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Anti-pcsk9 antibodies with ph-dependent binding characteristics

A technology of antibodies and binding fragments, applied in the direction of antibodies, specific peptides, drug combinations, etc.

Active Publication Date: 2015-04-22
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, if the antibody can only bind and neutralize one antigen before it is targeted for degradation in the host, relatively large quantities of the antibody must be administered to be therapeutically effective, and / or the antibody must be administered relatively frequently

Method used

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  • Anti-pcsk9 antibodies with ph-dependent binding characteristics
  • Anti-pcsk9 antibodies with ph-dependent binding characteristics
  • Anti-pcsk9 antibodies with ph-dependent binding characteristics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1. Production of human antibodies against human PCSK9

[0146] Human anti-PCSK9 antibodies were produced as described in US Patent No. 8,062,640. Table 1 lists the sequence identifiers of the heavy chain and light chain variable region amino acid sequence pairs and CDR amino acid sequences of selected anti-PCSK9 antibodies, and their corresponding antibody names. Nucleotide sequences are given with odd sequence identifiers corresponding to even sequence identifiers in Table 1. For example, SEQ ID NO: 1 is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2; SEQ ID NO: 3 is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4; and so on.

[0147] Table 1: Amino Acid Sequence Identifiers of Selected Anti-PCSK9 Antibodies

[0148]

[0149]

[0150]

[0151]

[0152] Any of the anti-PCSK9 antibodies listed in Table 1 (with the amino acid sequences of their corresponding heavy and chain variable regions and / or CD...

Embodiment 2

[0153] Example 2. Construction of histidine substitution mutants of human anti-PCSK9 antibodies

[0154] The anti-PCSK9 antibody designated 300N is known to have intermediate pH-dependent binding properties with decreased binding affinity for PCSK9 at acidic pH, and enhanced pharmacokinetics (see US Patent No. 8,062,640). In order to produce antibodies with even greater pH-dependent binding properties (i.e., binding decreases at low pH compared to neutral pH) and improved in vivo potency (e.g., longer antibody serum half-life, prolonged cholesterol-lowering activity, etc.) ) of the 300N variant to construct a series of variant antibodies. Specifically, a mutated version of 300N was constructed in which each amino acid within the complementarity determining region (CDR) of 300N was individually mutated to histidine. As shown in Table 1, the heavy chain variable region (HCVR) of the parental 300N antibody comprises the amino acid sequence of SEQ ID NO: 218, and the light chain ...

Embodiment 3A

[0162] Example 3A. Binding properties of variant anti-PCSK9 antibodies at neutral and acidic pH

[0163] The histidine-substituted variant antibody of Example 2 was tested for pH-dependent binding to human PCSK9 using a real-time plasmon resonance biosensor (Biacore T200) assay at 25°C, pH 5.75 or pH 7.2. The purpose of this experiment was to identify histidine substituted variant antibodies that exhibit reduced human PCSK9 binding at acidic pH relative to neutral pH.

[0164] The Biacore CM4 sensor chip is derivatized with a monoclonal mouse anti-human Fc antibody to capture human antibodies. After transient expression in Chinese hamster ovary (CHO) cells, histidine-substituted variant anti-PCSK9 antibodies from culture medium were captured on the surface of anti-human Fc sensors. Human PCSK9 (SEQ ID NO: 755) with a C-terminal myc-myc-hexahistidine tag (hPCSK9-mmH) at different concentrations ranging from 3.125 nM to 500 nM was mixed at a flow rate of 50 μl / min Injection wa...

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Abstract

The present invention provides antibodies and antigen-binding fragments thereof that specifically bind proprotein convertase subtilisin / kexin-9 (PCSK9) with greater affinity at neutral pH than at acidic pH. The antibodies of the invention may possess one or more amino acid changes as compared to antibodies that do not exhibit pH-dependent binding properties. For example, the present invention includes anti-PCSK9 antibodies which possess one or more histidine substitutions in one or more complementarity determining regions. The antibodies of the invention, with pH-dependent binding properties, remain in circulation and exhibit cholesterol lowering activity for prolonged periods of time in animal subjects as compared to anti-PCSK9 antibodies that do not exhibit pH-dependent binding properties. The antibodies of the invention are therefore useful for treating diseases and disorders related to elevated HDL cholesterol, wherein the antibodies of the invention can be administered to a patient at a lower dose and / or with less frequent dosing as compared to antibodies that do not exhibit pH-dependent binding properties.

Description

technical field [0001] The present invention relates to antigen binding molecules that specifically interact with proprotein convertase subtilisin / kexin type 9 (PCSK9) and the use of such molecules in the treatment of hypercholesterolemia and other related disorders characterized by elevated cholesterol levels. Background technique [0002] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a proprotein convertase belonging to the proteinase K subfamily of the secretory subtilase family. The encoded protein is synthesized as a soluble zymogen, which undergoes autocatalytic intramolecular processing in the endoplasmic reticulum. Circulating PCSK9 binds to and targets the low-density lipoprotein receptor (LDLR) on the surface of liver cells for destruction. This process reduces the liver's ability to bind and remove LDL cholesterol (LDL-C), thus causing LDL-C levels to increase. Antibodies that specifically bind PCSK9 and block its interaction with the LDL receptor hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40A61K39/395A61P3/06
CPCC07K2317/565C07K2317/21C07K2317/92C07K16/40C07K2317/94A61K2039/505C07K2317/76A61P3/06
Inventor G·D·扬科普洛斯N·J·帕帕多普洛斯A·J·墨菲N·斯塔尔
Owner REGENERON PHARM INC
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