This is called the transcription direction. The direction of addition from 5' to 3' in a newly synthesized RNA transcript is called the transcription direction. A DNA strand having the same sequence as the RNA transcript, which is 5' relative to the 5' end of the RNA transcript. The upper sequence region is called the "upstream sequence." It is 3' relative to the 3' end of the RNA transcript. The sequence region on the DNA strand that has the same sequence as the RNA transcript is called the "downstream sequence."

[0077] The term "regulatory sequence" refers to the expression and processing of the coding sequence to which the regulatory sequence is linked. This represents a polynucleotide sequence that can influence sing. The properties may depend on the host organism. In certain embodiments, the regulatory sequence for prokaryotes This may include the promoter, ribosome binding site, and transcription termination sequence. For example, eukaryotic The regulatory sequence for a substance is a promoter that includes one or more recognition sites for transcription factors. , may include transcription-enhancing sequences and transcription-termination sequences. “Regulatory sequences” may include leader sequences and / Alternatively, it may include fusion pair sequences.

[0078] The term "vector" is used to transmit protein-coding information into host cells. Any molecule or entity that is subjected to this (e.g., nucleic acids, plasmids, bacteriophages or viruses) It means (Rus).

[0079] The terms "expression vector" or "expression construct" are suitable for the transformation of host cells. , and the expression of one or more heterogeneous coding regions ligated to the nucleic acid sequence This represents a vector containing a nucleic acid sequence that induces and / or regulates (in conjunction with host cells). Expression constructs may affect or regulate transcription and translation, and if introns are present... , containing sequences that affect RNA splicing of functionally linked coding regions These are rare, but not limited to these.

[0080] As used herein, “operably connected” means the combination to which this term applies. This means that the components are in a relationship where they can perform their unique functions under appropriate conditions. For example, The regulatory sequence in a vector "operably linked" to the protein coding sequence is the regulatory sequence Under conditions compatible with the transcriptional activity, the expression of the protein-coding sequence is achieved. It is linked to the protein code sequence.

[0081] The term "host cell" refers to a cell that has been transformed with a nucleic acid sequence, or a cell that has been transformed with a nucleic acid sequence. This can be done, and thus it refers to cells that express the desired gene. This term is, As long as the target gene is present, whether the offspring are morphologically identical to the original parent cell, or This includes offspring of the parent cell, regardless of whether they have the same genetic composition as the parent cell.

[0082] The term "transfer of DNA" refers to the uptake of foreign or exogenous DNA by cells. When causal DNA is introduced into the cell membrane, the cell is "transformed." The import technology is well known in this art and is disclosed herein. For example, "Gra ham et al., 1973, Virology 52:456; ook et al., 2001, Molecular Cloning:A La Boratory Manual, see above; Davis et al., 1986. Basic Methods in Molecular Biology, Else See vier; Chu et al, 1981, Gene 13:197. This technology involves introducing one or more foreign DNA segments into a suitable host cell. It can be used for that purpose.

[0083] The term "transformation" refers to a change in the genetic properties of a cell, resulting in new DNA or RNA. Cells are transformed when they are modified to contain [a specific substance]. For example, translocation, shape By introducing new genetic material through genetic introduction or other techniques, its intrinsic state can be altered. Cells are transformed when they are genetically modified. By physically integrating into the chromosomes of the cell, the transformed DNA is incorporated into the cell. It can be recombined with DNA, or it is transiently maintained as an episomal element without being replicated. It can be transformed, or it can replicate independently as a plasmid. When the DNA present in the cell is replicated during cell division, the cell becomes "stablely transformed." It's possible.

[0084] The terms "polypeptide" or "protein" refer to the original protein, i.e., the natural protein. Macromolecules having the amino acid sequence of proteins produced by naturally occurring non-recombinant cells. The terms "polypeptide" or "protein" refer to genetically modified cells or groups. A molecule or prototype sequence produced by replacement cells that has the amino acid sequence of the original protein. Includes molecules having one or more amino acid deletions, additions and / or substitutions. This term refers to the corresponding amino acids and polyamino acids that exist naturally, one or more of which are present in the natural environment. It also includes amino acid polymers, which are chemical analogs of MAR. Specifically, "polypeptides" and The term "protein" refers to one or more amino acids from an antigen-binding protein. Includes PCSK9 antigen-binding proteins, antibodies, or sequences with deletions, additions, and / or substitutions. The term "polypeptide fragment" refers to a fragment of a protein that has an amino acid-rich base, compared to a full-length original protein. This represents a polypeptide having terminal deletions, carboxyl terminus deletions, and / or internal deletions. Such fragments contain modified amino acids compared to the original protein. In embodiments, the fragments are approximately 5 to 500 amino acid long. For example, the fragments are at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 25 The amino acid length may be 0, 300, 350, 400, or 450. Useful polypeptide fragments. It contains an immunologically functional fragment of the antibody (including the binding domain). PCSK9 In the case of conjugated antibodies, useful fragments include the CDR region, the variable domains of the heavy and / or light chains, and the anti- This includes, but is not limited to, only a portion of the body chain or its variable region containing two CDRs. I can't.

[0085] The term “isolated protein” as cited means that the protein in question is (1) normally (2) from the same source, ( For example, (3) cells from different species that contain virtually no other proteins (from the same species). (4) Polynucleotides, lipids, which are expressed by, (5) Separated from carbohydrates or other substances, at least about 50% polypeptides that do not exist together can bind to each other (by covalent or non-covalent interactions). (6) It means that it is isolated or does not exist in its natural state. Typically, it means "isolated The "proteins" make up at least about 5%, at least about 10%, and at least about It accounts for 25% or at least about 50%. Genomic DNA, cDNA, mRNA, or compound Other RNAs of origin, or any combination thereof, may be involved in the production of such isolated proteins. It can encode quality. Preferably, the isolated protein has therapeutic, diagnostic, and prophylactic properties. Proteins or polyps found in their natural environment may interfere with research or other uses. It is substantially free of butylene or other impurities.

[0086] The term "amino acid" includes its usual meaning in this field.

[0087] A "variant" of a polypeptide (e.g., an antigen-binding protein or antibody) is a different polypeptide. Compared to a peptide sequence, one or more amino acid residues are inserted into the amino acid sequence. Contains deleted and / or substituted amino acid sequences. Variants are fusion proteins. Includes quality.

[0088] The term "identity" refers to two sequences that are determined by juxtaposing and comparing them. The relationship between sequences of more than one polypeptide molecule or two or more nucleic acid molecules Represents "% identity." "% identity" means that the same residues between amino acids or nucleotides in the molecules being compared. This represents a percentage of the base molecule, calculated based on the smallest size among the molecules being compared. These calculations require a certain type of mathematical model or computer program (i.e., By the "algorithm", preferably, (if a gap exists) the juxtaposition of A gap is assigned. This is used to calculate the identity of juxtaposed nucleic acids or polypeptides. The methods that can be used include "Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Ox. ford university press;Biocomputing Infor matics and Genome Projects,(Smith,DW,e d.), 1993, New York: Academic Press; Compute r Analysis of Sequence Data,Part 1,(Grif fin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology,N ew York:Academic Press(1987);Sequence An alysis Primer,(Gribskov,M. and Devereux, J., eds.), 1991, New York: M. Stockton Press. ; and Carillo et al., 1988, SIAMJ.Applied Mat This includes the material described in "h., 48:1073".

[0089] When calculating % identity, the arrays being compared are chosen to give the greatest match between them. They are typically placed side by side. A computer program that can be used to measure % identity. An example of a RAM is the GCG program package which includes GAP (Devereux e t al., 1984, Nucl.AcidRes.12:387; cs Computer Group, University of Wisconsin (in, Madison, WI). The computer algorithm GAP is % sequence identity Used to juxtapose two polypeptides or polynucleotides to be measured. The columns are juxtaposed to obtain the best possible match for each amino acid or nucleotide. The "matched span" determined by the algorithm. Gap opening penalty (Calculated as 3 × average diagonal. "Average diagonal" is the diagonal of the comparison matrix used) This is the average. The "diagonal" is assigned to each perfect amino acid match in this comparison matrix. This is the score or number that can be increased.) and gap extension penalty (usually, gap open penalty) It is 1 / 10 of the penalty.) and ratios such as PAM250 or BLOSUM62 A comparison matrix is ​​used with the algorithm. In certain embodiments, a standard comparison Matrices are also used by the algorithm (regarding the PAM250 comparison matrix, Dayho ff et al., Atlas of Protein Sequence and Structure, 5:345-35; Regarding the BLOSUM62 comparison matrix, Heni koff et al.,1992,Proc.Natl.Acad.Sci USA, See 89:10915-10919 (1992).

[0090] The GAP program is used to measure the % identity of polypeptides or nucleotide sequences. Examples of parameters that can be used when performing this operation are as follows:

[0091] • Algorithm: Needleman et al., 1970, J.Mol.Bio l.,48:443-453 • Comparison matrix: Henikoff et al., 1992, BLOS obtained from the above UM62 • Gap penalty: 12 (no penalty for terminal gaps) • Gap length penalty: 4 • Similarity threshold: 0

[0092] Certain juxtaposition schemes for juxtaposing two amino acid sequences involve the short region of the two sequences. This may only result in a match, and there may be no significant relationship between the two full-length sequences. However, these small juxtaposed regions can have extremely high sequence identity. Therefore, target poly This results in a juxtaposition of at least 50 or other consecutive amino acids of a peptide. If desired, the selected juxtaposition method (GAP program) can be adjusted. Cut.

[0093] The 20 commonly used (e.g., naturally occurring) amino acids and so on used herein These abbreviations follow conventional usage. "Immunology-A Synthesis" (2nd Edition,ES Golub and DR Gren,E ds.,Sinauer Associates,Sunderland,Mass.( See (1991)) (which is incorporated herein by reference for all purposes). Please be informed. 20 stereoisomers of common amino acids (e.g., D-amino acids), α, α-dimorphs. Non-natural amino acids such as converted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids Mino acids can also be suitable components for the polypeptide of the present invention. Examples of non-conventional amino acids include It contains 4-hydroxyproline, γ-carboxyglutamic acid, and ε-N,N,N-trimethic acid. Luridine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-hol Myrmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine It contains nin and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The workspace notation used herein follows standard usage and convention. The left-hand direction is towards the amino terminus, and the right-hand direction is towards the carboxyl terminus.

[0094] Similarly, unless otherwise specified, the left end of a single-stranded polynucleotide sequence is the 5' end. In this case, the left-hand direction of the double-stranded polynucleotide sequence is called the 5' direction. The addition in the direction from 5' to 3' is called the direction of transcription. It has the same sequence as RNA, and The sequence region on the DNA strand that is 5' relative to the 5' end of an RNA transcript is called the "upstream sequence." It has the same sequence as RNA and is 3' relative to the 3' end of the RNA transcript. The sequence region on the A strand is called the "downstream sequence."

[0095] Conservative amino acid substitutions are typically performed through chemical peptide synthesis, rather than through synthesis within biological systems. This allows for the incorporation of amino acids that do not exist naturally. These include peptides. This includes cytoid mimics and other inverted or reversed forms of the amino acid moiety.

[0096] Naturally occurring residues can be classified into classes based on their common side-chain properties.

[0097] 1) Hydrophobic: norleucine, Met, Ala, VaI, Leu, Ile; 2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) Acidic: Asp, Glu; 4) Basicity: His, Lys, Arg; 5) Residues that affect chain directionality: Gly, Pro and 6) Aromatics: Trp, Tyr, Phe.

[0098] For example, a non-conservative substitution is the exchange of a member of one of these classes for a member of another class. This may include, for example, a human antibody homologous to a non-human antibody. It can be introduced into a region of the body or into a non-homologous region of a molecule.

[0099] In modifying antigen-binding proteins or PCSK9 proteins, certain methods are employed. Depending on their morphology, the hydropathy index of amino acids can be examined. Each amino acid has a hydrophobic and A hydrophobicity index is assigned based on the charge characteristics. The hydrophobicity index is: Soleusine (+4.5), Valine (+4.2), Leucine (+3.8), Phenylalanine N (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alani N (+1.8), Glycine (-0.4), Threonine (-0.7), Serine (-0.8) Tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histamine Zin (-3.2), glutamic acid (-3.5), glutamine (-3.5), asparagine Acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4) .5)

[0100] Hydrophobic and hydrophilic amino acid indicators for conferring interactive biological functions to proteins Its importance is understood in this field. Kyte et al, J.Mol.Bio l.,157:105-131(1982). Certain amino acids have similar hydrophobic and hydrophilic properties. It can be substituted with other amino acids that have an index or score, and still similar biological activity It is known to retain its properties. When changing it based on the hydrophobicity index, there is a certain In one embodiment, the substitution includes an amino acid whose hydrophobicity index is within ±2. Morphologically, it includes amino acid substitutions within ±1, and in some embodiments, within ±0.5. Includes amino acid substitutions within a certain limit.

[0101] Substitutions of similar amino acids, as in this case, produce biologically functional substances. When using proteins or peptides in immunological embodiments, it is especially important to use them in a hydrophilic manner. It is understood in this field that this can be effectively implemented. In this context, the local maximum plane of the protein is governed by the hydrophilicity of its adjacent amino acids. Homohydrophilicity correlates with its immunogenicity and antigenicity, that is, the biological properties of the protein. ru.

[0102] The following hydrophilic values ​​are 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), Glycerin Syn (0), Threonine (-0.4), Proline (-0.5±1), Alanine (-0.5 ), histidine (-0.5), cysteine ​​(-1.0), methionine (-1.3), barium ¹(-1.5), leucine(-1.8), isoleucine(-1.8), tyrosine(-2. 3) Phenylalanine (-2.5) and tryptophan (-3.4). Similar hydrophilicity values. When changing based on this, in one embodiment, the hydrophilicity value is within ±2. This includes amino acid substitutions, and in some embodiments, amino acid substitutions within ±1. In one embodiment, this includes amino acid substitutions within ±0.5. Based on hydrophilicity Furthermore, it is also possible to identify epitopes from the primary amino acid sequence. These regions are called "epitopes." It is also called the "pitope core region."

[0103] Examples of amino acid substitutions are shown in Table 1.

[0104] [Table 1]

[0105] The term "derivative" refers to a chemical modification other than the insertion, deletion, or substitution of amino acids (or nucleic acids). Represents a molecule containing ornaments. In certain embodiments, the derivative is a polymer, lipid or other Covalent modifications such as (but not limited to) chemical bonding with mechanical or inorganic parts Includes. In certain embodiments, chemically modified antigen-binding proteins are chemically It can have a longer circulating half-life than unmodified antigen-binding proteins. In the species embodiment, the chemically modified antigen-binding protein is used in the desired cells, tissues, and It may have improved targeting ability to veterinary and / or organs. In some embodiments, The derivative antigen-binding proteins are polyethylene glycol and polyoxyethylene glycol. or polypropylene glycol, etc. (but not limited to these), one or more It is covalently modified to include the attachment of the water-soluble polymer shown above. For example, U.S. Patent No. 4.64 No. 0,835, No. 4,496,689, No. 4,301,144, No. 4,670 See issues 417, 4,791,192 and 4,179,337. In certain embodiments, the derivative antigen-binding protein is monomethoxypolyethylene. Glycols, dextran, cellulose or other carbohydrate-based polymers, poly -(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopoly Mer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated Polyols (e.g., glycerol) and polyvinyl alcohol and such polymers A mixture of (but not limited to) one or more polymers including .

[0106] In certain embodiments, the derivative is a polyethylene glycol (PEG) subunit. It is shared in the form of a certain embodiment. The rimer is bonded 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 derivatives of one of the derivatives. Or it is randomly attached to the side chains or more. In certain embodiments, antigen-binding PEG is used to improve the therapeutic effect on proteins. In certain embodiments In some cases, PEG is used to improve the therapeutic efficacy against humanized antibodies. Such methods are, for example, U.S. Patent No. 6,133,426 (see reference to all relevant documents). It is incorporated for the purpose of the target. (This is discussed in the text.)

[0107] Peptide analogs are non-peptide drugs that have properties similar to those of the template peptide. These are commonly used in the pharmaceutical industry. These types of non-peptide compounds are "Peptide mimetics" It is referred to as "tics". (Fauchere, J. Adv. Drug Res.) 15:29(1986); Veber and Freidinger TINS ​​p. 392 (1985); and Evans et al. J. Med. Chem., 30: 1229 (1987) (for all purposes, incorporated herein by reference) . ) . Such compounds are often studied with the help of computerized molecular modeling. They are developed by... Peptide mimes that are structurally similar to therapeutically useful peptides are similar... It can be used to produce therapeutic or preventive effects. Generally, peptide mimes are human Model polypeptides such as antibodies (i.e., polypeptides that have biochemical properties or pharmacological activity) Although structurally similar to (Petido), by methods well known in this field, -CH2NH-, -CH 2S-, -CH2-CH2-, -CH=CH- (cis and transform), -COCH2-, A bond selected from -CH(OH)CH2- and -CH2SO- is placed as needed. It has one or more replaced peptide bonds. One or more of the consensus sequence The systematic substitution of the above amino acids with the same type of D amino acid (for example, replacing L-lysine) D-lysine is used in certain embodiments to produce more stable peptides. It is possible. Furthermore, constraints including consensus sequence or substantially identical consensus sequence mutations. The peptide is then subjected to intramolecular cyclization by methods known in this field, for example, by cyclizing the peptide. By adding internal cysteine ​​residues that can form disulfide crosslinks, It can be produced (Rizo and Gierasch Ann. Rev. Biochem) .61:387(1992), incorporated herein for all purposes by reference. (It can be done.)

[0108] The biological substances used herein in relation to polypeptides, nucleic acids, host cells, etc. The term "naturally occurring" refers to a substance found in its natural state or in its natural state. It describes the form of the substance being released.

[0109] The term "antigen-binding protein" ("ABP") used herein refers to an identified antigen-binding protein. This refers to any protein that binds to a target antigen. In this application, the specified target antigen is , PCSK9 protein or a fragment thereof. "Antigen-binding protein" includes antibodies and This includes, but is not limited to, binding sites (such as immunologically functional fragments). Chibodi is another example of an antigen-binding protein. Antibodies or "Immunologically functional fragments" of immunoglobulin chain (heavy chain or light chain) antigen-binding proteins. The term (or simply "fragment") refers to at least some of the amino acids present in a full-length chain. Although it lacks the necessary components, the portion of the antibody that can still specifically bind to the antigen (how that portion can bind) Regardless of whether they were obtained or synthesized, the species of antigen-binding protein containing ) Such fragments bind to the target antigen and, with respect to binding to a certain epitope, are complete. It is biologically active in that it can compete with other antigen-binding proteins, including antibodies. In some embodiments, the fragment is a neutralizing fragment. In some embodiments, One can block or reduce the potential interaction between LDLR and PCSK9. In one embodiment, such fragments are present in the full-length light chain or heavy chain. It also holds one CDR, and in some embodiments, a single heavy chain and / or light chain or so This includes a portion of these biologically active fragments, which can be produced using recombinant DNA technology. It is possible to enzymatically or chemically process antigen-binding proteins containing antibodies in their complete state. It can be produced by targeted cleavage. Immunologically functional immunoglobulin fragments include Fab, diabody (short to allow pairing between two domains on the same chain) A polypeptide identical to the light chain variable domain, linked via a short peptide linker. (The above heavy chain variable domain), Fab', F(ab')2, Fv, domain antibody and single-chain antibody This includes, but is not limited to, the bodies of humans, mice, rats, camelids, or rabbits. It may be derived from any mammalian source, including (but not limited to) ghs. Functional portions of antigen-binding proteins disclosed herein, for example, one or more CDRs These drugs are induced to specific targets in the body, possess dual therapeutic properties, or have an extended serum half-life. To create a therapeutic agent having [this property], it can be covalently bonded to a second protein or small molecule. As understood by the industry, antigen-binding proteins may contain non-protein components. Yes, it is possible. In some sections of this disclosure, examples of ABP are given as “number / letter / digit” (for example, It is described herein in the form of 25A7). In these cases, the exact name is the specific anti It represents the substance. That is, ABP named 25A7 is (in this specification, the same as) Unless explicitly taught otherwise (e.g., 25A7 and 25A7.3), This is not necessarily the same antibody as the one named 25A7.1. This is understood by those skilled in the art. Thus, in some embodiments, LDLR is not an antigen-binding protein. In this embodiment, the binding subsection of the LDLR is not an antigen-binding protein (e.g.) For example, EGFa). In some embodiments, PCSK9 is used to deliver in vivo Other molecules that transmit signaling are not antigen-binding proteins. Such embodiments are... It is explicitly identified as such.

[0110] Certain antigen-binding proteins described herein are antibodies, or are antibodies. In certain embodiments, the polypeptide structure of the antigen-binding protein is monochromatic. Ronal antibodies, bispecific antibodies, minibody antibodies, domain antibodies, synthetic antibodies (as specified herein, Sometimes referred to as "antibody mimetic," chimeric antibody, humanized antibody, human antibody, antibody fusion. The substance (which may be referred to as "antibody conjugate" in this specification) and each of these Based on antibodies containing (but not limited to) fragments of . In some embodiments In this context, ABP contains or consists of an avimer (a strongly binding peptide). These various antigen-binding proteins are further described herein.

[0111] The "Fc" region is the C of the antibody. H 1 and C H It contains two heavy chain fragments, each containing two domains. The heavy chain fragments are connected by two or more disulfide bonds, and C H Sparse domains They are fixed together by hydrological interactions.

[0112] "Fab fragment" is a C2 fragment consisting of one light chain and one heavy chain. H Includes 1 and variable regions. Fab The heavy chains of a molecule cannot form disulfide bonds with other heavy chain molecules.

[0113] The "Fab' fragment" consists of one light chain and a VH domain and C H It contains 1 domain, and An interchain disulfide bond is formed between the two heavy chains of the two Fab' fragments, resulting in F(ab')2 C H 1 and C H A single heavy chain that also includes the region between the two domains Includes some parts.

[0114] The "F(ab')2 fragment" is formed between two light chains and two heavy chains by interchain disulfide bonds. C H 1 and C H Two heavy chains containing a portion of the constant region between the two domains It contains. Therefore, the F(ab')2 fragment is formed by a disulfide bond between the two heavy chains. It consists of two Fab' fragments fixed together.

[0115] The "Fv region" includes variable regions derived from both heavy and light chains, but lacks a steady region. ru.

[0116] A "single-chain antibody" has its heavy chain and light chain variable regions connected by a flexible linker, and the antigen It is an Fv molecule that forms a single polypeptide chain that forms a binding region. Single-chain antibodies are used in the country International Patent Application Publication WO88 / 01649 and U.S. Patent No. 4,946,778 and No. 5 As discussed in detail in publications 260 and 203 (these disclosures are incorporated by reference). Yes, they are.

[0117] "Domain antibodies" are immunologically functional antibodies that contain only the variable region of the heavy chain or the variable region of the light chain. It is an active immunoglobulin fragment. In some cases, a bivalent domain antibody was produced. In order to do so, two or more V H The regions are covalently bonded using a peptide linker. Two V values ​​of the domain antibody H The region may target the same or different antigens.

[0118] A "bivalent antigen-binding protein" or "bivalent antibody" contains two antigen-binding sites. In several cases, the two binding sites have the same antigen specificity. Bivalent antigen-binding protein Cellular and bivalent antibodies may be bispecific. See below. "Multiple specificity" or "multiple" Bivalent antibodies other than "functional" antibodies are, in certain embodiments, typically identical to their It is understood that each of these has a connecting site.

[0119] A "multispecific antigen-binding protein" or "multispecific antibody" is a protein that binds two or more antigens or epidermal compounds. It targets tope.

[0120] "Dual-specific," "dual-functioning," or "dual-functional" antigen-binding proteins or antibodies These are hybrid antigen-binding proteins that each have two different antigen-binding sites or It is an antibody. A bispecific antigen-binding protein and an antibody are both bispecific antigen-binding proteins. It is a type of antibody that includes hybridoma fusion or Fab' fragment linkage (however, these It is not limited to this.) It can be made by various methods. For example, "Songsi vilai and Lachmann, 1990, Clin.Exp.Immun ol.79:315-321; Kostelny et al, 1992, J. See "Immunol. 148:1547-1553". Bispecific antigen-binding The two binding sites of a protein or antibody may be located on the same or different protein targets. It binds to different epitopes.

[0121] Antigen-binding proteins have a dissociation constant (K d ) is 10 -7 If M is less than or equal to the target antibody The term "specifically binds" to the source. ABP is K d 5 x 10 -9 If M or less It specifically binds the antigen with "high affinity", K d 5 x 10 -10 When M is less than or equal to It specifically binds the antigen with "extremely high affinity". In one embodiment, ABP is 1 0 -9 K below M d It has. In one embodiment, the off speed is 1 × 10 -5 Less than In another embodiment, ABP is about 10 -9 M and 10 -13 K between M d Human P In yet another embodiment, ABP is bonded to CSK9, and in another embodiment, ABP is 5 × 10 -10 The following K d in To bind. As will be understood by those skilled in the art, in some embodiments, antigen-binding fragments Any or all of these can specifically bind to PCSK9.

[0122] When an antigen-binding protein binds more firmly to a target than to a second target, It is "selective".

[0123] The "antigen-binding region" is a protein that specifically binds to a particular antigen (e.g., a paratope). It refers to a substance or part of a protein. For example, it interacts with an antigen and its specificity to the antigen. Antigen-binding protein containing amino acid residues that confer sex and affinity to antigen-binding proteins That part of the protein is called the "antigen-binding region." The antigen-binding region is usually one or It includes additional "complementary binding regions" ("CDRs"). Certain antigen-binding regions include one or This also includes the "framework" domain beyond that. "CDR" refers to antigen binding specificity and affinity. This is the amino acid sequence that contributes to the appropriate three-dimensional structure of the CDR. It can assist in maintenance and promote binding between the antigen-binding region and the antigen. Structurally, The framework region can be located between CDRs in the antibody. Examples of the CDR region are shown in Figures 2A to 2D, 3CCC-JJJ, and 15A to 15D. In some embodiments, the sequence of the CDR for the light chain of antibody 3B6 is as follows. As stated above. CDR1 TLSSGYSSYEVD (Sequence ID 279); CDR2 VDTGGIVGSKGE(Sequence ID 280);CDR3 GADHGSGTNFVVV (Sequence ID 281), FR is as follows: FR1 QPVLTQPLFASA SLGASVTLTC(Sequence ID 282);FR2 WYQQRPGKGPRFVMR( Sequence ID 283); FR3 GIPDRFSVLGSGLNRYLTIKNIQEEDE SDYHC (sequence number 284); and FR4 FGGGTKLTVL (sequence number 285). .

[0124] In one embodiment, a recombinant antigen-binding agent that binds PCSK9, for example, human PCSK9, is used. Proteins are provided. In this context, "recombinant antigen-binding protein" refers to recombinant technology. Prepared using the technique, that is, through the expression of recombinant nucleic acids as described herein. It is a protein. Methods and techniques for producing recombinant proteins are in this field. This is common knowledge.

[0125] The term "antibody" refers to any isotype of complete immunoglobulin or target antigen. This represents a fragment that can compete with a complete antibody in terms of specific binding, for example, This includes chimeric, humanized, fully human, and bispecific antibodies. "Antibody" refers to an antigen-binding protein. It is a type of antibody. A complete antibody consists of at least two full-length heavy chains and two full-length heavy chains. Naturally occurring in camelid animals, containing light chains in general, but in some cases only heavy chains. Antibodies, like those found in [unspecified source], may contain fewer chains. Antibodies can only be obtained from a single source. It is possible for this to be the case, or it may be a "chimera" (i.e., different parts of the antibody may be as follows): Furthermore, as described, it may be derived from two different antibodies. (Antigen-binding protein, Antibodies or binding fragments are obtained by recombinant DNA technology, or by enzymatically converting complete antibodies. It can be produced in a hybridoma by chemical cleavage. Unless otherwise stated, However, the term "antibody" can refer to an antibody that contains two full-length heavy chains and two full-length light chains, This includes derivatives, variants, fragments, and mutant proteins, examples of which are listed below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies and bispecific antibodies. Target antibodies, minibodies, domain antibodies, synthetic antibodies (referred to as "antibody mimics" in this specification) (This may be the case.) Chimeric antibodies, humanized antibodies, human antibodies, antibody fusion products (as specified herein) These are sometimes referred to as "antibody conjugates." ) and each of these fragments is included. In some embodiments, this term also encompasses peptide bodies.

[0126] Naturally occurring antibody structural units typically contain tetramers. Each of these tetramers It is typically composed of two identical pairs of polypeptide chains, each pair consisting of one full-length "light" chain. In certain embodiments, a chain of about 25 kDa and one full-length "heavy" chain (certain embodiments In its state, it has approximately 50 to 70 kDa. The amino-terminus of each chain is typically antigen-recognized. It typically contains a variable region of approximately 100 to 110 or more amino acids necessary for recognition. The carboxyl terminus of the chain typically defines the steady-state region that may be required for effector function. Human light chains are typically classified as κ and λ light chains. Heavy chains are μ, δ, γ, α, or ε. They are typically classified as such, with antibody isotypes being IgM, IgD, IgG, and IgA, respectively. And defined as IgE. IgG includes IgG1, IgG2, IgG3, and IgG4, etc. It has several subclasses (but is not limited to these). IgM is IgM1 and has subclasses such as IgM2 (but not limited to these). IgA is Similar subclasses include (but are not limited to) IgA1 and IgA2. Within the full-length light and heavy chains, the variable and steady regions are typically about 12 or more. The above amino acids are linked by the "J" region, and the heavy chain consists of approximately 10 or more amino acids. This also includes the "D" region. For example, "Fundamental Immunology, Ch. 7(Paul, W., ed., 2nd ed.Raven Press, NY (1989) (By reference, the whole is incorporated for all purposes.) See below. The variable region of each light / heavy chain pair typically forms an antigen-binding site.

[0127] The variable region is comprised of three hypervariable regions (also known as complementary determination regions or CDRs). Typical examples of linked, relatively preserved framework regions (FRs) with the same general structure. The CDR obtained from the two chains of each pair is typically parallelized by the framework domain. This may enable binding to specific epitopes. From the N-terminus to the C-terminus. The light chain and heavy chain variable regions are typically domains FR1, CDR1, FR2, and CDR2. , including FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically done by immunology. Definition of Kabat sequences of epidemiologically interesting proteins (National Institutes titutes of Health, Bethesda, Md. (1987 an d 1991)), or "Chothia & Lesk, J.Mol.Biol., 196:901-917 (1987); Chothia et al., Natu Follow re, 342:878-883 (1989).

[0128] In certain embodiments, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In one embodiment, the antibody light chain binds to the antigen in the absence of the antibody heavy chain. In this embodiment, the antibody-binding region binds to the antigen in the absence of the antibody light chain. In this embodiment, the antibody-binding region binds to the antigen in the absence of the antibody heavy chain. In this embodiment, each variable region specifically binds to the antigen in the absence of other variable regions. ru.

[0129] In certain embodiments, the definitive description of the CDR and the residues containing the antibody binding site are also described. The determination is made by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex. This is achieved. In certain embodiments, this is known to those skilled in the art, such as in X-ray crystallography. This can be achieved by any of the various technologies. In certain embodiments, CDR Various analytical methods can be used to identify or estimate the area. Examples of such methods include Kabat definitions, Chothia definitions, AbM definitions, and contact definitions. These are rare, but not limited to them.

[0130] The Kabat definition is a standard for numbering residues in antibodies and for identifying CDR regions. It is typically used for this purpose. For example, "Johnson & Wu, Nucleic See Acids Res., 28:214-8 (2000). The ia definition is similar to the Kabat definition, but the Chothia definition is a kind of structural rule The location of the region is taken into consideration. For example, "Chothia et al, J.Mo l.Biol., 196:901-17 (1986); Chothia et a See "l, Nature, 342:877-83 (1989)". AbM The definition is by the Oxford Molecular Group, which models antibody structures. It uses an integrated package of computer programs produced by [the program]. For example, Martin et al, Proc Natl Acad Sci (USA), 86:9268-9272 (1989); “AbM TM A Computer Pr gram for Modeling Variable Regions of A ntibodiies,”Oxford, UK; Oxford Molecular, Please refer to "Ltd." The AbM definition is based on publicly available databases and PROTEINS. Structure, Function and Genetics Suppl., 3:194-198 (1999) "Samudrala et al, "Ab Initio Protein Structure Prediction Usin "ga Combined Hierarchical Approach," by Using the combination of Ab-Initio methods described, the quaternary structure of the antibody is obtained from the primary sequence. The structure is modeled. Contact definition is based on the analysis of available complex crystal structures. For example, , “MacCallum et al, J. Mol.Biol., 5:732-4 See "5 (1996)".

[0131] By convention, the CDR region in the heavy chain is typically referred to as H1, H2, and H3, and is the amino terminus. They are numbered sequentially from the carboxyl terminus. The CDR regions in the light chain are typically L1, L2 These are also referred to as L3, and are numbered sequentially from the amino terminus to the carboxyl terminus.

[0132] The term "light chain" refers to a light chain of full length and a variable region sufficient to confer binding specificity. It contains a fragment having a column. The full-length light chain has a variable region domain, V L and steady-state domain Hmm, C L It includes. The variable region domain of the light chain is located at the amino terminus of the polypeptide. The chain includes a κ chain and a λ chain.

[0133] The term "heavy chain" refers to a heavy chain of full length and a variable region sufficient to confer binding specificity. It includes its fragments having columns. The full-length heavy chain has variable region domain V. H and three steady-state regions Domain C H 1, C H 2 and C H Includes 3. V H The domain is at the amino terminus of the polypeptide. , and C H The domain is located at the carboxyl terminus, C H 3 is the carboxyl part of the polypeptide. Closest to the end. The heavy chain is IgG (IgG1, IgG2, IgG3 and IgG4 subtypes). (including IgA1 and IgA2 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE It could be any isotype.

[0134] Bispecific or bifunctional antibodies typically consist of two different heavy / light chain pairs and two different types of antibodies. It is an artificial hybrid antibody that has a binding site. A bispecific antibody is a hybrid Various methods such as the fusion of M or the linking of Fab' fragments (but not limited to these) Therefore, it can be produced. For example, "Songsivilai et al., Clin." Exp. Immunol., 79:315-321 (1990); y et al., J. Immunol., 148:1547-1553 (19 See "92)".

[0135] Some mammalian species also produce antibodies that consist of only a single heavy chain.

[0136] Each immunoglobulin chain consists of approximately 90 to 110 amino acids. Typically composed of several "immunoglobulin domains" with distinctive folding patterns. These domains are the basic units that make up antibody polypeptides. In humans, IgA and IgD isotypes contain four heavy chains and four light chains, while IgG and IgE isotypes contain four heavy chains and four light chains. The type contains two heavy chains and two light chains, while the IgM isotype contains five heavy chains and five It contains a light chain. The heavy chain C region may be one or more required for the effector function. It typically contains the domain. The number of heavy chain constant region domains depends on the isotype. Ig G heavy chains are, for example, C H 1, C H 2 and C H It contains three C region domains known as 3. The antibodies provided may contain any of these isotypes and subtypes. In certain embodiments of the invention, the anti-PCSK9 antibody is an IgG2 or IgG4 subtype. It is an antibody.

[0137] The term "variable region" or "variable domain" refers to the area from approximately 120 degrees from the amino terminus in the heavy chain. It typically contains 130 amino acids and approximately 100 to 110 amino-terminal amino acids in its light chain. , represents a portion of the light chain and / or heavy chain of the antibody. In certain embodiments, different antibodies can be used. The variable region of an antibody can have significantly different amino acid sequences, even among antibodies of the same species. The specificity of the antibody against its target is typically determined.

[0138] The terms "neutralizing antigen-binding protein" or "neutralizing antibody" refer to a protein that binds to a ligand and has a ligand. These inhibitors interfere with or reduce the biological effects of ligands, respectively, by antigen-binding proteins or anti- This represents the body. This can be achieved, for example, by directly blocking the binding site on the ligand, or by It binds to Gand and, through indirect means (such as structural or energetic changes in the ligand), This can be done by changing the ligand binding ability. In some embodiments... In this context, this term refers to something that prevents the protein to which it is bound from performing its biological function. Antigen-binding proteins can also be represented. Antigen-binding proteins (e.g., antibodies or immunologically active) When evaluating the binding and / or specificity of the active fragment, an excess of antibody (in vitro competition) (When used in a binding assay) at least about 1 to 20, 20 to 30%, 30 From 40%, 40 to 50%, 50 to 60%, 60 to 70%, 70 to 80%, 80 From 85%, 85 to 90%, 90 to 95%, 95 to 97%, 97 to 98%, 98 When reducing the amount of binding pairs bound to the ligand by 99% or more, the antibody or The fragment can significantly inhibit the binding of the ligand to its binding pair. Several implementations In this state, in the case of the PCSK9 antigen-binding protein, such a neutralizing molecule is PCS The ability of K9 to bind LDLR can be reduced. In some embodiments, The neutralization capacity is determined and / or described through a competitive assay. Several embodiments In this context, neutralization ability is IC 50 or EC 50 Described in terms of value. Several embodiments In this case, ABP27B2, 13H1, 13B5 and 3C4 are non-neutralizing ABP, and 3 B6, 9C9, and 31A4 are weak neutralizing agents, while the remaining ABPs in Table 2 are strong neutralizing agents. In some embodiments, the antibody or antigen-binding protein binds to PCSK9. And prevents PCSK9 from binding to LDLR (or prevents PCSK9 from binding to LDLR) Neutralizes by reducing its ability. In some embodiments, an antibody or ABP It binds to PCSK9, and while binding PCSK9 to LDLR, the LDLR's PCSK 9. Neutralization is achieved by inhibiting or reducing mediating degradation. Therefore, in some embodiments... In this case, neutralized ABP or antibody still allows PCSK9 / LDLR binding, CSK9 inhibits (or reduces) the subsequent degradation of LDLR.

[0139] The term "target" refers to a molecule that can be bound by an antigen-binding protein or Represents a part of a molecule. In one embodiment, the target has one or more epitopes. It is possible. In one embodiment, the target is an antigen. "Antigen-binding protein" The use of the term "antigen" in this context refers to a protein sequence containing an antigen that is bound by an antibody. It simply indicates that something is possible. In this context, the protein is foreign, or an immune response. The ability to induce this is not required.

[0140] Antigen-binding proteins that compete for the same epitope (e.g., neutralizing antigen-binding proteins) The term "competing" when used in the context of "quality or neutralizing antibody" refers to the test. The antigen-binding protein (e.g., an antibody or an immunologically functional fragment thereof) is common. A reference antigen-binding protein (e.g., rigor) to the antigen (e.g., PCSK9 or a fragment thereof) Assays that prevent or inhibit (e.g., reduce) the specific binding of an antibody (or reference antibody). This refers to competition between antigen-binding proteins as measured by [method]. Multiple competitive binding assays are used to determine whether or not it competes with other antigen-binding proteins. Several types, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect Enzyme-enzyme assays (EIAs), sandwich competition assays (e.g., Stahli et al, 1983, Methods in Enzymology 9:242 -See 253); Solid-phase direct biotin-avidin EIA (e.g., Kirkland et al.) al, 1986, J.Immunol.137:3614-3619), solid phase Direct labeling assays, solid-phase direct labeling sandwich assays (e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Ma nual, Cold Spring Harbor Press); I-125 sign The solid-phase direct labeling RIA used (e.g., Morel et al, 1988, Molec .Immunol.25:7-15); Solid-phase direct biotin-avidin EIA (e.g., C Heung, et al., 1990, Virology 176:546-55 See reference 2); and directly labeled RIA (Moldenhauer et al., 1990, (See Scand.J.Immunol.32:77-82) can be used. Typically, such assays are performed by binding to a solid surface or cell having one of these properties. Purified antigen, unlabeled test antigen-binding protein, and labeled reference antigen-binding protein This involves using proteins. Competitive inhibition is performed in the presence of the antigen-binding protein used for testing. It is measured by measuring the amount of a label bound to the body surface or cells. Usually, the test antibody The proto-binding protein is present in excess. Antigen-binding proteins are identified by competitive assays. The quality (competitive antigen-binding protein) binds to the same epitope as the reference antigen-binding protein. Antigen-binding proteins and steric interference occur, but the reference antigen-binding protein binds It contains antigen-binding proteins that bind to adjacent epitopes that are sufficiently close to the epitope being bound. Further details regarding methods for measuring competitive bonding are provided in the examples herein. It is provided. Normally, when there is an excess of competitive antigen-binding proteins, at least 40 to 45%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, or 75% or more, reference antigen binding to common antigen. It inhibits (e.g., reduces) the specific binding of proteins. In some cases, At least 80 to 85%, 85 to 90%, 90 to 95%, 95 to 97%, or 97% Or, the binding is inhibited to an even greater extent.

[0141] The term "antigen" refers to an antigen-binding protein (e.g., an antibody or its immunological functional fragment). Represents a molecule or part of a molecule that can be bound by selective binding factors such as (etc.). In the embodiment, the antigen is used to produce an antibody that can bind to the antigen. It can be used in substances. Antigens interact with different antigen-binding proteins (e.g., antibodies). It may have one or more epitopes that can interact with each other.

[0142] The term "epitope" refers to an antigen-binding protein such as an antibody or T cell receptor. It contains all determinants that can be bound to it. The epitope is an antigen-binding agent that targets its antigen. This is the region of an antigen that is bound by a protein; if the antigen is a protein, then antigen binding occurs. Contains specific amino acids that are in direct contact with proteins. Most frequently, epitopes are proteins. Although it exists qualitatively, in some cases it can also exist on other types of molecules such as nucleic acids. The epitope determinant is a molecule such as an amino acid, sugar side chain, phosphoryl or sulfonyl group. It may contain chemically active surface groups, and may have specific three-dimensional structural characteristics and / or specific It can have specific electrical characteristics. Generally, antibodies specific to a particular target antigen are In complex mixtures of proteins and / or macromolecules, preferential treatment is given to epitopes on target antigens. To recognize something.

[0143] As used herein, "substantially pure" means that the species of molecule described exists. It is the dominant species, that is, on a molar basis, compared to any other species in the same mixture. This means they are more abundant than individual species. In certain embodiments, they are substantially pure These molecules constitute at least 50% (based on molar concentration) of all polymer species in which the target species exists. .) is a composition comprising. In other embodiments, a substantially pure composition is in the composition Contains at least 80%, 85%, 90%, 95%, or 99% of all polymer species present. In other embodiments, conventional detection methods are used to detect hybrids in the composition. This is not possible, and therefore the composition becomes substantially homogeneous, consisting of a single detectable polymer species. The target species is purified up to this point.

[0144] The term "factor" as used herein refers to a chemical compound, a mixture of chemical compounds, or a raw material. It is used to describe extracts made from physical polymers or biological materials.

[0145] As used herein, the terms “labeled” or “labeled” mean, for example, “emitted.” Incorporation of linearly labeled amino acids or labeled avidin (e.g., fluorescent marker or light (Streptavidin containing enzyme activity detectable by scientific or dye-based methods) Therefore, the biotin portion that can be detected is attached to the polypeptide, making it detectable. This represents the incorporation of a marker. In one embodiment, the label or marker is also therapeutic. It is possible. Various methods for labeling polypeptides and glycoproteins are known in this field. Yes, and it is possible to use them. Examples of labeling for polypeptides include the following: These are rare, but not limited to, radioactive isotopes or radionuclides (for example, 3 H, 14 C , 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I) Fluorescent labeling (For example, FITC, rhodamine, lanthaniderin photomaterial), enzyme labeling (for example, Western W Sabiperoxidase, β-galactosidase, luciferase, alkaline phosphatase Ze), chemiluminescence, biotinyl group, a predetermined polypeptide recognized by a secondary reporter Epitope (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal-binding site) Main, epitope tag). In certain embodiments, to reduce the possibility of steric hindrance Therefore, the signs are attached by spacer arms of various lengths.

[0146] As used herein, the term “biological sample” means a living organism or a previously living organism. This includes, but is not limited to, all quantities of substances obtained from existing materials. These organisms include humans, mice, monkeys, rats, rabbits and other animals, but these include Not limited to these. Such substances include blood, serum, urine, cells, organs, tissues, bone, bone marrow, and This includes, but is not limited to, lymph nodes and skin.

[0147] As used herein, the terms “drug composition” (or drug or medicine) refer to patient Chemical compounds, combinations that, when administered appropriately, can induce the desired therapeutic effect. This term refers to an object, composition, drug, or pharmaceutical product. A drug composition does not necessarily require two or more types of components. Do not.

[0148] The term "therapeutic effective dose" refers to the amount determined to produce a therapeutic response in mammals. This represents the amount of PCSK9 antigen-binding protein. Such a therapeutically effective amount is known to those skilled in the art. This can be easily determined.

[0149] As used herein, the term "modulator" refers to a substance that alters the activity or function of a molecule or This is a compound that modifies something. For example, the modifier is the activity observed in the absence of the modifier. Or, compared to the scale of its function, it causes an increase or decrease in the scale of a certain activity or function of the molecule. It is possible. In certain embodiments, the modifier is at least one activity of the molecule Or it is an inhibitor that reduces the scale of function. Typical activities and functions of a certain molecule include: This includes, but is not limited to, binding affinity, enzyme activity, and signal transduction. Typical inhibitors include proteins, peptides, antibodies, peptide bodies, carbohydrates, or small organic compounds. Peptibodies include, but are not limited to, molecules. For example, U.S. Patent Act 6,6 It is described in patent no. 60,843 (corresponding to PCT application WO01 / 83525).

[0150] The terms "patient" and "subject" are used interchangeably, and include human and non-human animal subjects as well as Subjects with previously diagnosed diseases, subjects without previously recognized diseases, medical treatment This includes individuals who have received treatment, and individuals at risk of developing the disease.

[0151] The terms "to treat" and "treatment" refer to therapeutic measures, preventive measures, and actions taken when the subject is a disease or This includes applications that reduce the risk of developing other risk factors. Treatment aims for a complete cure of the disease. It does not require [specific method / function], but encompasses embodiments that reduce symptoms or underlying risk factors. ru.

[0152] The term "prevent" does not imply the need to completely eliminate the probability of an event. No. Rather, the term "prevent" means that in the presence of a compound or method, the occurrence of an event is certain. This indicates that the rate will decrease.

[0153] Recombinant DNA, oligonucleotide technology, and tissue culture and transformation (e.g., electroporation) Standard techniques for enzymatic reactions and purification can be used. The technology is provided in accordance with the manufacturer's specifications, or as is commonly practiced in this field, or in accordance with this specification. It can be carried out as described in the book. The aforementioned techniques and procedures are in this field. In accordance with well-known methods, and as cited and discussed throughout this specification, various general As described in the and more specific references, this can generally be done. For example, “Sambrook et al, Molecular Cloning:A Laboratory Manual (2d ed., Cold Spring H arbor Laboratory Press, Cold Spring Harb or, NY (1989)) (by reference, for all purposes herein) See (which is incorporated into). Unless otherwise specified, in this specification The life sciences described are used in relation to analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry. The terminology and these research techniques and methods are well known and commonly used in this field. It is used for chemical synthesis, chemical analysis, preparation, formulation and delivery of pharmaceuticals and for the treatment of patients. Standard technologies can be used.

[0154] Antigen-binding protein for PCSK9 Proprotein converterase subtilisin kexin type 9 (PCSK9) is a low-density protein serine proteins involved in regulating the levels of poprotein receptor (LDLR) proteins It is an ase (Horton et al, 2007; Seidah and Pr (at, 2007). PCSK9 is a serine protease subtilisin (S8) familia. Lee's prohormone-proprotein converterase (Seidah et al. .,2003). The typical human PCSK9 amino acid sequence is shown in Figure 1A (underlined). The "pro" domain of the protein is shown in the diagram. (Figure 1B shows the signal sequence in bold and the underlined part.) The prodomains with the notation are shown in the diagram. ) are represented as sequence numbers 1 and 3. The typical human PCSK9 coding sequence is represented as sequence number 2 (Figure 1B). As described herein, the PCSK9 protein is the full-length PCSK9 protein It can also contain qualitative fragments. The structure of the PCSK9 protein is divided into two groups. Recently resolved (Cunningham et al., Nature Struct) ural & Molecular Biology, 2007, and Pipe r et al., Structure, 15:1-8, 2007) (All references (By this reference, the whole is incorporated herein.) PCSK9 has a signal sequence, N-terminal phase It includes a rod domain, a subtilisin-like catalytic domain, and a C-terminal domain.

[0155] Antigen-binding proteins (ABPs) that bind to PCSK9, including human PCSK9, are described in this specification. It is described in the book. In some embodiments, the antigen-binding protein provided is As described herein, including one or more complementarity-determining regions (CDRs) It is a polypeptide. In the same antigen-binding protein, CDR is the appropriate antigen for CDR. Embedded within a "framework" domain that directs CDR so that coupling properties are achieved. In some embodiments, the antigen-binding proteins provided herein are It is possible to interfere with, block, reduce, or modulate the interaction between PCSK9 and LDLR. Such antigen-binding proteins are described as "neutralizing." In some embodiments, And even if the antigen-binding protein is neutralizing and bound to PCSK9, PC Coupling between SK9 and LDLR is still possible. For example, in some embodiments, AB P does not block the LDLR binding site on PCSK9, and PCSK to LDLR To prevent or reduce the adverse effects of 9. Therefore, in some embodiments, ABP is P This method leads to LDLR degradation without the need to suppress the binding interaction between CSK9 and LDLR. Adjust or change the capabilities of PCSK9. Such ABP "neutralize non-competitively". It can be specifically described as "ABP". In some embodiments, neutralized ABP is P Bound to PCSK9 in a position and / or manner that prevents CSK9 from binding to LDLR. Such ABP can be specifically described as "competitively neutralizing" ABP. Neutralizing substances all result in a greater amount of free LDLR present in the target substance. This results in more LDLR being bound to LDL (this This further reduces the amount of LDL in the subject. Next, this reduces the serum cyanide present in the subject. This leads to a decrease in resterol levels.

[0156] In some embodiments, the antigen-binding proteins provided herein are PCS It is possible to inhibit the activity (including binding) mediated by K9. Several implementations In this state, the antigen-binding proteins that bind to these epitopes are, in particular, PCSK It inhibits the interaction between 9 and LDLR and other physiological effects mediated by PCSK9. In some embodiments, the antigen-binding protein is a fully human antibody against PCSK. The body, etc., is that of a human being.

[0157] 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 is bound into the prodomain of PCSK9. ABP selectively binds to the mature form of PCSK9. In some embodiments, A BP is in a format in which PCSK9 cannot be combined with LDLR, or in a format in which it cannot be combined efficiently. Then, it binds to the catalytic domain. In some embodiments, the antigen-binding protein is a catalyst. It does not bind to the C-terminus of the domain. In some embodiments, the antigen-binding protein contacts It does not bind to the N-terminus of the medium domain. In some embodiments, ABP is PCSK9 It does not bind to the N-terminus or C-terminus of the protein. In some embodiments, ABP is the present invention It binds to one of the epitopes to which the antibody is bound, as described in the details. In some embodiments, this involves competition between the antibody disclosed herein and other antibodies. It can be measured by a combined assay. In some embodiments, ABP is shown in Table 2. It binds to an epitope that is conjugated by one of the antibodies listed. Several implementations In this state, the antigen-binding protein binds to the specific three-dimensional structure of PCSK9, P It prevents CSK9 from interacting with LDLR. In some embodiments, ABP is It binds to the V domain of PCSK9. In some embodiments, ABP binds to PCSK9 It binds to the V domain and prevents (reduces) PCSK9 from binding to LDLR. In several embodiments, ABP binds to the V domain of PCSK9 and P to LDLR. Without interfering with (or reducing) CSK9 binding, LDL mediated through PCSK9 It inhibits or reduces harmful activity against R.

[0158] The antigen-binding proteins disclosed herein have a variety of applications. Some proteins, for example, are used in specific binding assays, such as PCSK9, and especially human PCSK9. or in affinity purification of its ligand, and other antagonists of PCSK9 activity Useful in screening assays for identifying antigens. Antigen-binding protein Some of these inhibit the binding of PCSK9 to LDLR, or are mediated by PCSK9. It is useful for inhibiting the activity of [unclear].

[0159] Antigen-binding proteins are used in a variety of therapeutic applications, as described herein. It can be used. For example, in some embodiments, the PCSK9 antigen-binding protein The substance is a cholesterol-related condition, such as hypercholesterolemia, as further described herein. Conditions associated with PCSK9, such as cholesterol-related disorders (or "serum cholesterol-related disorders"). It is useful in treating symptoms. Other uses for antigen-binding proteins include, for example, P Screening for the diagnosis of CSK9-related disorders or symptoms and for determining the presence or absence of PCSK9 A leaning assay is included. Several antigen-binding proteins are described herein. This is useful in treating symptoms and / or lesions, as it is associated with PCSK9 activity.

[0160] In some embodiments, the antigen-binding protein provided is one or more C Includes DRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs). In some embodiments... The antigen-binding protein is inserted into (a) the polypeptide structure and (b) the polypeptide structure. It contains one or more CDRs that are inserted and / or linked. The polypeptide structure is, It can take on a variety of different forms. For example, polypeptide structures are naturally occurring antibodies. A body framework or a fragment or variant thereof, or including such a thing It can be synthesized, or, due to its properties, can be completely synthesized. Examples of various polypeptide structures are shown below. Further details are provided.

[0161] In certain embodiments, the polypeptide structure of the antigen-binding protein is an antibody. or monoclonal antibodies, bispecific antibodies, minibody antibodies, domain antibodies, synthetic antibodies (honmei In detailed documents, these are sometimes referred to as "antibody mimics," chimeric antibodies, humanized antibodies, or antibody fusions. Compounds (which may be referred to as "antibody conjugates" in this specification) and each of them Derived from antibodies containing (but not limited to) part or fragment of several. In this case, the antigen-binding protein is an immunological fragment of the antibody (e.g., Fab, F ab', F(ab')2 or scFv). Various structures are further described and defined herein. It is justified.

[0162] Some of the antigen-binding proteins provided herein are specific to human PCSK9. It binds to and / or selectively. In some embodiments, the antigen-binding protein is distributed Human PCSK9 protein having and / or residues 153 to 692 of column 3 It binds specifically and / or selectively to the sequence. In some embodiments, ABP binds to the sequence Specifically affecting human PCSK9 having and / or residues 31 to 152 of residue number 3. And / or selectively coupled. In some embodiments, the ABP is shown in Figure 1A. It selectively binds to the human PCSK9 protein (SEQ ID NO: 1) in several embodiments. In this context, the antigen-binding protein is at least a fragment and / or signature of the PCSK9 protein. It specifically binds to full-length PCSK9 proteins with or without a null sequence.

[0163] In embodiments in which antigen-binding proteins are used for therapeutic purposes, the antigen-binding protein The substance inhibits, interferes with, or modulates one or more biological activities of PCSK9. This is possible. In one embodiment, the antigen-binding protein specifically targets human PCSK9. Binding and / or (for example, measuring binding in an in vitro competitive binding assay) (by) at least about 20% to 40%, 40 to 60%, 60 to 80%, 80% It substantially inhibits the binding of human PCSK9 to LDLR by 85% or more. Some of the antigen-binding proteins provided are antibodies. In some embodiments... And ABP is 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 K is smaller than M. d It has (a stronger bond). In some embodiments Furthermore, ABP was found to be less than 1 μM and 1000 μM in relation to blocking the binding of LDLR to PCSK9. From nM to 100nM, from 100nM to 10nM, from 10nM to 1nM, from 1000pM 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, ICs from 200 pM to 100 pM, 100 pM to 10 pM, and 10 pM to 1 pM 50 of Possesses (D347Y, high affinity variant).

[0164] An example of the IgG double-chain constant domain of the anti-PCSK9 antibody of the present invention is shown in SEQ ID NO: 154, Figure [Figure]. It has the amino acid sequence shown in 3KK.

[0165] An example of the IgG quadruple-chain constant domain of the anti-PCSK9 antibody of the present invention is shown in SEQ ID NO. 155, Figure [Figure]. It has the amino acid sequence shown in 3KK.

[0166] An example of the κ light chain constant domain of an anti-PCSK9 antibody is shown in SEQ ID NO: 157, Figure 3KK. It has the following amino acid sequence.

[0167] An example of the λ light chain constant domain of an anti-PCSK9 antibody is shown in SEQ ID NO: 156, Figure 3KK. It has the following amino acid sequence.

[0168] The variable region of an immunoglobulin chain consists of three hypervariable regions (more frequently, "complementarity-determining regions"). A relatively preserved framework domain linked by (or referred to as CDR). It generally exhibits the same overall structure including the region (FR). It is obtained from the two chains of each heavy / light chain pair described above. The CDRs identified are specific epitopes on the target protein (e.g., PCSK9) and specific To form a structure that connects to N, it is typically parallelized by framework domains. From terminal to C-terminus, both naturally occurring light chain and heavy chain variable regions are of the following nature: This usually corresponds to the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and F R4. In order to assign numbers to the amino acids that occupy positions in each of these domains, a numbering system is used. A stem has been devised. This numbering system is called "Kabat Sequences o f Proteins of Immunological Interest (19 87 and 1991, NIH, Bethesda, MD)” or “Choth ia & Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:878-88 It is defined in "3".

[0169] Various heavy and light chain variable regions are provided herein, as shown in Figures 2A to 3JJ and 3 These are illustrated in LL to 3BBB. In some embodiments, these variable regions Each of them, in order to form a complete antibody heavy chain and light chain, the heavy chain and light chain constant regions mentioned above It can be attached to the region. Furthermore, in order to form a complete antibody structure, in this way... It is possible to combine each of the heavy chain and light chain sequences that have been fabricated.

[0170] Some specific examples of the variable regions of the light and heavy chains of the provided antibodies and their corresponding regions. The amino acid sequences are summarized in Table 2.

[0171] [Table 2]

[0172] Similarly, each of the typical variable heavy chains listed in Table 2 is required to form an antibody. It can be combined with any of the typical variable light chains shown in 2. Table 2 is the main explanation. The document shows typical light and heavy chain pairs found in some of the antibodies disclosed. In some cases, the antibody is obtained from at least one of the sources listed in Table 2. It contains one variable heavy chain and one variable light chain. In other examples, an antibody has two identical light chains and two It contains the same heavy chain. For example, an antibody or antigen-binding protein contains a heavy chain and a light chain. It may contain two heavy chains or two light chains. In some embodiments, antigen-binding The protein is obtained from at least one of the sequences listed in Table 2, 1, 2 and / or Three heavy and / or light CDRs (the CDRs for these sequences are shown in Figures 2A to 3D and Figure 2A to 3D) Other embodiments are outlined in 3CCC to 3JJJ and 15A to 15D. Includes (and / or consists of). In some embodiments, six CDRs (C from light chains) DR1 to 3 (CDRL1, CDRL2, CDRL3) and CDR1 to 3 from the heavy chain ( CDRH1, CDRH2, and CDRH3) are part of ABP. In some embodiments In this context, 1, 2, 3, 4, 5 or more CDRs are included in the ABP. In this embodiment, one heavy and one light CDR obtained from the CDRs in the sequence in Table 2 However, the CDRs for the sequences included in ABP (Table 2 are outlined in Figures 2A to 3D) (In some embodiments, for example, in Figures 2A to 2D and 3A to 3D) , as well as illustrated in other embodiments in 3CCC to 3JJJ and 15A to 15D Further sections (such as those listed below) are also included in ABP. Heavy and light chains are listed in Table 2. Examples of CDR and FR for this are shown in Figures 2A to 3D (and Figures 3CCC to 3JJJ and 1 Other embodiments are outlined in 5A to 15D. A variable light chain may be present depending on the case. The sequence (including CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4) is You can choose from the following: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 3 5, 36, 37, 38, 39, 40, 42, 44 and 46. Heavy chains may be present in some cases. Variant arrangement (including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4). You can choose from the following: 74, 85, 71, 72, 67, 87, 58, 52 , 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and 60. In Figures 2A to 2D In some entries, variations in the sequence or alternative boundaries between CDR and FR have been identified. These alternatives are identified by "vl" after the ABP name. Most of the differences are inherently small, so only the sections with differences are shown in the table. Remaining light or heavy chain It is understood that the section is the same as that shown for the basic ABP in other panels. Therefore, only the differences are shown in Figure 2C, so for example, in Figure 2C 1 9H9vl has the same FR1, CDR1, and FR2 as 19H9 in Figure 2A. Nucleic acid distribution For the three columns (ABP26E10, 30B9, and 31B12), further different nucleic acid distributions are applied. The columns are provided in the figure. As will be understood by those skilled in the art, the preparation of antibodies or ABP In reality, you will need to use one or fewer of these arrays. In this embodiment, it is necessary for only one specific heavy chain or light chain nucleic acid to be present, or all It doesn't need to exist.

[0173] In some embodiments, ABP encodes any of the protein sequences in Table 2. It is encoded by a nucleic acid sequence that can do this.

[0174] In some embodiments, ABP is a form of PCSK9 coupled to LDLR (e.g.) , selectively binds to the autocatalytic form of the molecule. In some embodiments, Antigen-binding proteins have a catalytic domain at the c-terminus (for example, 5,5 to 10,1 in the c-terminus). Many soils: 0 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40 It does not bind to acids. In some embodiments, the antigen-binding protein has a catalytic domain. n-terminus (for example, 5 in the n-terminus, 5 to 10, 10 to 15, 15 to 20, 20 to 2) 5, does not bind to many amino acids (25 to 30, 30 to 40). In some embodiments In this context, ABP binds to amino acids 1 through 100 of the mature form of PCSK9. In some embodiments, ABP is amino acid 31 to 100, 100 to 200. , 31 to 152, 153 to 692, 200 to 300, 300 to 400, 452 683, 400 to 500, 500 to 600, 31 to 692, 31 to 449 and / or binds to amino acids (and / or amino acid sequences consisting of these) between 600 and 692. In some embodiments, ABP binds to the catalytic domain. In this state, neutralizing and / or non-neutralizing ABP binds to the prodomain. Several implementations In this state, ABP binds to both the catalyst and the prodomain. In some embodiments... ABP then binds to the catalytic domain and interacts with the prodomain on the catalytic domain. It blocks the area. In some embodiments, ABP is Piper et al. (Structur e15:1 to 8 (2007) (including the 3D representation within it, the entire work is referenced by...) As outlined in the (incorporated herein), the locations where the prodomains interact. Alternatively, it binds to the catalytic domain on the surface. In some embodiments, ABP is catalytic It binds to the main and restricts the mobility of the prodomain. In some embodiments, ABP It binds to the catalytic domain without binding to the prodomain. In some embodiments In ABP, the prodomain is reoriented to bind PCSK9 to LDLR. It binds to the catalytic domain without binding to the prodomain, while preventing it from doing so. In this embodiment, ABP is derived from residue 149 around the prodomain in Piper et al. It binds in the same epitope as 152. In some embodiments, ABP is V. Domaine It connects to a groove on the (as outlined in Piper et al.). Several embodiments In this case, ABP binds to histidine-rich patches near the groove on the V domain. In that embodiment, such an antibody (binding to the V domain) is not neutralizing. In one embodiment, such an antibody that binds to the V domain is neutralizing. In some embodiments, neutralizing ABP prevents the binding of PCSK9 to LDLR. In the application form, neutralizing ABP inhibits the decomposition of PCSK9 of LDLR, while also affecting LDLR. It does not interfere with PCSK coupling (e.g., ABP31A4). In some embodiments, A BP is bound to at least one histidine molecule, as illustrated in Figure 4 of the paper by Piper et al. or block. In some embodiments, ABP is a catalyst triple structure in PCSK9. Block it.

[0175] In some embodiments, the antibody is more effective than wild-type PCSK9 compared to variant PCSK9. It selectively binds to protein 9 (e.g., D374Y). In some embodiments, These antibodies are (K d (When measured via [a specific method]) Barrier is at least twice as strong as the wild type. Combined with, preferably 2 to 5, 5 to 10, 10 to 100, and 100 to 100 It binds to mutants 0, 1,000 to 10,000 times or more more strongly than the wild type. In one embodiment, the antibody inhibits wild-type PCSK9 from interacting with LDLR. Beyond its ability to do so, variant D374YPCSK9 interacts with LDLR. Selectively inhibits. In some embodiments, these antibodies are (IC50 Measured via (Assuming a specific condition) The variant has a stronger ability to bind to LDLR than the wild type. Blocking the ability to do so (for example, at least twice as strong as the wild type, preferably more so than the wild type) Mutants 2 to 5, 5 to 10, 10 to 100, 100 to 1000 times or more strongly. (to). In some embodiments, the antibody is wild-type PCSK9 and PCSK9 Varian It binds to and neutralizes both forms (such as D374Y) at a similar level. Several embodiments In this process, the antibody binds to PCSK9, and the LDLR variant binds to PCSK9. This prevents it. In some embodiments, variants of LDLR are less than human LDLR. They are at least 50% identical. It is noteworthy that the variants of LDLR are known to those skilled in the art. (For example, BrownMS et al., “Calcium cages, acid bath s and recycling receptors” Nature 388:62 9-630, 1997). In some embodiments, ABP is heterozygous familial. Effective LDLR in hypercholesterolemia (where loss-of-function variants of LDLR are present). You can increase its level.

[0176] In some embodiments, ABP is P as shown in Figure 1A and / or Figure 1B. For CSK9 morphology, at least 50%, 50 to 60%, 60 to 70%, 70 to 80% PCSK9 with a percentage identity of 80 to 90, 90 to 95, 95 to 99 or higher. It binds to the variant of (but does not block). In some embodiments, ABP is At least 5 of the mature forms of PCSK9 shown in Figure 1A and / or Figure 1B 0%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95 It binds to PCSK9 variants with 99% or more identity (but blocks (Not). In some embodiments, the ABP is shown in Figure 1A and / or Figure 1B. For the form of PCSK9, at least 50%, 50 to 60%, 60 to 70%, 70 P is a percentage identity of 80%, 80% to 90%, 90% to 95%, 95% to 99%, or higher. It binds to a variant of CSK9 and prevents it from interacting with LDLR. Several embodiments In this context, ABP is at least equivalent to the mature form of PCSK9 shown in Figure 1B. 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 95, 9 It binds to PCSK9 variants with 5 to 99% or more identity, and LDLR and It prevents interaction. In some embodiments, a variant of PCSK9 is 47 The variant in 4th place is 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 (as in cynomolgus monkeys and mice) threonine. In light of the cross-reactivity data, this antibody readily binds to the above variant. It is thought that...

[0177] In some embodiments, ABP is conjugated by one of the antibodies listed in Table 2. It binds to the epitope. In some embodiments, the antigen-binding protein is PC It binds to a specific three-dimensional state of SK9, preventing PCSK9 from interacting with LDLR. Give.

[0178] Humanized antigen-binding proteins (e.g., antibodies) As described herein, the antigen-binding protein for PCSK9 is This may include humanized antibodies and / or parts thereof. Important practical applications of such strategies This is the "humanization" of the mouse humoral immune system.

[0179] In certain embodiments, humanized antibodies are substantially non-immunogenic in humans. In one embodiment, the humanized antibody is derived from another species from which the humanized antibody was derived. The antibody has substantially the same affinity for the target. For example, U.S. Patent No. 5,530, U.S. Patent No. 101, U.S. Patent No. 5,693,761, U.S. Patent No. 5,693,762, U.S. Patent No. Please refer to Permit No. 5,585,089.

[0180] In certain embodiments, the immunogenicity is reduced while the intrinsic properties of the antigen-binding domain are Amino acids in the antibody variable domain that can be modified without reducing affinity are identified. For example, See U.S. Patent Nos. 5,766,886 and 5,869,619.

[0181] In certain embodiments, modification of an antibody by a method known in the art is performed against the target. To achieve increased binding affinity and / or immunity of the antibody in the recipient They are typically designed to reduce virulence. In certain embodiments, they are analogous antigens. Humanization to remove glycosylated sites in order to increase the affinity of antibodies against The antibody is modified. For example, "Co et al., Mol.Immunol., 3 See 0:1361-1367 (1993). In certain embodiments, "Reshaping", "Super Chimera", or "Veneering / Resurfacing" Technologies such as "Vaswamm" are used to produce humanized antibodies. i et al., Annals of Allergy, Asthma, & I mmunol.81:105(1998);Roguska et al, Prot. Engineer, 9:895-904 (1996) and U.S. 6,072,0 See issue 35. In certain such embodiments, such technology is foreign Reducing the number of residues typically reduces the immunogenicity of an antibody, but the antibody It does not interfere with anti-idiotype and anti-allotype responses after repeated administration. Some other methods for reducing it include, for example, "Gilliland et al., It is described in "J. Immunol., 62(6):3663-71 (1999)". It is.

[0182] In certain cases, humanized antibodies result in reduced antigen-binding ability. In this state, the humanized antibody is "reverse-mutated". In certain such embodiments, Humanized antibodies are designed to contain one or more amino acid residues found in the donor antibody. It mutates. For example, "Saldanha et al., Mol Immunol See 36:709-19 (1999).

[0183] In certain embodiments, the complementarity of the light chain and heavy chain variable regions of antibodies against PCSK9 Transferring the decision region (CDR) to a framework region (FR) obtained from the same or a different species. It is possible. In certain embodiments, the light chain and heavy chain of the antibody against PCSK9 The variable region CDR can be implanted into the consensus human FR. In order to produce FR, in certain embodiments, the consensus amino acid sequence is identified. To achieve this, several FR sequences obtained from human heavy or light chain amino acid sequences are juxtaposed. In some embodiments, the FR of the antibody against the PCSK9 heavy or light chain is different from that of the heavy or light chain. It is substituted with FR obtained from the light chain. In certain embodiments, it is an anti-PCSK9 While rare amino acids in the FR of the body's heavy and light chains are not substituted, the remaining FR amino acids... A substitution occurs. Rare amino acids are present in FR at positions where they are not normally found. It is a specific amino acid. In certain embodiments, it is obtained from an antibody against PCSK9. The transplanted variable region, along with the constant region of the antibody against PCSK9, is different from the constant region. It can be used. In certain embodiments, the transplanted variable region is a single-stranded Fv It is part of the antibody. CDR implantation is, for example, U.S. Patent No. 6,180,370, U.S. Patent U.S. Patent No. 6,054,297, U.S. Patent No. 5,693,762, U.S. Patent No. 5,859,2 U.S. Patent No. 05, U.S. Patent No. 5,693,761, U.S. Patent No. 5,565,332, U.S. Patent In U.S. Patent No. 5,585,089 and U.S. Patent No. 5,530,101, and Jones et al., Nature, 321:522-525 (1986); hmann et al, Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-153 6 (1988), Winter, FEBS Letts., 430:92-94 (1998) (For all purposes, incorporated herein by reference.) It is stated.

[0184] Human antigen-binding proteins (e.g., antibodies) As described herein, the antigen-binding protein that binds to PCSK9 is human Certain embodiments may include (i.e., fully human) antibodies and / or parts thereof. In this, the nucleotide sequences encoding heavy chain and light chain immunoglobulin molecules, and heavy chain and Amino acid sequences containing light chain immunoglobulin molecules, particularly sequences corresponding to variable regions, are provided. In certain embodiments, the complementarity determination region (CDR), in particular, CDR1 to CD A sequence corresponding to R3 is provided. According to certain embodiments, such an immunoglobulin A hybridoma cell line expressing the molecule is provided. According to certain embodiments, this A hybridoma cell line expressing such monoclonal antibodies is provided. Morphologically, hybridoma cell lines are at least one of the cell lines listed in Table 2. For example, selected from 21B12, 16F12 and 31H4. In certain embodiments Purified human monoclonal antibodies against human PCSK9 are provided.

[0185] In the expectation that mice will produce human antibodies in the absence of mouse antibodies, the human Ig gene... Using giant fragments of this locus, it is possible to modify mouse strains that lack mouse antibody production. Large human Ig fragments preserve vast variable genetic diversity and appropriate regulation of antibody production and expression. This can be achieved through: diversification and selection of antibodies, and immunological tolerance to human proteins. By utilizing the mouse mechanism due to the lack of [something], the regenerated [something] in these mouse strains The human antibody repertoire is highly effective against all target antigens (including human antigens). It is possible to provide affinity-to-human antibodies. Using hybridoma technology, the desired specificity can be achieved. Antigen-specific human MAbs possessing these properties can be produced and selected. A certain typical method This is covered by WO98 / 24893, U.S. Patent No. 5,545,807, EP546073 and E It is described on page 546073.

[0186] In certain embodiments, along with the human variable region, a constant region obtained from a non-human species is also obtained. The area can be used.

[0187] Cloning and reconstructing megabase-sized human gene loci in yeast artificial chromosomes (YACs) The ability to build and introduce them into the mouse germline results in extremely large and This will elucidate the functional components of roughly mapped gene loci and provide useful models for human diseases. An approach to constructing the mouse gene locus is given. Furthermore, the mouse gene locus is compared to those of human average. The use of such techniques for substitution with equivalents allows for the expression of developing human gene products and and control, their interactions with other systems, and their involvement in the induction and progression of disease. Insights into this matter may be provided.

[0188] Human antibodies have some of the problems associated with antibodies that have variable and / or constant regions in mice or rats. Avoiding contact. The presence of such mouse or rat-derived proteins allows for rapid antibody response. This can lead to elimination, or to the patient generating an immune response against the antibody. This can be done. To avoid the use of antibodies derived from mice or rats, rodents, other In order for mammals or animals to produce fully human antibodies, rodents, other mammals or animals By introducing functional human antibody gene loci, it is possible to produce fully human antibodies.

[0189] Humanized antibodies begin by containing non-human antibody amino acid sequences, Antibodies having at least some of these non-human antibody amino acid sequences substituted with antibody sequences It is a body. This is a human being in which antibodies are encoded by a gene (or encoded by) It is different from human antibodies (which can do this).

[0190] antigen-binding protein variants Other antibodies provided may be the combinations of variable heavy and variable light chains shown in Table 2 or Formed by subparts, the sequence in Table 2 (the entire sequence or a subpart of the sequence (for example, For one or more CDR)) amino acid sequences, at least 50% each, 50 to 60, 60 to 70, 70 to 80%, 80 to 85%, 85 to 90%, 90 Variable light chains having identity of 95%, 95 to 97%, 97 to 99%, or more than 99% The above ABP variants include and / or variable heavy chains. In some cases, this While eel antibodies contain at least one heavy chain and one light chain, in other cases... The variant form consists of two identical light chains and two identical heavy chains (or their subparts). ) contains. In some embodiments, variations that affect bonding and affect bonding Identify antibody compartments that can be modified by observing fluctuations that do not appear to be affected. To that end, Figures 2A to 3D (and other figures in 13A to 13J and 15A to 15D) Sequence comparison can be used in the application form. For example, by comparing similar sequences... This involves identifying modifiable compartments (e.g., specific amino acids) and preserving the functionality of ABP. Identify how they can be modified while (or improving) them. It is possible. In some embodiments, variants of ABP are shown in Figures 13A, 13 Consensus bases illustrated in C, 13F, 13G, 13H, 13I and / or 13J This includes the arrangement, and variation is possible at the positions identified as variable in the figure. Figure 13 The CDRs shown in A, 13C, 13F, and 13G are based on the Chothia method (structure loop). Based on the location of the region. For example, “Standard conformations fo r the canonical structures of immunoglob ulins” Bissan Al-Lazikani, Arthur M. Les k and Cyrus Chothia, Journal of Molecule r Biology, 273(4):927-948, 7 November (1 See 997). ) and Kabat method (based on sequence variability. For example, Seque nces of Proteins of Immunological Intere st, Fifth Edition.NIH Publication No. 91 See -3242, Kabat et al., (1991). Defined based on lid combinations. Each residue determined by either method is C The final list of DR residues included (as shown in Figures 13A, 13C, 13F, and 13G) (It is.) The CDRs in Figures 13H, 13I, and 13J were obtained solely by the Kabat method. Unless otherwise stated, the defined consensus sequences in Figures 13H to 13J, CD, etc. R and FR are denotations CDR and FR for ABP referenced in Figure 13. To justify and regulate.

[0191] In certain embodiments, the antigen-binding protein is SEQ ID NOs: 74, 85, 71, 72 ,67,87,58,52,51,53,48,54,55,56,49,57,50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and An amino acid sequence selected from at least one of 60 sequences that is at least 90% identical to an amino acid sequence. It contains a heavy chain that includes a variable region containing an acid sequence. In certain embodiments, the antigen-binding protein The components are: SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48 , 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, Selected from at least one of the sequences 76, 77, 78, 83, 69, 81, and 60. It contains a heavy chain that includes a variable region containing an amino acid sequence that is at least 95% identical to the amino acid sequence. In one embodiment, the antigen-binding protein is sequence numbers 74, 85, 71, 72, 6 7, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91 , 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and 60 An amino acid sequence selected from at least one of the sequences and an amino acid sequence that is at least 99% identical It contains a heavy chain that includes a variable region containing an array.

[0192] In some embodiments, the antigen-binding protein is SEQ ID NOs: 74, 85, 71, 72 ,67,87,58,52,51,53,48,54,55,56,49,57,50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and One or more CDRs obtained from at least one CDR in the sequence of 60 and less They all contain sequences that are 90%, 90-95%, and / or 95-99% identical. In the embodiment, 1, 2, 3, 4, 5, or 6 CDRs (each with the above sequence and at least There are cases where they are 90%, 90 to 95%, and / or 95 to 99% identical.

[0193] In some embodiments, the antigen-binding protein is SEQ ID NOs: 74, 85, 71, 72 ,67,87,58,52,51,53,48,54,55,56,49,57,50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and One or more FRs obtained from at least one FR in the sequence of 60 and at least Includes sequences that are 90%, 90-95%, and / or 95-99% identical. Several implementations Morphologically, 1, 2, 3, or 4 FRs (each with at least 90% of the above sequence, 9 There exists a 0 to 95% and / or 95 to 99% identical value.

[0194] In certain 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 and 46 An amino acid sequence selected from at least one of the sequences and an amino acid sequence that is at least 90% identical It includes a light chain containing a variable region containing columns. In certain embodiments, the antigen-binding protein is Sequence numbers 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, An amino acid sequence selected from at least one of sequences 39, 40, 42, 44, and 46 and Includes a light chain containing a variable region with at least 95% identical amino acid sequences. A certain type of implementation. In this state, antigen-binding proteins are sequence numbers 5, 7, 9, 10, 12, 13, 15, 1 6, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32 at least of the sequences 33, 35, 36, 37, 38, 39, 40, 42, 44 and 46 A variable region containing an amino acid sequence that is at least 99% identical to one of the selected amino acid sequences. Includes a light chain.

[0195] In some embodiments, the antigen-binding protein is sequence numbers 5, 7, 9, 10, 12 , 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44 and 46 One or more CDRs obtained from at least one CDR in the sequence and at least 9 Includes sequences that are 0%, 90-95%, and / or 95-99% identical. Several implementations In this state, 1, 2, 3, 4, 5, or 6 CDRs (each containing the above sequence and at least 9 There are cases where 0%, 90 to 95%, and / or 95 to 99% are identical.

[0196] In some embodiments, the antigen-binding protein is sequence numbers 5, 7, 9, 10, 12 , 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44 and 46 One or more FRs obtained from at least one FR in the sequence and at least 90% , including sequences that are 90 to 95% and / or 95 to 99% identical. In some embodiments, In this, 1, 2, 3, or 4 FRs (each with at least 90% of the above sequence, 90% from There are cases where they are 95% and / or 95 to 99% identical.

[0197] In light of this disclosure, a person skilled in the art can use well-known techniques to perform the following in accordance with this specification. In certain embodiments, the appropriate variant of ABP can be determined. The company disrupts the activity by targeting areas that appear to be unimportant to the activity. It is possible to identify the appropriate regions of molecules that can be altered without doing so. In certain embodiments, This allows us to identify conserved molecular residues and parts among similar polypeptides. In certain embodiments, in regions that may be important for biological activity or structure Even without destroying biological activity or adversely affecting polypeptide structure, preserved amino acids It can be used for acid substitution.

[0198] Furthermore, those skilled in the art will recognize that residues in similar polypeptides that are important for activity or structure are the same A structure-function study can be examined to determine the structure. In light of such a comparison, similar tan The amino acid residues in the protein that are important for the activity or structure of the protein The importance of amino acid residues can be predicted. Those skilled in the art can predict the importance of such predicted important residues. Chemically similar amino acid substitutions can be selected for amino acid residues.

[0199] Those skilled in the art can analyze the three-dimensional structure and the amino acid sequence related to that structure in similar ABPs. It is also possible to do so. In light of such information, a person skilled in the art can determine the three-dimensional structure of an antibody. The juxtaposition of amino acid residues can be predicted. In certain embodiments, proteins Amino acid residues expected to be present on the surface may be involved in important interactions with other molecules. Therefore, a person skilled in the art would select such residues in such a way that no abrupt changes occur. This can be done. Furthermore, those skilled in the art can perform a single amino acid substitution in each desired amino acid residue. A test variant containing can be prepared. The variant can then be made public to those skilled in the art. This can be screened using an intelligence activity assay. Such variants are It can be used to gather information about the appropriate variant. For example, Changes in amino acid residues result in disrupted activity, undesirably reduced activity, or incompatibility. If it is discovered that this leads to excessive activity, variants with such changes should be avoided. In other words, based on the information collected from such standardized experiments, The company identifies amino acids, either alone or in combination with other mutations, that should not be further substituted. This can be easily determined.

[0200] Numerous scientific publications focus on predicting secondary structures. (Moult J., Cur) r.Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113( 2):211-222 (1974); Chou et al., Adv.Enzy mol.Relat.AreasMol.Biol., 47:45-148 (197 8); Chou et al., Ann.Rev.Biochem., 47:25 1-276 and Chou et al., Biophys.J., 26:36 See 7-384 (1979). Furthermore, to aid in predicting the secondary structure, Currently, computer programs are available. One method for predicting secondary structure is phase It is based on same-sex modeling. For example, it has more than 30% sequence identity or 40% Two polypeptides or proteins with a similarity exceeding a certain degree are often structurally similar. It has topology. Recent developments in protein structure databases (PDBs) have shown that polypeptides Increase in secondary structure, such as the number of folds that may exist within the structure of a cytoplasm or protein. This resulted in predictability. ("Holm et al., Nucl.Acid.Re") See s., 27(1):244-247 (1999). The existence of a limited number of folding patterns in a protein, and once a certain level If the structures of more than 100% of the data are elucidated, structural predictions will become dramatically more accurate. (Brenner et al., Curr. Op. Struct. Biol) ., 7(3):369-376 (1997).

[0201] Further methods for predicting secondary structures include "threading" (J ones, D., Curr.Opin.Struct.Biol., 7(3):3 77-87 (1997); Sippl et al, Structure, 4( 1):15-19 (1996), "Profile Analysis" (Bowie et al, Science, 253:164-170(1991); Gribskov et al. al, Meth.Enzym., 183:146-159 (1990); ibskov et al, Proc.Nat.Acad.Sci.USA, 84( 13):4355-4358 (1987)) and "evolutionary connections (evolutiona (ry linkage)" (Holm, above (1999), and Brenner, This includes the above (see 1997).

[0202] In certain embodiments, the antigen-binding protein variant contains the parent polypeptide A Compared to the amino acid sequence, the number and / or type of glycosylation sites have been altered. It contains sylated variants. In certain embodiments, the protein variant is solid It contains more or fewer N-linked glycosylation sites than other proteins. The sylation site is in the sequence: Asn-X-Ser or Asn-X-Thr (denoted as X). An amino acid residue can be any amino acid residue except proline. This is possible. The amino acid residue substitutions required to create this sequence add an N-linked carbohydrate chain. This provides new candidate sites for [the purpose]. Alternatively, substitutions that remove this sequence can lead to the existing N-bonding. Remove the type of carbohydrate chain. One or more N-linked glycosylation sites (typically, Naturally occurring elements are removed, and one or more new N-bond sites are created. Reconstitution of N-linked carbohydrate chains is also provided. Further preferred antibody variants include parental Compared to the amino acid sequence, one or more cysteine ​​residues are deleted, or another amino acid is present. It contains cysteine ​​variants that are substituted with (for example, serine). Soluble inclusions When antibodies must be refolded into a biologically active three-dimensional structure after isolation, etc. Cysteine ​​variants may be useful. Cysteine ​​variants are unique proteins. Generally, it has fewer cysteine ​​residues than the quality and is derived from cysteine ​​that does not form pairs. To maximize the interaction, the number of elements is typically even.

[0203] According to certain embodiments, amino acid substitutions (1) improve sensitivity to proteolysis. (2) Amino acid substitutions that reduce (3) (4) Amino acid substitutions that change binding affinity in order to form protein complexes, (4) amino acid substitutions that alter affinity and / or other substitutions for such polypeptides This refers to amino acid substitutions that impart or modify physicochemical or functional properties. Depending on the application method, one or more amino acid substitutions (in one embodiment, a conserved amino acid Acid substitution is a process that, in some embodiments, forms intermolecular contacts within naturally occurring sequences. It can be produced (in the polypeptide portion outside the main body). In certain embodiments, it can be conserved. Amino acid substitutions may typically not substantially alter the structural characteristics of the parent sequence. (For example, substituted amino acids should not tend to cleave helices that occur in the parent sequence.) (It should not tend to disrupt or disintegrate other types of secondary structures that characterize the parent sequence.) Examples of polypeptide secondary and tertiary structures recognized in this field include "Proteins, S Structures and Molecular Principles (Crei ghton, Ed., W. H. Freeman and Company, N. ew York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, NY (19 91)); and Thornton et al, Nature, 354:10 5 (1991)" (each incorporated herein by reference) It is being done.

[0204] In some embodiments, the variant is the nucleic acid of ABP disclosed herein. It is a variant of the sequence. Those skilled in the art can identify, evaluate, and To produce these, and for nucleic acid sequences that can encode these protein variants Therefore, we understand that the above considerations can be used. Thus, encoding a protein variant Nucleic acid sequences (and those encoding ABP in Table 2, but which are explicitly disclosed herein) A nucleic acid sequence different from the acid sequence is assumed. For example, the ABP variant is sequence number 15. 2, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 10 2, 103, 104, 105, 106, 107, 108, 109, 110, 111, 11 2, 113, 114, 115, 116, 117, 118, 119, 120, 121, 12 2, 123, 124, 125, 126, 127, 128, 129, 130, 131, 13 2, 133, 134, 135, 136, 137, 138, 139, 140, 141, 14 Note in 2, 143, 144, 145, 146, 147, 148, 149, 150, 151 At least one nucleic acid sequence or sequence numbers 152, 153, 92, 93, 94 are listed. , 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, Encoded by nucleic acid sequences in 146, 147, 148, 149, 150 and 151 At least one to six CD-Rs (and various combinations thereof) and at least 80, 80 to 85, 85 to 90, 90 to 95, 95 to 97, 97 to 99 or any other. It can possess the above-mentioned identity.

[0205] In some embodiments, under strict conditions, a nucleic acid sequence encoding a specific ABP (or (The nucleic acid sequence itself) is one of the nucleic acid sequences that encode proteins in Table 2 (Sequence ID 1 52, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 1 02, 103, 104, 105, 106, 107, 108, 109, 110, 111, 1 12, 113, 114, 115, 116, 117, 118, 119, 120, 121, 1 22, 123, 124, 125, 126, 127, 128, 129, 130, 131, 1 32, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 42, 143, 144, 145, 146, 147, 148, 149, 150 and 151 How can (but not limited to) selectively form hybrids? In that case, the antibody (or the nucleic acid sequence encoding the antibody) is a variant. In one embodiment... The appropriate moderately strict conditions are 5×SSC;0.5%SDS, 1.0mMEDTA( Pre-wash in a pH 8.0 solution, 50°C, -65°C, 5×SSC, overnight hybridization In the case of hybrid formation or interspecific homology, hybrid formation is performed at 45°C using 0.5×SSC. Next, together with 2×, 0.5×, and 0.2× SSC containing 0.1% SDS, This includes two washes at 65°C for 20 minutes. Such hybrid-forming DNA sequences are also available. Due to degeneracy, the antibody polypeptide encoded by the hybrid-forming DNA sequence and and the nucleotide sequences that encode the amino acid sequences encoded by these nucleic acid sequences Similarly, this falls within the scope of the present invention. In some embodiments, variants of the CDR include: One or more CDRs in the above sequence (Figures 2A to 3D and Figures 3CCC to 3JJJ) And in light of the other embodiments in 15A to 15D, each CDR can be easily determined. ) can form a hybrid nucleic acid sequence and an amino acid sequence encoded by the nucleic acid sequence The column is included. The usage of "selectively hybridize" as quoted in this context The term means that it binds in a detectable and selective manner. Polynucleotide according to the present invention Oligonucleotides and their fragments exhibit a large amount of detectable binding to nonspecific nucleic acids. Selective hybrid formation occurs on nucleic acid chains under conditions that minimize hybrid formation and washing. As is well known in the art and as discussed herein, selective H Highly stringent conditions can be used to achieve hybrid formation conditions. The nucleus between the polynucleotide, oligonucleotide, and fragment of the present invention and the target nucleic acid sequence Acid sequence homology is at least 80%, and more typically at least 85%, 90%. The two amino acid sequences have preferably increasing homology of 95%, 99%, and 100%. If there is partial or complete identity between them, then the two amino acid sequences are homologous. For example, 85% homology is achieved when the two sequences are juxtaposed to obtain the greatest match. In this case, it means that 85% of the amino acids are identical. In order to maximize the match (when matched) A gap is allowed in either of the two sequences. Gap length of 5 or less. Preferably, 2 or less is more preferable. Alternatively and preferably as specified herein. When this term is used in the context of the variant data matrix and 6 or more Using the program ALIGN which employs gap penalties, 5 (in units of standard deviation) Having more juxtaposition scores means two protein sequences (or at least 30 amino acids) The polypeptide sequences obtained from these are homologous. "Dayhoff, M. O., in Atlas of Protein Sequence and St. ructure, pp.101-110 (Volume 5, National Biomedical Research Foundation (1972) and See "Appendix 2 to this volume, pp. 1 to 10." Using the ALIGN program... When optimally juxtaposed, the amino acids in two sequences or parts thereof are greater than 50%. If the ratio is equal to 50%, then the two sequences or parts thereof are more preferably homologous. In this book, the term "corresponding" refers to all or part of a reference polynucleotide sequence. In contrast, polynucleotide sequences are homologous (i.e., identical and strictly evolutionary). (Not related to) or the polypeptide sequence is identical to the reference polypeptide sequence. It is used to mean this. In contrast, the term "complementary" is used herein. In this case, the complementary sequence is homologous to all or part of the reference polynucleotide sequence. It is used to mean that. For example, the nucleotide sequence "TATAC" is a reference sequence. It corresponds to the column "TATAC" and is complementary to the reference array "GTATA".

[0206] Preparation of antigen-binding proteins (e.g., antibodies) In certain embodiments, an antigen-binding protein (such as an antibody) is an antigen (e.g., P It is produced by immunization using CSK9). In certain embodiments, the antibody is completely The full-length PCSK9, the soluble form of PCSK9, and the catalytic domain only are shown in Figure 1A. Using the mature form of PCSK9, the splice variant form of PCSK9, or fragments thereof It can be produced by immunization. In certain embodiments, the antibody of the present invention is It may be polyclonal or monoclonal, and / or a recombinant antibody. In certain embodiments, the antibody of the present invention is capable of producing, for example, human antibodies. These are human antibodies prepared by immunization of lancegenic animals (e.g., PCT publicly available). (See application WO93 / 12227).

[0207] In certain embodiments, the unique properties of the antibody, such as its affinity for its target, are used. Certain strategies can be used to manipulate the system. Such strategies include antibody barriers. To produce an antibody, site-specific or non-site-specific polynucleotide molecules encoding the antibody are used. This includes, but is not limited to, the use of artificial mutation introduction. In this state, after such fabrication, the desired change (e.g., increased or decreased affinity) Screening will be performed for antibody variants that exhibit the following characteristics.

[0208] In certain embodiments, the amino acid residue targeted in the mutation introduction strategy is CDR These are amino acid residues within. In certain embodiments, the framework region of the variable domain. The amino acids in the region are targeted. In certain embodiments, such a framework The region has been shown to contribute to the target binding properties of certain antibodies. For example, "Hud Son, Curr. Opin. Biotech., 9:395-402 (1999 Please refer to the document and its bibliography.

[0209] In certain embodiments, random or site-targeted mutagenesis is performed on high-frequency mutations in CDRs. Restriction to heterosites (regions corresponding to areas susceptible to mutation during the somatic cell affinity maturation process). By doing so, antibody variants can be screened more effectively and more efficiently. Ibrary is created. For example, "Chowdhury & Pastan, Nat See "ure Biotech., 17:568-572 (1999)" and the references within it. Please refer to the literature. In certain embodiments, certain direct and inverted repeats, Consensus sequences of species, certain secondary structures, and certain palindromes, etc. (However, this (Not limited to these.) To identify high-frequency mutation sites, certain types of DNA elements are used. This can be used, for example, to identify high-frequency mutation sites. Such DNA elements include a quaternary nucleotide sequence containing purine (A or G) followed by guanine (G). A pyrimidine (C or T) followed by either adenosine or thymidine (A or T) (that is, This includes, but is not limited to, A / GGC / TA / T. (High-frequency variants) Another example of a DNA element that can be used to identify a position is the serine codon AG- It is C / T.

[0210] Preparation of fully human ABP (e.g., antibodies) In certain embodiments, to produce monoclonal antibodies, phage dispers Ray technology is used. In certain embodiments, such technology is used for complete human monocles. Ronal antibodies are produced. In certain embodiments, a single Fab or Fv antibody fragment is used. The encoded polynucleotide is expressed on the surface of the phage particle. For example, Hoog enboom et al., J. Mol. Biol., 227:381 (19 91); Marks et al., J Mol Biol 222:581 (1 991); See U.S. Patent No. 5,885,793. In certain embodiments Phages "screen" to identify antibody fragments that have affinity for the target. Therefore, certain types of such processes occur on the surface of filamentous bacteriophages. Display of the antibody fragment repertoire and binding of the antibody fragment repertoire to the target This mimics immune selection through the subsequent selection of phages. In certain types of such methods, A high-affinity functional neutralizing antibody fragment is isolated. (This is discussed in more detail below.) In such embodiments, the complete repertoire of human antibody genes is peripheral blood lymph It is created by cloning the human V gene that has been naturally rearranged from a sphere. For example, “Mullinax et al, Proc Natl Acad Sci ( See "USA), 87:8095-8099 (1990)."

[0211] According to certain embodiments, a significant portion of the inserted human antibody-producing genome is present. Through the use of transgenic mice in which the production of endogenous mouse antibodies is lost, this development Antibodies are prepared. Then, these mice are subjected to human immunoglobulin molecules and antibodies. It can produce this, but lacks the production of mouse immunoglobulin molecules and antibodies. The techniques used to achieve the results are those disclosed herein in the patents, applications and references. Disclosed in the referenced documents. In certain embodiments, PCT publication application WO98 / 24 893 or “Mendez et al, Nature Genetics, 15: 146-156 (1997) (For all purposes, incorporated herein by reference) Methods such as those disclosed in (included) can be used.

[0212] Generally, a fully human monoclonal ABP (e.g., antibody) specific to PCSK9. It can be prepared as follows: Transidence containing human immunoglobulin genes A genetically modified mouse is immunized with the target antigen (e.g., PCSK9) and expresses an antibody. Lymphocytes (such as B cells) are obtained from the sac. Immortalized hybridoma cell lines are prepared. To achieve this, these recovered cells are fused with myeloid cell lines and specifically target the antigen of interest. To identify hybridoma cell lines that produce heterologous antibodies, such hybrids M cell lines are screened and selected. In certain embodiments, PCSK9 is used This provides the production of hybridoma cell lines that produce specific antibodies.

[0213] In certain embodiments, fully human antibodies in vitro spleen cells (B or T cells) The mice were exposed to the antigen in a nasal passage, and then immunodeficient mice (e.g., SCID or nod / ) were subjected to further exposure. It is produced by reconstituting cells exposed in SCID. For example, "Br ams et al, J.Immunol.160:2051-2058 (1998 ); Carballido et al., Nat.Med., 6:103-10 See 6 (2000). In certain kinds of approaches, SCID Transplantation of human fetal tissue into mice (SCID-hu) promotes long-term hematopoiesis and human T cell growth. This brings about... For example, "McCune et al., Science, 241:1 532-1639 (1988); Ifversen et al., Sem.Im See "munol., 8:243-248 (1996)". In certain cases... In this context, humoral immune responses in such chimeric mice are related to the simultaneous formation of human T cells in animals. It depends on the length. For example, "Martensson et al., Immunol., See 83:1271-179 (1994). In a certain approach Human peripheral blood lymphocytes are then transplanted into SCID mice. For example, "Mosier See et al., Nature, 335:256-259 (1988). In certain embodiments, Staphylococcus enterotoxin A (SEA) Such transplants can be performed using stimulants such as ) or anti-human CD40 monoclonal antibodies. When the cells are processed, a higher level of B cell production is detected. For example, "Ma rtensson et al., Immunol., 84:224-230 (1 995); Murphy et al, Blood, 86:1946-1953 See (1995).

[0214] Therefore, in certain embodiments, a fully human antibody is used to control recombinant DNA in host cells. It can be produced by expression or by expression in hybridoma cells. Other embodiments In this process, antibodies are produced using the phage display technology described herein. It is possible.

[0215] The antibodies described herein are XenoM as described herein. ouse (R) Prepared through the use of technology. Then, such mice are subjected to human immunotherapy. It can produce globulin molecules and antibodies, and mouse immunoglobulin molecules and antibodies. It lacks life. The techniques used to achieve the same thing are the background techniques of this specification. This is disclosed in the patents, applications, and references disclosed in this section. However, in particular, A preferred embodiment of transgenic production in mice and the antibodies obtained therefrom is described in 19 U.S. Patent Application 08 / 759,620, filed on December 3, 1996, and June 1, 1998 International patent application WO98 / 24893 was published on the 1st and published on December 21, 2000. WO00 / 76310 (These disclosures are incorporated herein by reference) It is disclosed in "Mendez et al, Nature Geneti". CS, 15:146-156 (1997) (Its disclosure is incorporated herein by reference) (See also: It is included.)

[0216] Through the use of such technologies, fully human monoclonal antibodies against various antigens can be produced. It has been produced in particular by the mouse XenoMouse. (R) The lineage is the target antigen (for example, Lymphatic cells (such as B cells) are immunized by PCSK9 and then regenerate from highly immunized mice. The collected and recovered lymphocytes are used to prepare immortalized hybridoma cell lines. It is fused with a myeloid cell line. A hybrid that produces antibodies specific to the target antigen is formed. To identify M cell lines, these hybridoma cell lines were screened and selected. This involves creating multiple hybridoma cell lines that produce antibodies specific to PCSK9. Methods for doing so are provided herein. Furthermore, heavy chains and light chains of such antibodies Nucleotide and amino acid sequence analysis of antibodies produced by such cell lines Property determination is provided herein.

[0217] XenoMouse (R) The lineage was filed on January 12, 1990. U.S. Patent Application No. 07 / 466,008, filed November 8, 1990, under the 07 / 6 Patent No. 10,515, filed on July 24, 1992, No. 07 / 919,297, 199 Application No. 07 / 922,649 filed on June 30, 2012, and application filed on March 15, 1993. Filed as No. 08 / 031,801 on August 27, 1993, as No. 08 / 112,8 Patent No. 48, filed on April 28, 1994, No. 08 / 234,145, January 1995 Filing No. 08 / 376,279 on the 20th, and No. 0 on April 27, 1995 Patent No. 8 / 430,938, filed on June 5, 1995, No. 08 / 464,584, 1 Application No. 08 / 464,582 filed on June 5, 1995, filed on June 5, 1995 Filed on June 5, 1995, under patent no. 08 / 463,191, under patent no. 08 / 462,837 Application No. 08 / 486,853, filed on June 5, 1995 Application No. 08 / 486,857 filed on June 5, 1995 Patent No. 08 / 462,513, filed on June 5, 1995, and filed on June 5, 1996. Filing No. 08 / 724,752 on October 2nd, and Filing No. 08 / 724,752 on December 3rd, 1996 Patent No. 08 / 759,620, filed on November 30, 2001, U.S. Publication No. 2003 / 0 U.S. Patent No. 093820 and U.S. Patent Nos. 6,162,963, 6,150,584, and Nos. 6,114,598, 6,075,181, 5,939,598 and Japan National Patent Nos. 3068180B2, 3068506B2, and 3068507 Discussed and described in B2. European Patent E, licensed and published on June 12, 1996. P0463151B1, International Patent Application WO94 / 026, published on February 3, 1994. 02. International patent application WO96 / 34096, published on October 31, 1996, 199 WO98 / 24893 was released on June 11, 2008, and on December 21, 2000. See also WO00 / 76310. The entire disclosure of each of the above patents, applications and references. The body is incorporated herein by reference.

[0218] In another approach, GenPharmInternational, Inc. Other companies used the "mini-locus" approach. In the mini-locus approach, I The foreign Ig locus is mimicked by including fragments (each gene) from the g locus. Therefore, one or more V H One or more genes, D H A gene, one or more More than J H The gene, the μ constant region, and usually a second constant region (preferably the γ constant region) , formed within a structure for insertion into the animal. This approach is used by Surani et al. U.S. Patent No. 5,545,807 in relation to Lonberg and Kay, respectively. U.S. Patent No. 5,545,806, No. 5,625,825, No. 5,625,12 No. 6, No. 5,633,425, No. 5,661,016, No. 5,770,429 No. 5,789,650, No. 5,814,318, No. 5,877,397 , No. 5,874,299 and No. 6,255,458, Krimpenfort et al. U.S. Patent Nos. 5,591,669 and 6,023.010 against Berns, Berns et al., U.S. Patent Nos. 5,612,205, 5,721,367 and No. 5,789,215 and Choi & Dunn, and GenPharm U.S. Patent No. 5,643,763, filed on August 29, 1990, against the International United States Patent. Patent application No. 07 / 574,748, filed on August 31, 1990, for the same patent application No. 07 / 575 Patent No. 962, filed on December 17, 1991; Patent No. 07 / 810,279, 1992 Filed on March 18, patent no. 07 / 853,408, filed on June 23, 1992. Patent No. 07 / 904,068, filed on December 16, 1992, is the same as patent No. 07 / 990,86 Application No. 0, filed on April 26, 1993, No. 08 / 053,131, filed on July 2, 1993. Filing No. 08 / 096,762 on the 2nd, and Filing No. 0 on November 18, 1993 Patent No. 8 / 155,301, filed on December 3, 1993, No. 08 / 161,739, 1 Filing No. 08 / 165,699 on December 10, 1993, and published on March 9, 1994. Application No. 08 / 209,741 (These disclosures are incorporated herein by reference) It is described in: European Patent 0 546 073 B1, International Patent Application WO9 2 / 03918, WO92 / 22645, WO92 / 22647, WO92 / 22670 , WO93 / 12227, WO94 / 00569, WO94 / 25585, WO96 / 1 4436, WO97 / 13852 and WO98 / 24884 and U.S. 5,981 See also No. 175 (the entirety of these disclosures is incorporated herein by reference). sea ​​bream. Taylor et al., 1992, Chen et al., 1993 ., Tuaillon et al., 1993, Choi et al., 1 993, Lonberg et al., (1994), Taylor et a l., (1994) and Tuaillon et al., (1995), Fis. hwild et al., (1996) (The entirety of these disclosures is referenced by this specification.) (It will be incorporated into the book.) See further reference.

[0219] Kirin is formed when microcell fusion, a large piece of a chromosome, or an entire chromosome is introduced into it. The production of human antibodies from mice was also demonstrated. European Patent Applications No. 773288 and 84396 See Section 1 (these disclosures are incorporated herein by reference). The Kirin Tc mouse and the Medarex mini-locus (Humab) mouse KM is the result of crossbreeding. TM Mice have been created. These mice are Kirin mice. The human IgH transchromosome and the Genpharm mouse κ chain transgene have (Is Hida et al., Cloning Stem Cells, (2002) 4:91-102).

[0220] Human antibodies can also be induced by in vitro methods. A suitable example is phage dispensing. Ray (CAT, Morphosys, Dyax, Biosite / Medarex, Xo ma, Symphogen, Alexion (formerly Proliferon), Affime d) These include ribosome displays (CAT), yeast displays, etc. Not limited to this.

[0221] In some embodiments, the antibodies described herein are human IgG quadrilaterals and It has a double IgG chain. The antibody may also be of other human isotypes, including IgG1. When measured using various techniques, antibodies have high affinity, and typically , about 10 -6 From about 10 -13 M or less K d It has.

[0222] As is understood, antibodies are expressed in cell lines other than hybridoma cell lines. This can be done. To transform appropriate mammalian host cells, a specific antibody encoding a specific antibody can be used. A column can be used. Transformation involves, for example, adding polynucleotides to the virus (or (Packaged in a viral vector) and the virus (or vector) or U.S. 4 Nos. 399,216, 4,912,040, 4,740,461 and 4 ,959,455 (These patents are incorporated herein by reference.) As illustrated, host cells can be transduced using transfusion methods known in this field. Any known method for introducing polynucleotides into host cells, including injection. Obtained by [method]. The transformation technique used depends on the host to be transformed. Mammals Methods for introducing heterologous polynucleotides into cells are well known in this field, Translocation mediated by quistran, calcium phosphate precipitation, and mediated by polyblen. Transfusion mediated by protoplast fusion, electroporation, and polynucleotides in liposomes This includes encapsulation and direct microinjection of DNA into the nucleus.

[0223] Mammalian cell lines that can be used as hosts for expression are well known in this field, Inies hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells ) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), human epithelium Kidney 293 cells and numerous other cell lines, etc. (but not limited to these), USA Many immortalized figures are available from the Type Culture Collection (ATCC). Cell lines are included. Particularly preferred cell lines are those in which any of the cells have high expression levels and constitutively This can be selected through a determination of whether or not to produce an antibody with PCSK9 binding properties.

[0224] In certain embodiments, antibodies and / or ABP are used to reduce the number of the following hybridomas. Each of the following: 21B12, 31H4, 16F12, listed in Table 2 or open in the examples. It is produced by any other hybridoma shown. In certain embodiments, Therefore, antigen-binding proteins are less than approximately 1 nM, for example, 100 pM to 100 pM. 0 pM to 10 pM, 10 pM to 1 pM and / or 1 pM to 0.1 pM or less The dissociation constant (K D ) is used to connect to PCSK9.

[0225] In certain embodiments, the antigen-binding protein is IgG1, IgG2, IgG3, At least one immunoglobulin of IgG4, IgE, IgA, IgD, and IgM isotypes Contains phosphorus molecules. In certain embodiments, the antigen-binding protein is human κ light chain and / or containing human heavy chains. In certain embodiments, the heavy chains include IgG1, IgG2, and IgG 3. IgG4, IgE, IgA, IgD, or IgM isotype. Certain types of In the application morphology, antigen-binding proteins are cloned for expression in mammalian cells. In certain embodiments, the antigen-binding proteins are IgG1, IgG2, and I. Any of the steady-state ranges of gG3, IgG4, IgE, IgA, IgD, and IgM isotypes Includes the outer steady-state region.

[0226] In certain embodiments, the antigen-binding protein is a human λ light chain and a human IgG double chain. Includes. In certain embodiments, the antigen-binding protein is human λ light chain and human IgG. It contains a quadruple chain. In certain embodiments, the antigen-binding protein is a human λ light chain and human Contains IgG1, IgG3, IgE, IgA, IgD, or IgM heavy chains. In other embodiments, The antigen-binding protein includes human κ light chains and human IgG double chains. In this state, the antigen-binding protein includes human κ light chain and human IgG quadruplex. In this embodiment, the antigen-binding protein is human κ light chain and human IgG1, IgG3, I It contains gE, IgA, IgD, or IgM heavy chains. In certain embodiments, antigen-binding tangent The protein is neither in the constant region for IgG2 isotype nor for IgG4 isotype. It includes a variable region of an antibody that is not a constant region but is linked to a constant region. In certain embodiments, Therefore, antigen-binding proteins have been cloned for expression in mammalian cells.

[0227] In certain embodiments, hybridoma strains: 21B12, 31H4 and 16F12 Conservative modifications (and encoding) of the heavy and light chains of antibodies obtained from at least one of the The corresponding modifications to the nucleotides are found in hybridoma strains 21B12, 31H4 and 1 Antibodies against PCSK9 that have similar functional and chemical characteristics to antibodies obtained from 6F12 It produces the body. In contrast, in certain embodiments, the mechanism of antibodies against PCSK9 Substantial modification of functional and / or chemical features is (a) the structure of the molecular skeleton in the region of substitution, e.g. For example, (b) sheet or helix stereostructure, (c) molecular charge or hydrophobicity at the target site. (c) The effect of the heavy and light chain amino acid combinations on sex or side chain size maintenance differs significantly. This can be achieved by selecting the replacement in the column.

[0228] For example, "conservative amino acid substitution" refers to a substitution that is relative to the polarity or charge of the amino acid residue at that position. Substituting specific amino acid residues with non-specific residues so that there is little or no effect. This may include the previously described "alanine scanning mutation introduction". As shown, any intrinsic residue in the polypeptide can be substituted with alanine.

[0229] The desired amino acid substitution (whether conserved or non-conserved) is this At the point in time when such substitutions are desired, it can be determined by those skilled in the art. In this state, as described herein, important residues of the antibody against PCSK9 To identify, or to increase or decrease the affinity of antibodies against PCSK9 Amino acid substitutions can be used.

[0230] In certain embodiments, the antibody of the present invention is used in cell lines other than hybridoma cell lines. It can be expressed. In certain embodiments, suitable mammalian host cells can be transmuted. To replace it, a sequence encoding a specific antibody can be used. Certain embodiments According to the information, transformation involves, for example, introducing polynucleotides into the virus (or viral vector). - Packaged inside with a virus (or vector) or U.S. 4,399,216 Nos. 4,912,040, 4,740,461 and 4,959,455 (These patents are incorporated herein by reference for all purposes.) By transferring host cells using transmutation methods known in this field, such as those exemplified by [example], Any known method for introducing polynucleotides into host cells, including transduction. Obtained by the method. In certain embodiments, the transformation method used is transformed It may depend on the host to which it is introduced. The method is well known in this field, and involves dextran-mediated translocation and phosphate transfer. Calcium precipitation, polyblen-mediated translocation, protoplast fusion, electroporation This includes the encapsulation of polynucleotides in liposomes and the direct microinjection of DNA into the nucleus. However, it is not limited to these.

[0231] Mammalian cell lines that can be used as hosts for expression are well known in this field, Inies hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells ) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2) and numerous Other cell lines, etc. (but not limited to these), American type culture... This includes many immortalized cell lines available from the collection (ATCC), but this These are not limited to the above. In certain embodiments, the cell line is such that any cell has a high expression level. It can be selected through a determination of whether it has the ability to produce antibodies with constitutive HGF-binding properties. Appropriate expression vectors for mammalian host cells are well known.

[0232] In certain embodiments, the antigen-binding protein is one or more polypeptides. Includes. In certain embodiments, one or more ABP components or ABP itself In order to express polynucleotide molecules that encode polypeptides containing various substances, Either an expression vector or a host system can be used. Such systems include recombinant bacteria Bacteria transformed with teriophages, plasmids, or cosmid DNA expression vectors, etc. Microorganisms; yeast transformed with a yeast expression vector; virus expression vector (e.g., Insect cell lines infected with culovirus, viral expression vectors (e.g., cauliflower) If transmuted by mosaic virus (CaMV, tobacco mosaic virus, TMV) or transformed with a bacterial expression vector (e.g., Ti or pBR322 plasmid) This includes, but is not limited to, phytocellular systems or animal cell systems.

[0233] In certain embodiments, one or more ABP components or ABP itself The polypeptides included are recombinantly expressed in yeast. Certain such embodiments are Follow the manufacturer's instructions for commercially available expression systems, such as Pichia Expressio. Use n System (Invitrogen, San Diego, CA) In certain embodiments, such a system uses a prepro-α sequence to induce secretion. It depends on. In certain embodiments, the transfer of the insert occurs when it is induced with methanol. It is driven by the alcohol oxidase (AOX1) promoter.

[0234] In certain embodiments, one or more ABP components or ABP itself The secreted polypeptides are purified from the yeast growth medium. In certain embodiments, The method used to purify polypeptides from yeast growth medium is used for bacteria and mammals This is the same method used to purify polypeptides from the supernatant of physical cells.

[0235] In certain embodiments, one or more ABP components or ABP itself The nucleic acid encoding the polypeptide is pVL1393(PharMingen, Sa It is cloned into baculovirus expression vectors such as n Diego, CA. In certain embodiments, such vectors are used in sF9 protein-free medium. Infect with Spodoptera frugiperda cells To produce recombinant polypeptides, follow the manufacturer's instructions (PharMingen). It can be used in this way. In certain embodiments, the polypeptide is heparin- Purification and concentration from such culture media are performed using a Farose column (Pharmacia). It can be done.

[0236] In certain embodiments, one or more ABP components or ABP itself The polypeptides included are expressed within insect systems. A certain type of insect system is required for polypeptide expression. This is well known to those skilled in the art. In one such system, Spodoptera fulgiperda cells Expressing foreign genes within the larvae of *Trichoplusia* or *Trichoplusia*. As a vector for Autographa California The nuclear polyhedron disease virus (AcNPV) is used. In this state, nucleic acid molecules encoding polypeptides are present in the non-essential genes of the virus. For example, by inserting it into a polyhedrin gene and under the control of the promoter for that gene. It can be placed. In certain embodiments, by successfully inserting nucleic acid molecules Non-essential genes are then inactivated. In certain embodiments, their inactivation is not detectable. This results in possible characteristics. For example, inactivation of the polyhedrin gene leads to a lack of coat protein. This leads to the production of viruses.

[0237] In certain embodiments, S. fulgiperda cells or Trichoprusia larvae are infected Recombinant viruses can be used to achieve this. For example, "Smith et al." l., J. Virol., 46:584 (1983); Engelhard et al, Proc.Nat.Acad.Sci.(USA), 91:3224- See "7 (1994)".

[0238] In certain embodiments, one or more ABP components are produced in bacterial cells. Polypeptides containing either ABP or ABP itself are produced in bacteria as insoluble inclusion bodies. In certain embodiments, host cells containing such inclusion bodies are collected by centrifugation. Then, it was washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM MEDTA, and in the room Warm water for 15 minutes with 0.1 mg / mL lysozyme (Sigma, St. Louis, MO) The solution is processed. In certain embodiments, the solubilized solution is clarified by ultrasonic treatment, and the cells The crushed material is settled by centrifugation at 12,000 × g for 10 minutes. Certain embodiments In this case, the precipitate containing polypeptides was 50 mM Tris, pH 8, and 10 mM E. The mixture was resuspended in DTA, topped with 50% glycerol, and centrifuged at 6000 × g for 30 minutes. In certain embodiments, the precipitate is Mg ++ and Ca ++ Standard that does not include It can be resuspended in a typical phosphate-buffered saline (PBS) solution. In certain embodiments... By fractionating the precipitate resuspended in the modified SDS polyacrylamide gel, The polypeptide is further purified (for example, Sambrook et al., (See note). In certain embodiments, to visualize the protein, 0.4MKCl It is possible to immerse such a gel inside, the protein is cleaved, and SDS It can be electrolytically eluted into a gel-running buffer that does not contain it. According to certain embodiments, Lutathione-S-transcriptase (GST) fusion protein is found in bacteria as a soluble protein and It is produced by GS. In certain embodiments, such a GST fusion protein is GS Purified using the TPurification Module (Pharmacia). .

[0239] In certain embodiments, certain polypeptides, for example, one or more It is desirable to "refold" the polypeptide containing the ABP component or ABP itself. i. In certain embodiments, such polypeptides are discussed herein. It is produced using a certain recombinant system. In certain embodiments, a desired tertiary structure is produced In order to form and / or generate disulfide bonds, the polypeptide is "Refolded" and / or "oxidized". In certain embodiments, such The structure and / or binding are related to certain biological activities of polypeptides. In terms of form, refolding can be achieved using any of the many methods known in this art. A typical method involves using a chaotropic agent to raise the pH to typically above 7. This includes, but is not limited to, exposure to solubilized polypeptide agents. The chaotropic agent is guanidine. In certain embodiments, refolding / acid The solution also contains a reducing agent and an oxidized form of the reducing agent. In certain embodiments, Therefore, reducing agents and their oxidized forms are specific oxidation-reducing agents that cause disulfide shuffling. It exists in a ratio that generates the original potential. In certain embodiments, such shuffling This enables the formation of cysteine ​​crosslinks. Typical redox pairs include cysteine / cysteine. Glutathione / Dithiobis(GSH), Copper Chloride, Dithiothreitol DTT / Dithio It contains DTT and 2-mercaptoethanol (bME) / dithio-bME, but It is not limited to these. In certain embodiments, in order to increase the efficiency of refolding, Cosolvents are used. Typical cosolvents include glycerol and polyethylene of various molecular weights. This includes, but is not limited to, glycols and arginine.

[0240] In certain embodiments, one or more ABP components or ABP itself The polypeptides contained are substantially purified. Certain protein purification techniques are known to those skilled in the art. Yes. In certain embodiments, protein purification is performed by separating the non-polypeptide fraction from the polypeptide fraction. This includes coarsely fractionating the nitrate fraction. In certain embodiments, the polypeptide is chroma Purification is performed using tography and / or electrophoresis techniques. Typical purification methods include sulfur Precipitation with ammonium phosphate; Precipitation with PEG; Immunoprecipitation; Centrifugation after thermal denaturation; Affi Nity chromatography (e.g., protein A-Sepharose), ion exchange chromatography Mathography, exclusion chromatography and reverse-phase chromatography; gel filtration; hydro Chromatography such as (but not limited to) roxyapatite chromatography Electrophoresis; isoelectric focusing; polyacrylamide gel electrophoresis and similar techniques This includes, but is not limited to, combinations of other technologies. In certain embodiments, Polypeptides are then analyzed by high-performance protein liquid chromatography, or by high-pressure liquid chromatography. It is purified by chromatography (HPLC). In certain embodiments, It is possible to change the process or omit certain steps, but the substantially refined This still provides a suitable method for preparing polypeptides.

[0241] In certain embodiments, the degree of purity of a polypeptide preparation is quantified. Certain methods for doing so are known to those skilled in the art. Certain typical methods are known to those skilled in the art. The binding activity of the polypeptides in the preparation is measured and analyzed by SDS / PAGE. This includes, but is not limited to, evaluating the quantity of the polypeptide preparation. A typical method for evaluation is to calculate the binding activity of the preparation, and the binding of the preparation. This involves comparing the activity with the binding activity of the initial extract. In certain embodiments, this The result of the calculation is expressed as the "purification factor." The unit used to express the amount of binding activity The position depends on the assay being performed.

[0242] In certain embodiments, one or more ABP components or ABP itself The polypeptides included are partially purified. In certain embodiments, partial purification is performed. By using fewer purification steps, or by using different forms of the same general purification scheme. This can be achieved by using a state. For example, in certain embodiments, Cation exchange column chromatography performed using an HPLC instrument is a low-pressure chromatography process. It generally yields a higher "purification ratio" than the same technique using tography. In terms of application methods, methods that result in a lower degree of purity are also available in the total recovery of polypeptides. This may have advantages in maintaining polypeptide binding activity.

[0243] In certain cases, the electrophoretic transfer of polypeptides is performed under different conditions of SDS / PAGE. Along with this, it can fluctuate (sometimes significantly). For example, "Capaldi et al., B Refer to "iochem.Biophys.Res.Comm.,76:425(1977)". Please examine the purified or partially purified polypeptide under different electrophoretic conditions. It is understood that the apparent molecular weight of butylene can vary.

[0244] Typical epitope An epitope to which an anti-PCSK9 antibody binds is provided. In some embodiments, The epitopes conjugated by the antibodies disclosed in the specification are particularly useful. In the embodiments thereof, what is the epitope conjugated by the antibody described herein? Antigen-binding proteins that bind to any of these are useful. In some embodiments, Table 2 and the following are shown. The epitopes conjugated by any of the antibodies listed in Figures 2 and 3 are particularly useful. Yes. In some embodiments, the epitope is located on the catalytic domain PCSK9. .

[0245] In certain embodiments, an anti-PCSK9 antibody or an antigen-binding protein against PCSK9 To inhibit (e.g., reduce) the binding of nucleotides, the PCSK9 epitope is used. This is possible. In certain embodiments, an anti-PCSK9 antibody or an antigen against PCSK9 is used. The PCSK9 epitope can be used to reduce the binding of binding proteins. In certain embodiments, an anti-PCSK9 antibody or an antigen-binding tangent to PCSK9 is used. The PCSK9 epitope can be used to substantially inhibit protein binding. .

[0246] In certain embodiments, antibodies or antigen-binding proteins that bind to PCSK9 are isolated. To achieve this, the PCSK9 epitope can be used. In certain embodiments, To produce antibodies or antigen-binding proteins that bind to PCSK9, PCSK9 epi A tope can be used. In certain embodiments, an antibody that binds to PCSK9 can be used. Alternatively, to produce an antigen-binding protein, a PCSK9 epitope or PCSK9 epitope Sequences containing the pc can be used as immunogens. In certain embodiments, PC The SK9 epitope can be administered to animals, and antibodies that bind to PCSK9 can then be administered to animals. It can be obtained from an object. In certain embodiments, the PCSK9 meeting with LDLR To interfere with normal PCSK9-mediated activity, such as synthesis, PCSK9 epididymitis is used. Sequences containing a tope or PCSK9 epitope can be used.

[0247] In some embodiments, the antigen-binding protein disclosed herein is N-terminal Specifically binds to the terminal prodomain, subtilisin-like catalytic domain, and / or C-terminal domain. In some embodiments, the antigen-binding protein is located in the substrate-binding groove of PCSK-9. (as described in Cunningham et al., the whole is referred to herein by reference) It will be incorporated into it.

[0248] In some embodiments, the material contains residues that come into contact with or are embedded by the antibody. The domain / region mutates specific residues in PCSK9 (e.g., wild-type antigen) and inhibits The original binding protein may be mutated or variant PCSK9 protein may be able to bind. It can be identified by measuring whether or not it is possible. A large number of individual mutations are created. By doing so, mutations can affect the binding between antigen-binding proteins and antigens. Identifying residues that play a direct role in binding or residues that are in close proximity to the antibody. This can be done. Based on the knowledge of these amino acids, residues that come into contact with antigen-binding proteins or antibodies It is possible to elucidate the domain or region of the antigen containing residues coated by [the specified substance]. Such domains can contain binding epitopes for antigen-binding proteins. One concrete example of a common approach is the arginine / glutamate scanning protocol. Use the call (e.g., Naneviz, T., et al., 1995, J. Biol.Chem., 270:37, 21619-21625 and Zupnick, A., et al, 2006, J. Biol.Chem. See 28L:29, 20464-20473). Generally, arginine And glutamic acid is charged, bulky, and therefore the area of ​​the antigen into which the mutation has been introduced Because it has the potential to disrupt the binding between antigen-binding proteins and antigens within the region, The amino acids in wild-type polypeptides are converted to arginine and glutamic acid (typically individually). It is substituted for arginine. The arginine present in the wild-type antigen is substituted for glutamic acid. Various individual mutants like these are obtained, and to determine which residues affect binding The collected combined results are then analyzed.

[0249] Example 39 is a PC against the PCSK9 antigen-binding protein provided herein. One such arginine / glutamate scan of SK9 is described. A series of changes Differentiated PCSK9 antigens were generated, and each mutant antigen possesses a single mutation. Various PCSK9 The binding of each variant PCSK9 antigen to ABP was measured, and the selected ABP was compared to wild-type PCSK9 antigen. This was compared to the ability to bind 9 (sequence number 303).

[0250] Binding between antigen-binding protein and variant PCSK9 used herein Changes (e.g., decrease or increase) are (e.g., Bia as described below in the examples) When measured by known methods such as core testing or bead-based assays. ) Binding affinity, EC 50 Changes in and / or (e.g., antigen-binding protein concentration versus antigen concentration) B in the degree plot max (The decrease in antigen-binding proteins reveals the totality of antigen-binding proteins) This means that there is a change in binding ability (e.g., a decrease). Significant changes in binding indicate mutation. The received residue is directly involved in binding to the antigen-binding protein, or the binding protein is anti- This suggests that when it binds to the original protein, it is in close proximity to the binding protein.

[0251] In some embodiments, a significant decrease in binding is observed between antigen-binding proteins and mutant PCS. Binding affinity between K9 antigens, EC 50 and / or ability of antigen-binding protein and wild-type P CSK9 coupling (for example, shown in SEQ ID NO: 1 and / or SEQ ID NO: 303) Compared to that, over 10%, over 20%, over 40%, over 50%, over 55%, over 60%, over 65%, This means a decrease of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%. It has a taste. In certain embodiments, the binding decreases to below the detectable limit.

[0252] In some embodiments, antigen-binding proteins to variant PCSK9 proteins The binding is between the antigen-binding protein and the wild-type PCSK9 protein (e.g., SEQ ID NO: 1 and Less than 50% (e.g., 40%) of the bindings observed between / or the protein of SEQ ID NO: 303 When the bond is significantly low (35%, 30%, 25%, 20%, 15%, or 10%), The following is proven. Measurement of such binding can be performed using various binding assays known in this field. It can be carried out using this method. One specific example of such an assay is described in Example 39. ru.

[0253] In some embodiments, wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or ) is used. Variant PC in which a residue in column number 303) is replaced with arginine or glutamic acid. An antigen-binding protein is provided that exhibits significantly lower binding to the SK9 protein. In some embodiments, wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or) Compared to number 303), the following mutations were observed: R207E, D208R, R185E, R439E , E513R, V538R, E539R, T132R, S351R, A390R, A41 3R, E582R, D162R, R164E, E167R, S123R, E129R, A One of 311R, D313R, D337R, R519E, H521R and Q554R or Any of the above (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 244) The binding of antigen-binding proteins to the variant PCSK9 protein is significantly reduced. To decrease or increase. In the abbreviated notation used herein, the form is: wild type remaining Base: Position in polypeptide: A mutant residue, as shown in SEQ ID NO: 1 or SEQ ID NO: 303 The residues are numbered in this manner.

[0254] In some embodiments, wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or ) is used. Compared to column number 303, the following positions as shown in sequence number 1: 207, 208 ,185,181,439,513,538,539,132,351,390,413 , 582, 162, 164, 167, 123, 129, 311, 313, 337, 519 , one or more of 521 and 554 (for example, 1, 2, 3, 4, 5 or more) ) Mutant PCSK9 proteins with this mutation exhibit reduced antigen-binding protein binding. or increases. In some embodiments, wild-type PCSK9 protein (e.g., sequence Compared to number 1 or sequence number 303), the following positions as shown in sequence number 1: 2 07, 208, 185, 181, 439, 513, 538, 539, 132, 351, 3 90, 413, 582, 162, 164, 167, 123, 129, 311, 313, 3 37, 519, 521 and 554, one or more (for example, 1, 2, 3, 4, 5 or For mutant PCSK9 proteins with mutations (or more), antigen-binding proteins The binding of the wild-type PCSK9 protein decreases or increases. In some embodiments, the binding of the wild-type PCSK9 protein ( For example, compared to SEQ ID NO: 1 or SEQ ID NO: 303, the following position in SEQ ID NO: 1: 207 ,208,185,181,439,513,538,539,132,351,390 , 413, 582, 162, 164, 167, 123, 129, 311, 313, 337 , 519, 521 and 554 one or more (for example, 1, 2, 3, 4, 5 or so For mutant PCSK9 proteins with more than this number of mutations, antigen-binding proteins are used. The ratio decreases or increases substantially.

[0255] In some embodiments, wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or ) is used. Compared to sequence number 303, the following mutation: R207E in sequence number 1 or sequence number 303, D208R, R185E, R439E, E513R, V538R, E539R, T132 R, S351R, A390R, A413R, E582R, D162R, R164E, E1 67R, S123R, E129R, A311R, D313R, D337R, R519E, Having one or more H521R and Q554R (for example, 1, 2, 3, 4, 5, etc.) In the mutant PCSK9 protein, ABP binding is significantly reduced or increased.

[0256] In some embodiments, wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or ) is used. Compared to sequence number 303, the following mutation: R207E in sequence number 1 or sequence number 303, D208R, R185E, R439E, E513R, V538R, E539R, T132 One or more of R, S351R, A390R, A413R and E582R (for example, ABP binding to mutant PCSK9 proteins having (1, 2, 3, 4, 5, etc.) It decreases or increases significantly. In some embodiments, the coupling decreases. In terms of morphology, the decrease in binding is due to EC 50 This is observed as a change in several embodiments. , EC 50 The changes in EC 50 This represents an increase in the numerical value (and therefore a decrease in the number of connections).

[0257] In some embodiments, wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or ) is used. Compared to column number 303), the following mutations were observed: D162R, R164E, E16 in sequence number 1. 7R, S123R, E129R, A311R, D313R, D337R, R519E, H Having one or more of 521R and Q554R (e.g., 1, 2, 3, 4, 5, etc.) In the mutant PCSK9 protein, ABP binding is significantly reduced or increased. In one embodiment, the bond is reduced. In some embodiments, the reduction in bond is B max This is observed as a change in B. In some embodiments, B max The shift is AB This is the decrease in the maximum signal caused by P. In some embodiments, the epitope Regarding some amino acids, B max It decreases by at least 10%, for example, by the following amount , that is, 20, 30, 40, 50, 60, 70, 80, 90, 85, 98, 99 or A 100% reduction in at least one of the embodiments is due to the residue being an epitope. It can be shown that it is a part of something.

[0258] The variant forms listed immediately before this are shown in Sequence ID No. 1 or Sequence ID No. 303. Although it is cited in reference to the wild-type sequence, in the allele variant of PCSK9, It is understood that the amino acids at the indicated positions can differ. Such alleles of PCSK9 It is also conceivable that antigen-binding proteins exhibit significantly lower binding to morphology. Therefore, several In that embodiment, all of the above embodiments are the pure wild-type shown in Figure 1A. It can be compared not to the sequence itself, but to the allele sequence.

[0259] In some embodiments, at a selected position in the wild-type PCSK9 protein Regarding variant PCSK9 proteins in which a residue is mutated to any other residue, The binding of the proto-binding protein is significantly reduced. In some embodiments, the following have been identified. For the position, the arginine / glutamate substitutions described herein are used. In some embodiments, alanine is used for the identified location.

[0260] As mentioned above, residues directly involved in binding or residues covered by antigen-binding proteins. These can be identified from the scanning results. Therefore, these residues are antigen-binding proteins. Sequence ID 1 (or Sequence ID 303 or Sequence ID 3) contains a binding region to which the substance binds. It may provide indicators of the main or region. As is evident from the results summarized in Example 39. In some embodiments, the antigen-binding protein is SEQ ID NO: 1 or SEQ ID NO: 303 The amino acids are: 207, 208, 185, 181, 439, 513, 538, 539, 13 2, 351, 390, 413, 582, 162, 164, 167, 123, 129, 31 In a domain containing at least one of 1, 313, 337, 519, 521, and 554 Binds. In some embodiments, the antigen-binding protein is SEQ ID NO: 1 or SEQ ID NO: 303 amino acids: 207, 208, 185, 181, 439, 513, 538, 539 , 132, 351, 390, 413, 582, 162, 164, 167, 123, 129 , a region containing at least one of 311, 313, 337, 519, 521 and 554 Combine.

[0261] In some embodiments, the antigen-binding protein is SEQ ID NO: 1 or SEQ ID NO: 303 Amino acids: Contains at least one of 162, 164, 167, 207 and / or 208. It binds to the region. In some embodiments, two or more of the identified residues (for example, 2, 3, 4, or 5) is part of the region joined by the ABP. In some embodiments, Therefore, ABP competes with ABP21B12.

[0262] In some embodiments, the antigen-binding protein is SEQ ID NO: 1 or SEQ ID NO: 303 It binds to a region containing at least one amino acid 185. In some embodiments, ABP competes with ABP31H4.

[0263] In some embodiments, the antigen-binding protein is SEQ ID NO: 1 or SEQ ID NO: 303 In the region containing amino acids 439, 513, 538 and / or 539, Binds. In some embodiments, two or more identified residues (e.g., 2, 3, or 4) ) is part of the region joined by the ABP. In some embodiments, the ABP is It competes with the ABP31A4.

[0264] In some embodiments, the antigen-binding protein is SEQ ID NO: 1 or SEQ ID NO: 303 Amino acids: 123, 129, 311, 313, 337, 132, 351, 390 and / or It binds to a region containing at least one of 413. In some embodiments, identification Two or more of the affected residues (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) are linked by ABP. It is part of the region to be combined. In some embodiments, ABP competes with ABP12H11. To combine.

[0265] In some embodiments, the antigen-binding protein is SEQ ID NO: 1 or SEQ ID NO: 303 In the region containing at least one of amino acids 582, 519, 521 and / or 554 Binds. In some embodiments, two or more identified residues (e.g., 2, 3, or 4) ) is part of the region joined by the ABP. In some embodiments, the ABP is It competes with ABP3C4.

[0266] In some embodiments, the antigen-binding protein is SEQ ID NO: 1 or SEQ ID NO: 303 It binds to the region in the fragment or full-length sequence. In other embodiments, antigen-binding protein The substance binds to polypeptides consisting of these regions. (SEQ ID NO: 1 or SEQ ID NO: 303) The notation " " indicates that one or both of these sequences may be used or related. This term does not imply that only one term should be used.

[0267] As stated above, the above description refers to a specific amino acid position by referring to Sequence ID No. 1. However, throughout this specification, generally, the Pr starting at position 31 as described in Sequence ID No. 3 is used. The o / Cat domain is referenced. Sequence ID 1 and sequence number are as described below. Number 303 lacks the signal sequence of PCSK9. Therefore, among these various disclosures Any comparison should take this difference in numbering into account. In particular, any of the A in sequence number 1 The amino acid position also corresponds to the 30th amino acid in the protein of sequence number 3. For example, the sequence Corresponding to position 207 of sequence number 1 and position 237 of sequence number 3 (full-length sequence and generally as specified herein) (Numbering system used within). Table 39.6 shows sequence number 1 (and / or sequence number 303). The above references how they correspond to sequence number 3 (including the signal sequence). To summarize, therefore, as stated in Sequence ID No. 1 (and / or Sequence ID No. 303) Each of the embodiments described herein refers to the corresponding location described herein, with reference to Sequence ID No. 3. It is listed.

[0268] In some embodiments, ABP21B12 is composed of residues 162 to 167 (for example, It binds to an epitope containing residues D162-E167 in column number 1. In some embodiments, In this context, ABP12H11 is derived from residues 123 to 132 (for example, S123- in SEQ ID NO: 1). It binds to an epitope including T132). In some embodiments, ABP12H11 This is an epitope containing residues 311 or 313 (for example, A311-D313 in SEQ ID NO: 1). It binds to. In some embodiments, ABP contains any one of these strands of the sequence. It can bind to an epitope.

[0269] Competing antigen-binding proteins In another embodiment, with respect to specific binding to PCSK9, as described herein An antigen-binding protein that competes with one of the exemplified antibodies or functional fragments that bind to the epitope. Quality is provided. Such antigen-binding proteins are antigen-binding proteins as exemplified herein. It can also bind to the same epitope as one of the synthetic proteins, or to an overlapping epitope. Antigen-binding proteins that compete for or bind to the same epitope as the antigen-binding protein and The fragments are expected to exhibit similar functional properties. Exemplary antigen-binding proteins and fragments. These are the heavy chain and light chain variable region domains and are included in Table 2 and / or Figures 2 to 3 and 15. This includes those mentioned above, such as those with a CD-R. Therefore, as a specific example, provided Antigen-binding proteins include, (a) The six CDRs listed for the antibodies listed in Figures 2 to 3 and 15 all; (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 This includes antibodies or antigen-binding proteins that compete with those possessing the characteristic [of the characteristic].

[0270] Certain therapeutic uses and pharmaceutical compositions In certain cases, PCSK9 activity correlates with the disease status of many individuals. For example, In certain cases, excessively high or low PCSK9 activity can lead to hypercholesterolemia. It correlates with certain symptoms. Therefore, in certain cases, it modulates PCSK9 activity. This may be therapeutically useful. In certain embodiments, neutralization of PCSK9 The target antigen-binding protein has at least one PCSK9 activity (e.g., binding to LDLR). This method is used to regulate elevated serum cholesterol levels. To treat and / or prevent diseases associated with or related to elevated cholesterol levels. It can prevent and / or reduce the risk of disease.

[0271] As will be understood by those skilled in the art, in light of this disclosure, fluctuating cholesterol, LD Related to L or LDLR levels, fluctuating cholesterol, LDL or LDLR levels May be affected by accompanying or fluctuating cholesterol, LDL, or LDLR levels. Diseases can be addressed by various embodiments of antigen-binding proteins. In embodiments, "cholesterol-related diseases" (including "serum cholesterol-related diseases") Examples of symptoms include elevated total serum cholesterol, elevated LDL, and elevated triglycerides. The following may present with elevated VLDL and / or low HDL: high cholesterol Bloody syndrome, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease This includes one or more of the following: dyslipidemia, Alzheimer's disease, and dyslipidemia in general. Treatment with ABP, or in combination with one or more other drugs. Some non-limiting examples of primary and secondary dyslipidemia include metabolic syndrome. Diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemia Lorleinemia (heterozygous hypercholesterolemia, homozygous hypercholesterolemia, home Diagnostic apolipoprotein B-100 deficiency, polygenic hypercholesterolemia; remnan Remnant removal disease, hepatic lipase deficiency, The following: binge eating, hypothyroidism, drugs including estrogen and progestin therapy, β Blockers and thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary Biliary cirrhosis, glycogen storage disease, liver cancer, cholestasis, acromegaly, islet cell tumor, growth hormone alone This includes dyslipidemia secondary to either deficiency or alcohol-induced hypertriglyceridemia. ABP is seen in, for example, coronary heart disease, coronary artery disease, peripheral artery disease, and stroke (ischemic). Atheromas (and hemorrhagic), angina pectoris or cardiovascular disease, acute coronary syndrome, myocardial infarction, etc. It may also be useful in preventing or treating atherosclerotic diseases. In some embodiments, ABP refers to non-fatal heart attacks, fatal and non-fatal strokes, and certain types of heart surgery. Hospitalization for heart failure, chest pain and / or previous heart attack in patients with heart disease, Cardiovascular phenomena and / or signs of occluded arteries due to established heart disease, such as previous cardiac surgery. It is useful in reducing the risk of chest pain having AB. In some embodiments, AB Methods P and the method can be used to reduce the risk of recurrent cardiovascular events.

[0272] As will be understood by those skilled in the art, the use of statins is generally effective (or treatable). The application of the antigen-binding protein of the present invention may also be beneficial for preventable diseases or illnesses. Furthermore, in some embodiments, inhibition or increased LDLR synthesis was observed. Diseases or illnesses in which expression may be beneficial can also be treated by various embodiments of antigen-binding proteins. It can be treated. Furthermore, as will be understood by those skilled in the art, the use of anti-PCSK9 antibodies It may be particularly useful in the treatment of diabetes. Diabetes is a risk factor for coronary heart disease. Furthermore, insulin increases the expression of PCSK9. In other words, diabetes Individuals who have this condition have elevated plasma lipid levels (which may be associated with high PCSK9 levels), Lowering plasma lipid levels may be beneficial. This is according to Costet et al. .(“Hepatic PCSK9 Expression is Regulated by Nutritional Status via Insulin and S terol Regulatory Element-binding Protei n 1C”, J.Biol.Chem., 281:6211-6218, 2006 (These entirety are incorporated herein by reference.) For more details, see General It is discussed in detail.

[0273] In some embodiments, antigen-binding proteins are used in diabetes, abnormal aortic aneurysms, and aterostomy. In individuals with arteriosclerosis and / or peripheral vascular disease, their serum cholesterol levels It is administered to reduce antigenicity to a safer range. In some embodiments, antigenicity Synthetic proteins are for patients at risk of developing any of the diseases described herein. It is administered to those who smoke, have high blood pressure, or are young. In some embodiments, ABP is administered to those who smoke, have high blood pressure, or are young It is administered to smokers with a family history of heart attacks.

[0274] In some embodiments, the subject is at moderate risk or ABP is administered to the subject if they have a higher risk. In some embodiments In this case, if the target LDL cholesterol level is greater than 160 mg / dL, ABP The drug is administered to the target. In some embodiments, the target's LDL cholesterol level is If it is greater than 130 (and according to the 2004 NCEP treatment goals, the subject is moderate risk If there is a risk of 1 or a moderately high risk, ABP is administered. In some embodiments, If the target LDL cholesterol level is greater than 100 (and 2004 NCEP treatment ABP is administered according to the treatment objectives (if the subject is at high risk or extremely high risk). It will be done.

[0275] Depending on the individual characteristics of each patient, the physician will select appropriate treatment indicators and target lipid levels. It is possible. One well-accepted guideline for the treatment of hyperlipidemia The standard is "National Cholesterol Detective Agency for the Detection, Evaluation and Treatment of High Blood Cholesterol in Adults." Third report of the Expert Committee on the Nursing Care Program (NCEP) (Adult Treatment Panel III), Final Report, National Institutes of Health, NIH Publication No. 02-5215 (2002) (Third R eport of the National Cholesterol Educat ion Program (NCEP) Expert Panel on Detect tion, Evaluation, and Treatment of the H igh Blood Cholesterol in Adults (Adult T reatment Panel III) Final Report, Nation al Institutes of Health, NIH Publication No. 02-5215 (2002) is the title, and the entire printed publication is referenced. By reference, it is incorporated herein.

[0276] In some embodiments, the antigen-binding protein to PCSK9 has an abnormally high threshold. It is used to reduce the amount of PCSK9 activity from a normal or bell level. In this embodiment, the antigen-binding protein to PCSK9 is used to treat hypercholesterolemia. For the treatment or prevention of hypercholesterolemia and / or other conditions In the preparation of pharmaceuticals for resterol-related diseases (such as those described herein) It is used in certain embodiments. The antigen-binding protein for PCSK9 is To treat or prevent symptoms such as hypercholesterolemia in which PCSK9 activity is normal. It is used. In such symptoms, for example, a decrease in PCSK9 activity below normal is It can provide therapeutic effects.

[0277] In some embodiments, two or more antigen-binding proteins for PCSK9 are used in PC It is used to regulate SK9 activity.

[0278] In certain embodiments, one or more antigen-binding proteins to PCSK9 This includes administering a therapeutically effective dose of a certain quality and another therapeutic agent, such as for hypercholesterolemia. A method for treating resterol-related disorders is provided.

[0279] In certain embodiments, the antigen-binding protein against PCSK9 is administered alone. In certain embodiments, the antigen-binding protein for PCSK9 is at least It is administered before the administration of another therapeutic agent. In certain embodiments, against PCSK9 The antigen-binding protein is administered simultaneously with at least one other therapeutic agent. In one embodiment, the antigen-binding protein for PCSK9 is at least one It is administered after the administration of other therapeutic agents. In another embodiment, antigen binding to PCSK9. The protein is administered before the administration of at least one other therapeutic agent. (Antigen-binding protein) Therapeutic agents (separate from quality) should include at least one other cholesterol-lowering agent (serum and / or systemic). This includes, but is not limited to, cholesterol-containing agents or drugs. In some embodiments, The drug increases LDLR expression, increases serum HDL levels, and raises LDL levels. It has been observed that these drugs lower or reduce triglyceride levels. Typical drugs This includes statins (atrovastatin, cerivastatin, fluvastatin, lovastatin, Mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin) Nicotinic acid (niacin) (NIACOR, NIASPAN (sustained-release niacin)) SLO-NIACIN (sustained-release niacin), fibric acid (LOPID (Gemphy) Brozil), TRICOR (fenofibrate), bile acid chelating agent (QUESTRAN( Cholestyramine, Coleseveram (WELCHOL), COLESTID (Colestipol) Cholesterol absorption inhibitors (ZETIA (ezetimibe)), statins and nicotine Acid combination (ADVICOR (LOVASTATIN and NIASPAN), absorption) Combinations of statins with inhibitors (VYTORIN (ZOCOR and ZETIA) and / Or, lipid modifiers are included, but are not limited to these. In some embodiments, AB P stands for PPARγ agonist, PPARα / γ agonist, squalene synthase inhibitor. CETP inhibitors, antihypertensive drugs, antidiabetic drugs (sulfonylurea, insulin, GLP-1 analogues) (e.g., DDPIV inhibitors), ApoB modifiers, MTP inhibitors and / or occlusive arteriosclerosis It is used in combination with chemotherapy. In some embodiments, ABP is used with statins, oncos Certain cytokines such as tatin M, estrogen and / or berberine Combined with agents that increase the level of LDLR protein in the target, such as plant components of a species. It is made to be. In some embodiments, ABP is (certain antipsychotics, certain HI V protease inhibitors, high fructose, sucrose, cholesterol, or certain fats Dietary factors such as acids, as well as certain nuclear receptor jaws for RXR, RAR, LXR, and FXR. Drugs that increase serum cholesterol levels in the target population (such as agonists and antagonists) It is combined with statins and / or insulin. In some embodiments, ABP is combined with statins and / or insulin. It is combined with drugs that increase the level of PCSK9 in the target, such as phosphorus. Combinations allow ABP to mitigate undesirable side effects from other medications. Obtain. As will be understood by those skilled in the art, in some embodiments, ABP is obtained from other drugs. It is combined with an agent / compound. In some embodiments, ABP and other agents are combined simultaneously. It is administered to [the body]. In some embodiments, ABP and other drugs are not administered simultaneously, and AB P is administered before or after the drug is administered. In some embodiments, the target is preventive During the same period, between the onset and / or treatment of the disease, the levels of ABP and (LDLR) Take any of the other medications (to increase the levels).

[0280] The pharmaceutical composition of the present invention is used in combination therapy (i.e., in combination with other drugs). ) can be administered. In certain embodiments, combination therapy is at least 1 An antigen-binding agent that can bind PCSK9 in combination with two anticholesterol agents. Contains protein. The drug contains chemical compositions, antibodies, and antigens that are synthetically prepared in vitro. This includes, but is not limited to, bonding regions and their combinations and connections. In one embodiment, the drug is an agonist, an antagonist, or an allosteric modifier. It can act as a substance or toxin. In certain embodiments, the drug is It acts to inhibit or stimulate a target (e.g., the activation or inhibition of a receptor or enzyme). This promotes increased expression of LDLR or reduces serum cholesterol levels. It can be done.

[0281] In certain embodiments, the antigen-binding protein for PCSK9 is cholesterol Administer before, simultaneously with, and after treatment with cholesterol-lowering agents (serum and / or total cholesterol). It is possible. In certain embodiments, the antigen-binding protein for PCSK9 is Hypercholesterolemia, heart disease, diabetes and / or any cholesterol-related disorder It can be administered prophylactically to prevent or reduce the onset of symptoms. In certain embodiments, Therefore, the antigen-binding protein for PCSK9 is used in the treatment of existing hypercholesterolemia symptoms. It can be administered for the following purposes. In some embodiments, ABP is used for the disease and / or It delays the onset of symptoms associated with the disease. In some embodiments, ABP helps to slow down cholesterol It is provided to individuals who lack any or part of the symptoms of any one of the ulcer-related diseases.

[0282] In certain embodiments, the antigen-binding protein to PCSK9 is high cholesterol It is used in conjunction with therapeutic agents to treat various cholesterol-related diseases such as hypercholesterolemia. In certain embodiments, two or three are used in light of the desired level of symptoms and treatment. One or more drugs can be administered. In certain embodiments, in the same formulation By including it in certain embodiments, such drugs can be administered together. In this context, such drugs and antigen-binding proteins for PCSK9 are present in the same preparation. They can be given together by including them. In certain embodiments, such The medications can be administered together by being formulated separately and included in a treatment kit. In certain embodiments, such drugs and antigen-binding proteins for PCSK9 The substances can be administered together by being formulated separately and included in a treatment kit. In certain embodiments, such drugs can be administered separately. In terms of form, when administered by gene therapy, protein agents and / or PCSK The gene encoding the antigen-binding protein for 9 can be included in the same vector. In certain embodiments, the antigen-binding agent for the protein agent and / or PCSK9 is used. Protein-coding genes can be regulated by the same promoter region. In the application form, the protein preparation and / or the antigen-binding protein for PCSK9 are coated The genes involved can exist in separate vectors.

[0283] In certain embodiments, the present invention relates to a pharmaceutically acceptable diluent, carrier, or solubilizer. Along with emulsifiers, preservatives and / or auxiliary agents, the antigen-binding protein for PCSK9 The present invention provides a pharmaceutical composition containing the following:

[0284] In certain embodiments, the present invention relates to a pharmaceutically acceptable diluent, carrier, or solubilizer. Along with emulsifiers, preservatives and / or adjuvants, the antigen-binding protein for PCSK9 and The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one further therapeutic agent.

[0285] In certain embodiments, the antigen-binding protein for PCSK9 is at least 1 It can be used in conjunction with anti-inflammatory agents. In certain embodiments, PCSK Antigen-binding proteins for 9 are used in conjunction with at least one immunotherapy agent. It is possible. Typical therapeutic agents for inflammatory and immune diseases include cyclooxygenase type 1 ( COX-1 and cyclooxygenase type 2 (COX-2) inhibitors, 38kDa cell division promoters Small molecule regulators of factor-activated protein kinase (p38-MAPK), involved in inflammatory pathways. Small molecule regulators of intracellular molecules (such intracellular molecules include jnk, IKK, NF) -κB, ZAP70, and lck are included, but are not limited to these. This is not limited to these. Some typical anti-inflammatory drugs include, for example, "CA.Dinar ello & LLMoldawer Proinflammatory and Anti-Inflammatory Cytokines in Rheumatoi d Arthritis:A Primer for Clinicians Thir d Edition (2001) Amgen Inc.Thousand Oaks It is stated in "CA".

[0286] In certain embodiments, the drug composition comprises two or more different antigens against PCSK9. Contains binding proteins. In certain embodiments, the pharmaceutical composition contains two or more epitopes It contains two or more antigen-binding proteins for PCSK9 that bind to it. Several embodiments In this context, various antigen-binding proteins compete with each other for binding to PCSK9. i. In some embodiments, antigen binding is shown in Table 2 and Figures 2 and / or 3. Any of these proteins can be combined together in a pharmaceutical composition.

[0287] In certain embodiments, acceptable formulation materials are preferably the dosage used and It is not toxic to the user at the concentration. In some embodiments, the formulation material is subcutaneous and It is for intravenous administration. In certain embodiments, the pharmaceutical composition is, for example, the pH of the composition. , osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release, adsorption or It may contain formulation materials for modifying, maintaining, or preserving the rate of penetration. In one embodiment, suitable formulation materials include amino acids (glycine, glutamine, asphalt). Paragine, arginine, or lysine; antibacterial agents; antioxidants (ascorbic acid, sodium sulfite, etc.); Thorium or sodium bisulfite), buffer (borate, bicarbonate, Tris -HCl, citrate, phosphate or other organic acids); bulking agent (mannitol or glycerin); (e.g.); chelating agents (ethylenediaminetetraacetic acid (EDTA), etc.); complexing agents (caffeine) Polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclo Rodextrin, etc.); fillers; monosaccharides; disaccharides; and other carbohydrates (glucose, mannor). (e.g., dextrin or syrup); protein (serum albumin, gelatin, or immunoglobulin) etc.); colorants, flavorings and diluents; emulsifiers; hydrophilic polymers (polyvinylpyrrolidone, etc.) (Low molecular weight polypeptides); salt-forming counterions (such as sodium); preservatives (benzalkonium chloride); Conium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, meth Parabens, propylparabens, chlorhexidine, sulfic acid, or hydrogen peroxide, etc.; Solvent (glycerin, propylene glycol, or polyethylene glycol, etc.); sugar alcohol (such as mannitol or sorbitol); suspending agents; surfactants or humectants (pluronic acid); Poly PEG, sorbitan ester, polysorbate 20, polysorbate 80, etc. Sorbate, Triton, Tromethamine, Lecithin, Cholesterol, Tyroxapal, etc. ); Stability enhancers (such as sucrose or sorbitol); Isotonic enhancers (alkali metal herbs) (Logenated compounds, preferably sodium chloride or potassium chloride, mannitol sorbitol, etc.) This includes, but is not limited to, delivery vehicles; diluents; excipients and / or pharmacokinetics. No. (Remington's Pharmaceutical Sciences) 18 th Edition, AR Gennaro, ed., Mack P Ublishing Company (1995). In several embodiments, formulation This includes PBS, 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM Contains NaOAC, pH 5.2, and 9% sucrose.

[0288] In certain embodiments, antigen-binding proteins and / or therapeutic components against PCSK9 The child is coupled to a half-life extension vehicle known in this field. Such a vehicle includes polyethylene glycol, glycogen (e.g., glycosylated ABP), and dextrin. This includes, but is not limited to, trans. Such vehicles are, for example, U.S. patents. Application No. 09 / 428,082 (currently U.S. Patent No. 6,660,843) and published PC T Application WO99 / 25044 (These are, for all purposes, by reference to this specification.) It is included in the book.

[0289] In certain embodiments, the optimal pharmaceutical composition is, for example, the intended route of administration, delivery. The form and desired dosage are determined by those skilled in the art. For example, "Remingto Please refer to n's Pharmaceutical Sciences, as mentioned above. In certain embodiments, such compositions control the physical state, stability, and other aspects of the antibody of the present invention. This may affect the rate of in vivo release and the rate of in vivo removal.

[0290] In certain embodiments, the main vehicle or carrier in the pharmaceutical composition is aqueous or non-aqueous. It can be a common substance in compositions for parenteral administration. For example, in certain embodiments, it is a common substance in compositions for parenteral administration. A suitable vehicle or carrier that can be refilled with other materials is suitable for use with water for injection, physiological saline. It may be a solution or artificial cerebrospinal fluid. In some embodiments, physiological saline is isotonic. Contains phosphate-buffered physiological saline. In certain embodiments, neutral buffered physiological saline is used. Physiological saline or saline mixed with serum albumin is used in further typical vehicles. Yes. In certain embodiments, the pharmaceutical composition is Tris saturates at a pH of approximately 7.0 to 8.5. The solution contains a buffer or an acetate buffer with a pH of approximately 4.0 to 5.5, which contains sorbitol or appropriate It may include substitutes thereof. In certain embodiments, a selected set having the desired purity The product is in the form of a freeze-dried cake or aqueous solution, and may be formulated with a compounding agent ( Rmington's Pharmaceutical Sciences (as above) and mixed By combining, PCSK may or may not include at least one additional therapeutic agent. A composition containing an antigen-binding protein for 9 can be prepared for storage. In certain embodiments, at least one further therapeutic agent is added or not added. A composition containing an antigen-binding protein for PCSK9 may contain an appropriate excipient such as sucrose. It can be formulated as a freeze-dried substance using an agent.

[0291] In certain embodiments, pharmaceutical compositions can be selected for parenteral delivery. In certain embodiments, the composition is intended for delivery through the gastrointestinal tract, such as by inhalation or oral ingestion. It may be selected. The preparation of such compositions that are pharmaceutically acceptable is within the scope of the skills of those skilled in the art. It belongs to the category.

[0292] In certain embodiments, the formulation component is present at the administration site in an acceptable concentration. In certain embodiments, the pH is adjusted to a physiological pH or slightly lower, typically, A buffer solution is used to maintain the composition within a pH range of approximately 5 to 8.

[0293] In certain embodiments where parenteral administration is envisioned, the therapeutic composition is PCSK9 is used in a pharmacopoeia-acceptable vehicle, with or without the addition of further therapeutic agents. A parenterally acceptable, nonpyrogenic aqueous solution containing the desired antigen-binding protein for a given antigen, in the form of a nonpyrogenic aqueous solution. It may be in a certain state. In certain embodiments, the parenteral injection vehicle is at least one With or without further therapeutic agents, the antigen-binding protein against PCSK9 is properly It is sterile distilled water formulated as a preservative-treated sterile isotonic solution. In certain embodiments In this preparation, the preparation consists of injectable microspheres, biocorrosable particles, and polymeric compounds (poly (Lactic acid or polyglycolic acid, etc.), beads or liposomes, etc., for sustained or continuous release of the product Add a drug that can deliver (the product may then be delivered via depot injection). The formulation may include the desired molecule obtained. In certain embodiments, hyaluronic acid It can also be used and may have the effect of promoting a sustained period in circulation. In one embodiment, an implantable drug delivery device is used to introduce a desired molecule. It can be used.

[0294] In certain embodiments, pharmaceutical compositions can be formulated for inhalation. In certain embodiments, PC may or may not include at least one further therapeutic agent. Antigen-binding proteins for SK9 can be formulated as a dry inhalation powder. In certain embodiments, at least one further therapeutic agent is added or not added. Inhalation solutions containing an antigen-binding protein for PCSK9 are used as propellants for aerosol delivery. It can be formulated together with other materials, and in certain embodiments, the solution can be sprayed. Transpulmonary administration describes the transpulmonary delivery of chemically modified proteins in the PCT application PCT / Further details are provided in US94 / 001875.

[0295] In certain embodiments, the formulation is intended to be administered orally. In the administration method, at least one additional therapeutic agent is added to the administration method. In addition, the antigen-binding protein for PCSK9 is not included in solid dosage forms such as tablets and capsules. It can be formulated with or without carriers commonly used in the formulation. Certain implementations In this state, the capsule maximizes bioavailability and minimizes pre-systemic degradation. The formulation can be designed to release its active portion at a specific point within the digestive tract when administered. In certain embodiments, an antigen-binding protein and / or any To further enhance the absorption of therapeutic agents, at least one additional drug may be included. In certain embodiments, diluents, fragrances, low-melting-point waxes, vegetable oils, lubricants, and suspensions are used. Disintegrants, tablet disintegrants, and binders may also be used.

[0296] In certain embodiments, the pharmaceutical composition is mixed with a non-toxic excipient suitable for the manufacture of tablets. Adding or not adding at least one further therapeutic agent to the substance provides an anti-PCSK9 agent. It may contain an effective amount of the proto-binding protein. In certain embodiments, sterile water or The solution is prepared in unit dosage form by dissolving the tablets in another suitable vehicle. This is possible. In certain embodiments, suitable excipients include calcium carbonate and sodium carbonate. Inert substances such as thorium or sodium bicarbonate, lactose or calcium phosphate. Diluents, or binders such as starch, gelatin or gum arabic, or stearin It contains, but is not limited to, lubricants such as magnesium oxide, stearic acid, or talc. It is not something that should be done.

[0297] In a sustained-release or controlled-delivery formulation, with or without the addition of at least one further therapeutic agent. Further pharmaceutical compositions, such as formulations containing an antigen-binding protein against PCSK9, are available to those skilled in the art. This is self-evident. In certain embodiments, liposome carriers, biodegradable microparticles or multi Techniques for formulating various other sustained or controlled delivery methods such as porous beads and depot injections. The technique is also known to those skilled in the art. For example, the sustained release of porous polymer microparticles for the delivery of pharmaceutical compositions. See PCT application PCT / US93 / 00829 for details. Certain embodiments In this case, the sustained-release preparation is in the form of a molded product, such as a film or microcapsules. It may contain a semipermeable polymer matrix. The sustained-release matrix may contain polyester Hydrogel, polylactide (US Patent No. 3,773,919 and EP058,48) 1) Copolymer of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al.) al., Biopolymers, 22:547-556 (1983), poly(2- Hydroxyethyl methacrylate) (Langer et al., J. Biomed .Mater.Res.,15:167-277(1981) and Langer, Che m.Tech., 12:98-105 (1982)), ethylene vinyl acetate (Lange r et al., above) and poly-D(-)-3-hydroxybutyrate (EP133,98 8) may include. In certain embodiments, the sustained-release composition may also include liposomes. This can be prepared by any method known in this art. For example, Epp stein et al.,Proc.Natl.Acad.Sci.USA,82:3 688-3692(1985);EP036,676;EP088,046 and EP14 See 3,949.

[0298] Pharmaceutical compositions to be used for in vivo administration are typically sterile. Morphologically, this can be achieved by filtration through a sterile filtration membrane. In the embodiment, when the composition is freeze-dried, sterilization using this method is freeze-dried. This can be carried out either before or after drying and reconstitution. In certain embodiments, Parenteral administration compositions may be stored in a lyophilized form or in solution. Certain implementations In terms of form, parenteral compositions are generally contained in containers with sterile access ports, for example or an intravenous solution bag or container having a stopper that can be inserted with a subcutaneous injection needle. It will be placed inside.

[0299] In certain embodiments, after the pharmaceutical composition is prepared, it is used to create a solution, suspension, gel, or emulsion. John, as a solid, or as a dehydrated or freeze-dried powder, is stored in a sterile container. It is possible. In certain embodiments, such formulations may be used in an immediate-use form or before administration. It can be preserved in any of the reconstructed forms (e.g., freeze-dried form).

[0300] In certain embodiments, a kit for manufacturing single-dose medication units is provided. In certain embodiments, the kit includes a first container having dried protein and water It may contain both a single and a second container having a sexual formulation. In certain embodiments, a single and / or pre-filled syringes with multiple chambers (e.g., liquid syringes and lyophilized syringes) The kit contains lyosyringe.

[0301] In certain embodiments, with or without adding at least one further therapeutic agent, A pharmaceutical composition containing an antigen-binding protein against PCSK9 should be used in the treatment of a medical ...

Claims

1. It binds to the PCSK9 protein containing the amino acid sequence of Sequence ID No. 1, and P against LDLR. An isolated neutralizing antigen-binding protein that reduces the LDLR-lowering effect of CSK9.

2. The claims wherein the antigen-binding protein is an LDLR-noncompetitive neutralizing antigen-binding protein. An isolated neutralizing antigen-binding protein according to any one of the claims of the surrounding antigen-binding protein.

3. Claims wherein the antigen-binding protein is an LDLR competitively neutralizing antigen-binding protein. An isolated neutralizing antigen-binding protein according to any one of the claims of the antigen-binding protein.

4. An antigen-binding protein that selectively binds to PCSK9, the antigen-binding protein K is less than 100 pM ｄ An antigen-binding protein that binds to PCSK9.

5. K is an antigen-binding protein with a concentration of less than 10 pM. ｄ Antigen binding as claimed, which binds by An antigen-binding protein according to any one of the protein claims.

6. K is an antigen-binding protein with a concentration of less than 5 pM. ｄ The antigen-binding agent of the claims is bound by An antigen-binding protein according to any one of the claims.

7. An isolated antigen-binding protein that binds to the PCSK9 protein of Sequence ID No.

1. The binding between the isolated antigen-binding protein and the variant PCSK9 protein is simple 50% of the binding between the detached antigen-binding protein and the PCSK9 protein of SEQ ID NO: 1 remains unresolved. A fully formed antigen-binding protein.

8. The variant PCSK9 protein is shown in SEQ ID NO: 1, 207, 208, 185, 1 ８１、４３９、５１３、５３８、５３９、１３２、３５１、３９０、４１３、５８２、１ 62, 164, 167, 123, 129, 311, 313, 337, 519, 521 and The claims include at least one mutation in a residue at a position selected from the group consisting of 554. An isolated human antigen-binding protein according to any one of the claims of the range of antigen-binding proteins.

9. At least one mutation is present in R207E, D208R, E181R, R185E, R439 E, E513R, V538R, E539R, T132R, S351R, A390R, A4 Antigen-binding protein of the claims, selected from the group consisting of 13R and E582R. An isolated human antigen-binding protein according to any one of the claims.

10. At least one mutation is D162R, R164E, E167R, S123R, E129 From R, A311R, D313R, D337R, R519E, H521R and Q554R Isolation of any one of the claims of the antigen-binding protein of the patent claims, selected from the group. A human antigen-binding protein.

11. The antigen-binding protein that binds to the PCSK9 protein of Sequence ID No. 303 in the first form ( The antigen-binding protein binds to a variant of PCSK9 in a second manner, and the PCSK The 9 variants are 207, 208, 185, 181, 439, 513 of sequence number 303. ５３８、５３９、１３２、３５１、３９０、４１３、５８２、１６２、１６４、１６７、 Select from the group consisting of 123, 129, 311, 313, 337, 519, 521, and 554. The selected position has at least one point variation, and the first form is the first EC50, first The Bmax or the first EC50 and the first Bmax, the second form is the second EC 50, including the second Bmax or the second EC50 and the second Bmax, and the first form The value for this is different from the value for the second form.

12. The first form includes the first EC50, the second form includes the second EC50, and the dot variation Different models: R207E, D208R, E181R, R185E, R439E, E513R, V5 38R, E539R, T132R, S351R, A390R, A413R and E582R An antigen-binding protein of any one claim of the claims, selected from the group consisting of the following: Antigen-binding protein.

13. The antigen of the claims wherein the first EC50 differs from the second EC50 by at least 20%. The antigen-binding protein according to any one of the claims for the combined protein.

14. The antigen of the claims wherein the first EC50 differs from the second EC50 by at least 50%. The antigen-binding protein according to any one of the claims for the combined protein.

15. The second EC50 is greater than the first EC50, according to the claims, antigen-binding An antigen-binding protein according to any one of the claims.

16. The first EC50 is measured by a multi-bead binding assay, according to the claims antigen. Antigen-binding protein according to any one of the claims.

17. The second EC50 is greater than 1 μM, which of the claims is an antigen-binding protein? One of the antigen-binding proteins.

18. The antigen-binding protein is a neutralizing antigen-binding protein, according to the claims. An antigen-binding protein according to any one of the protein claims.

19. The claims relating to an anti-antigen binding protein, wherein the neutralizing antigen-binding protein is a competitive neutralizing antigen-binding protein. The antigen-binding protein according to any one of the claims.

20. The claims state that the neutralizing antigen-binding protein is a non-competitive neutralizing antigen-binding protein. Antigen-binding protein The antigen-binding protein according to any one of the claims.

21. The first form includes the first Bmax, and the second form includes a second Bm that is different from the first Bmax. ax is included, and the PCSK9 variants are D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521 A patent claim having at least one point mutation selected from the group consisting of R and Q554R. An antigen-binding protein in the range of any one of the claims.

22. The antigen-binding protein of the claims, wherein the second Bmax is approximately 10% of the first Bmax. An antigen-binding protein according to any one of the claims.

23. The antigenicity of the claims wherein the first Bmax differs from the second Bmax by at least 20%. The antigen-binding protein according to any one of the claims for the combined protein.

24. The antigenicity of the claims wherein the first Bmax differs from the second Bmax by at least 50%. The antigen-binding protein according to any one of the claims for the combined protein.

25. At a position that overlaps with the position where LDLR is coupled to PCSK9, the single unit that is coupled to PCSK9 Detached antibodies.

26. PCSK9 binds to and acts as the low-density lipoprotein receptor for PCSK9 against LDLR. A neutralizing antibody that reduces the LDLR-lowering effect.

27. PCSK9 binds to PCSK9 at positions within residues 31 to 447 of SEQ ID NO:

3. A neutralizing antibody that binds to the antibody.

28. The antibody according to claim 27, which binds to the epitopes in residues 31 to 447 of SEQ ID NO:

3. 。

29. At least one antigen-binding protein and pharmaceutical according to any one of claims 1 to 28 A pharmaceutical composition containing an excipient that is acceptable as such.

30. A nucleic acid molecule encoding an antigen-binding protein according to any one of claims 1 to 28.

31. Preparing host cells containing nucleic acid sequences encoding antigen-binding proteins; and Maintaining host cells under conditions that allow antigen-binding proteins to be expressed; An antigen-binding tag that binds to the PCSK9 protein containing the amino acid sequence of SEQ ID NO: 1, including the amino acid sequence of SEQ ID NO:

1. Protein (This antigen-binding protein reduces the LDLR-reducing effect of PCSK9 on LDLR) A method for producing (to cause).

32. Isolated neutralizing antigen that binds to the PCSK9 protein containing the amino acid sequence of SEQ ID NO: 1 This includes administering an effective amount of the binding protein to a target, wherein the neutralizing antigen-binding protein is L To reduce the LDLR-lowering effect of PCSK9 on DLR, the serum cholesterol levels of the subjects decreased. Methods to lower the level.

33. For patients who require treatment or prevention of symptoms accompanied by elevated serum cholesterol levels Isolated neutralizing antigen binds to the PCSK9 protein containing the amino acids of SEQ ID NO:

1. This includes administering an effective amount of protein (the neutralizing antigen-binding protein is applied to LDLR). It reduces the LDLR-lowering effect of PCSK9, and the elevated serum cholesterol levels in patients. Methods for treating or preventing symptoms accompanied by a roll level.

34. For individuals requiring treatment or prevention of symptoms accompanied by elevated serum cholesterol levels Simultaneously or sequentially with drugs that increase the availability of LDLR protein, isolated This includes administering an effective amount of the antigen-binding protein (the isolated antigen-binding protein) The substance binds to the PCSK9 protein containing the amino acid sequence of SEQ ID NO: 1, and the neutralizing antigen binds to the PCSK9 protein. The combined protein reduces the LDLR-reducing effect of PCSK9 on LDLR. A method for treating or preventing symptoms associated with elevated serum cholesterol levels in elephants.

35. Claims 31 to 31 include a statin as a drug that increases the availability of LDLR protein. The method of item 34.

36. Statins include atrovastatin, cerivastatin, fluvastatin, lovastatin, and mevastatin. Statins, pitavastatin, pravastatin, rosuvastatin, simvastatin and this A method according to one of claims 31 to 35, selected from a group consisting of several combinations thereof.

37. Antigens according to claims 1 to 22 in the preparation of a pharmaceutical for lowering serum cholesterol Use of binding proteins.

38. A medicine for treating or preventing symptoms accompanied by elevated serum cholesterol levels in the target population. Use of antigen-binding proteins according to claims 1 to 22 in the preparation of [the product].

Images