Anti-PCSK9 antibodies with pH-dependent binding characteristics

• US 9,540,449 B2
• Filed: 08/12/2013
• Issued: 01/10/2017
• Est. Priority Date: 08/13/2012
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Yancopoulos, George D., Papadopoulos, Nicholas J., Murphy, Andrew J., Stahl, Neil
• Primary Examiner(s): WEN, SHARON X
• Application Number:
  US13/964,159
• Publication Number:
  US 20140044730A1
• Time in Patent Office: 1,247 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance

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.

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds human proprotein convertase subtilisin/kexin type 9 (PCSK9), wherein the acidic/neutral K_D ratio for the antibody or antigen-binding fragment binding to PCSK9 at 25°

C. is greater than about 12.5 as determined by surface plasmon resonance;

wherein the antibody or antigen-binding fragment thereof comprises 3 heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and 3 light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3),

wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO;

220;

wherein the HCDR2 comprises the amino acid sequence of SEQ ID NO;

222;

wherein the HCDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NOs;

224 and 788;

wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO;

802;

wherein the LCDR2 comprises the amino acid sequence of SEQ ID NO;

230; and

wherein the LCDR3 comprises the amino acid sequence of SEQ ID NO;

232.

2. The isolated antibody or antigen-binding fragment of claim 1, wherein the acidic/neutral k_d ratio for the antibody or antigen-binding fragment binding to PCSK9 at 25°

C. is greater than about 7.5 as determined by surface plasmon resonance.

3. The isolated antibody or antigen-binding fragment of claim 1, wherein the acidic/neutral t1/2 ratio for the antibody or antigen-binding fragment binding to PCSK9 at 25°

C. is less than about 0.14 as determined by surface plasmon resonance.

4. The isolated antibody or antigen-binding fragment of claim 3, wherein the antibody or antigen-binding fragment thereof binds human proprotein convertase subtilisinfkexin type 9 (PCSK9) at 25°

C. and acidic pH with a dissociative half-life (t1/2) less than about 4.5 minutes, wherein the antibody or antigen-binding fragment thereof binds PCSK9 at 25°

C. and neutral pH with a t1/2 of greater than about 35 minutes.

5. The isolated antibody or antigen-binding fragment of claim 4, wherein the antibody or antigen-binding fragment thereof binds PCSK9 at 25°

C. and acidic pH with a dissociative half-life (t1/2) less than about 2 minutes, wherein the antibody or antigen-binding fragment thereof binds PCSK9 at 25°

C. and neutral pH with a t1/2 of greater than about 35 minutes.

6. The isolated antibody or antigen-binding fragment of claim 5, wherein the antibody or antigen-binding fragment thereof binds PCSK9 at 25°

C. and acidic pH with a dissociative half-life (t1/2) less than about 1.5 minutes, wherein the antibody or antigen-binding fragment thereof binds PCSK9 at 25°

C. and neutral pH with a t1/2 of greater than about 35 minutes.

7. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof blocks the interaction between human proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low density lipoprotein receptor (LDLR) at neutral pH with an IC₅₀ that is at least 36 times less than the PCSK9/LDLR blocking IC₅₀ value of the antibody or antigen-binding fragment thereof at acidic pH.

8. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof, when administered to a subject in a single dose of about 10 mg/kg, reduces serum LDL-C by at least 33% from baseline, and wherein the reduction in serum LDL-C is sustained for at least 26 days after administration.

9. The isolated antibody or antigen-binding fragment of claim 8, wherein the antibody or antigen-binding fragment thereof, when administered to a subject in a single dose of about 10 mg/kg, reduces serum LDL-C by at least 33% from baseline, and wherein the reduction in serum LDL-C is sustained for at least 33 days after administration.

10. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof, when administered to a subject in a single dose of about 10 mg/kg, reduces serum LDL-C by at least 15% from baseline, and wherein the reduction in serum LDL-C is sustained for at least 42 days after administration.

11. The isolated antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment thereof, when administered to a subject in a single dose of about 10 mg/kg, reduces serum LDL-C by at least 15% from baseline, and wherein the reduction in serum LDL-C is sustained for at least 55 days after administration.

12. The isolated antibody or antigen-binding fragment of claim 1, wherein the HCDR3 comprises the amino acid sequence of SEQ ID NO:

224.

13. The isolated antibody or antigen-binding fragment of claim 1, wherein the HCDR3 comprises the amino acid sequence of SEQ ID NO:

788.