ANTIBODIES WITH pH DEPENDENT ANTIGEN BINDING

Abstract

The present invention relates to antibodies with pH dependent binding to its antigen such that the affinity for antigen binding at physiological pH (i.e., pH 7.4) is greater than at endosomal pH (i.e., pH 6.0 or 5.5). In other words, the K D or k off ratio at pH 5.5/pH 7.4 or at pH 6.0/pH 7.4 is more than, or ranges between, 2, 3, 4, 8, 10, 16, 20, 30, 40, or 100 or more. Such pH dependent antibodies preferentially dissociate from the antigen in the endosome. This can increase antibody half life, as compared to antibodies with equivalent K D s at pH 7.4 but with no pH dependent binding, when the antigen is one that undergoes antigen-mediated clearance (e.g., PCSK9). Antibodies with pH dependent binding can decrease total antigen half life when the antigen undergoes reduced clearance when bound to antibody (e.g., IL6). Antibodies with pH dependent binding can also prolong the decrease in antigen which is not antibody-bound. This can be important when antagonizing a target antigen typically present at high levels (e.g., IgE, DKK1, C5 and SOST). In addition, such antibodies can increase antigen half life when the antigen is a receptor and the receptor has increased clearance when bound to antibody (e.g, GMCSF receptor).

It is claimed: 
     
         1 . An antibody with pH dependent binding which specifically binds an antigen with higher affinity at pH 7.4 than at pH 6.0, wherein the K D  ratio and/or the k off  ratio at pH 6.0/pH 7.4 and at 25° C. is more than, or ranges between, 2, 3, 4, 8, 10, 16, or more, and wherein:
 (1) the antibody with pH dependent binding has reduced plasma clearance in vivo when exposed to said antigen; 
 (2) the antigen is both membrane bound and soluble in vivo and wherein the antibody with pH dependent binding mediates increased localization to the cell membrane receptor; 
 (3) the amount of in vivo antigen not bound to the antibody with pH dependent binding is decreased for a prolonged time; 
 (4) the amount of in vivo antigen bound to antibody with pH dependent binding is decreased; and/or 
 (5) the antigen is a receptor and the receptor has reduced clearance in vivo when exposed to said antibody with pH dependent binding; 
 as compared to an antibody without pH dependent binding that has a similar affinity for the antigen at pH 7.4, but has a comparable K D  and/or k off  ratio at pH 6.0/pH 7.4 and at 25° C. that is less than 2. 
 
     
     
         2 . The antibody of  claim 1 , wherein the antigen is not the interleukin-6 receptor. 
     
     
         3 . The antibody of  claim 1 , wherein said antibody with pH dependent binding is an antibody drug conjugate, mediates antibody dependent cell-mediated cytotoxicity (ADCC), and/or mediates complement-dependent cytotoxicity (CDC). 
     
     
         4 . The antibody of  claim 1 , wherein pH dependent binding of the antibody has the K D  ratio or k off  ratio at pH 6.0/pH 7.4 and at 25° C. which is more than, or ranges between, 20, 30, 40 or 100 or more. 
     
     
         5 . The antibody of  claim 1 , wherein the pH dependent binding of the antibody to the antigen at pH 7.4 and at 25° C. has a K D  of about 0.01 nM to about 100 nM. 
     
     
         6 . The antibody of  claim 5 , wherein the pH dependent binding of the antibody to the antigen at pH 7.4 and at 25° C. has a K D  of about 0.1 nM to about 10 nM. 
     
     
         7 . The antibody of  claim 1 , wherein the pH dependent binding of the antibody to the antigen at pH 7.4 and at 25° C. has a k off  of about 1×10E-4 s-1 to about 1×10E-1 s-1. 
     
     
         8 . The antibody of  claim 7 , wherein the pH dependent binding of the antibody to the antigen at pH 7.4 and at 25° C. has a k off  of about 1×10E-3 s-1 to about 1×10E-1 s-1. 
     
     
         9 . The antibody of  claim 1 , wherein the antigen is PCSK9. 
     
     
         10 . The antibody of  claim 1 , wherein the antigen is IgE, C5, or DKK1. 
     
     
         11 . The antibody of  claim 10 , wherein the pH dependent binding of the antibody to the antigen at pH 7.4 and at 25° C. has a K D  ranging between 1.0 nM to about 10 nM or between 1.0 nM to about 100 nM. 
     
     
         12 . A method of extending interval dosing and/or decreasing the therapeutic dose for treating a patient with a therapeutic antibody, said method comprising administering to the patient a therapeutically effective amount of the antibody  claim 1 , wherein said antibody has an extended pharmacodynamic effect and/or half life as compared to an antibody that has similar affinity at pH 7.4, and at 25° C., but has a comparable K D  ratio and/or k off  ratio at pH 6.0/7.4 and at 25° C. that is less than 2. 
     
     
         13 . A method of making an antibody with prolonged half-life and/or pharmacodynamic effect by regulating antibody binding affinity in a pH dependent manner, said method comprising selecting for antibody CDR histidine residues or other residues that optimize the microenvironment affecting pKa, such that antibody antigen binding has a K D  ratio and/or k off  ratio at pH 6.0/pH 7.4 and at 25° C. that is more than, or ranges between, 2, 3, 4, 8, 10, 16, or more. 
     
     
         14 . An antibody library enriched for histidines in CDR residues or other residues that optimize the microenvironment affecting pKa. 
     
     
         15 . The method of  claim 13 , further comprising mutagenizing the antibody to achieve antibody affinity with a K D  at pH 7.4 and at 25° C. of at least 100 nM. 
     
     
         16 . An isolated antibody which specifically binds to human PCSK9 and comprises a heavy chain variable region (VH) complementary determining region one (CDR1), a VH CDR2, and a VH CDR3 from the VH amino acid sequence shown in SEQ ID NO: 4 or 5. 
     
     
         17 . The isolated antibody of  claim 16 , further comprising the light chain variable region (VL) CDR1, CDR2, and CDR3 of the VL amino acid sequence shown in SEQ ID NO: 3. 
     
     
         18 . The isolated antibody of  claim 17 , wherein the VH CDR1 has the amino acid sequence shown in SEQ ID NO: 6, the VH CDR2 has the amino acid sequence shown in SEQ ID NO: 7, the VH CDR3 has the amino acid sequence shown in SEQ ID NO: 9, the VL CDR1 has the amino acid sequence shown in SEQ ID NO: 10, the VL CDR2 has the amino acid sequence shown in SEQ ID NO: 11, and the VL CDR3 has the amino acid sequence shown in SEQ ID NO: 12. 
     
     
         19 . The isolated antibody of  claim 18 , wherein the VH region comprises SEQ ID NO: 5 and the VL region comprises SEQ ID NO: 3. 
     
     
         20 . An antibody or antigen-binding portion thereof, encoded by the plasmid deposited at the ATCC and having ATCC Accession No. PTA-10547, or PTA-10548, and/or PTA-10549. 
     
     
         21 . A host cell that recombinantly produces the antibody of  claim 16 . 
     
     
         22 . An isolated nucleic acid encoding the antibody or antigen-binding portion thereof of  claim 16  or as deposited at the ATCC and having ATCC Accession No. PTA-10547, PTA-10548, and/or PTA-10549. 
     
     
         23 . A method for reducing a level of LDL-cholesterol in blood of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody of  claim 16 .