Compound targeting il-23a and tnf-alpha and uses thereof

Abstract

The disclosure relates to compounds specific for IL23A and TNF-alpha, compositions comprising the compounds, and methods of use thereof. Nucleic acids, cells, and methods of production related to the compounds and compositions are also disclosed.

1 - 35 . (canceled) 
     
     
         36 . A method of purifying a bispecific compound comprising a first polypeptide and a second polypeptide, which bispecific compound binds to a first and a second target protein, which target proteins comprise TNF-alpha and IL-23A, wherein:
 (A) said first polypeptide of the bispecific compound comprises:
 (i) a light chain variable domain of a first immunoglobulin (VL1) specific for a first target protein; 
 (ii) a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second target protein; and 
 (iii) a hinge region, a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3); and 
   (B) said second polypeptide of the bispecific compound comprises:
 (i) a light chain variable domain of the second immunoglobulin (VL2) specific for said second target protein; and 
 (ii) a heavy chain variable domain of the first immunoglobulin (VH1) specific for said first target protein; 
   said method comprising the following steps:
 (i) applying a composition comprising said bispecific compound to a protein A cross-linked agarose matrix; 
 (ii) eluting the bispecific compound from the matrix using an sodium acetate buffer; 
 (iii) neutralizing the resultant sample; 
 (iv) applying the neutralized sample comprising the bispecific compound to a hydroxyapatite resin; and 
 (v) recovering the purified bispecific compound by eluting from the hydroxyapatite resin using one or more phosphate buffers. 
   
     
     
         37 . The method of  claim 36 , wherein the sodium acetate buffer is pH 3.5. 
     
     
         38 . The method of  claim 36 , which does not include any high salt washes. 
     
     
         39 . The method of  claim 36 , wherein the one or more phosphate buffer compositions include a ≈140 nM NaPhosphate 100 nM NaCl pH 7.0 and/or a ≈200 nM NaPhosphate 100 nM NaCl pH 7.0. 
     
     
         40 . The method of  claim 36 , wherein the hydroxyapatite elution results in a composition comprising >95% monomer. 
     
     
         41 . The method of  claim 36 , wherein the bispecific compound comprises a first polypeptide and a second polypeptide, wherein:
 (A) said first polypeptide comprises:
 (i) a light chain variable domain of a first immunoglobulin (VL1) specific for a first target protein; 
 (ii) a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second target protein; and 
 (iii) a hinge region, a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3); and 
   (B) said second polypeptide comprises:
 (i) a light chain variable domain of the second immunoglobulin (VL2) specific for said second target protein; 
 (ii) a heavy chain variable domain of the first immunoglobulin (VH1) specific for said first target protein; 
   (C), in said first and second polypeptides of (A) and (B):
 (i) said VL1 and VH1 associate to form a binding site that binds said first target protein; 
 (ii) said VL2 and VH2 associate to form a binding site that binds said second target protein; 
 (iii) said heavy chain constant region 2 (CH2) comprises a tyrosine at position 252, a threonine at position 254 and a glutamic acid a position 256, numbered according to the EU index as in Kabat for the CH2 of a conventional antibody; and 
 (iv) said first target protein is TNF-alpha and said second target protein is IL-23A or said first target protein is IL-23A and said second target protein is TNF-alpha, 
   and (D), in said first and second polypeptides of (A), (B) and (C):
 (i) said VL1 comprises the amino acid sequence of SEQ ID NO:2, said VH1 comprises the amino acid sequence of SEQ ID NO:1, said VL2 comprises the amino acid sequence of SEQ ID NO:8 and said VH2 comprises the amino acid sequence of SEQ ID NO:7; or 
 (ii) said VL1 comprises the amino acid sequence of SEQ ID NO:4 or 6, said VH1 comprises the amino acid sequence of SEQ ID NO:3 or 5, said VL2 comprises the amino acid sequence of SEQ ID NO:8 and said VH2 comprises the amino acid sequence of SEQ ID NO:7; or 
 (iii) said VL1 comprises the amino acid sequence of SEQ ID NO:8, said VH1 comprises the amino acid sequence of SEQ ID NO:7, said VL2 comprises the amino acid sequence of SEQ ID NO:2 and said VH2 comprises the amino acid sequence of SEQ ID NO:1; or 
 (iv) said VL1 comprises the amino acid sequence of SEQ ID NO:8, said VH1 comprises the amino acid sequence of SEQ ID NO:7, said VL2 comprises the amino acid sequence of SEQ ID NO:4 or 6 and said VH2 comprises the amino acid sequence of SEQ ID NO:3 or 5. 
   
     
     
         42 . The method of  claim 41 , wherein in (D) (ii) said VL1 comprises the amino acid sequence of SEQ ID NO:4, said VH1 comprises the amino acid sequence of SEQ ID NO:3, said VL2 comprises the amino acid sequence of SEQ ID NO:8 and said VH2 comprises the amino acid sequence of SEQ ID NO:7. 
     
     
         43 . The method of  claim 41 , wherein in (D) (ii) said VL1 comprises the amino acid sequence of SEQ ID NO:6, said VH1 comprises the amino acid sequence of SEQ ID NO:5, said VL2 comprises the amino acid sequence of SEQ ID NO:8 and said VH2 comprises the amino acid sequence of SEQ ID NO:7. 
     
     
         44 . The method of  claim 41 , wherein in (D) (iv) said VL2 comprises the amino acid sequence of SEQ ID NO:4, said VH2 comprises the amino acid sequence of SEQ ID NO:3, said VL1 comprises the amino acid sequence of SEQ ID NO:8 and said VH1 comprises the amino acid sequence of SEQ ID NO:7. 
     
     
         45 . The method of  claim 41 , wherein in (D) (iv) said VL2 comprises the amino acid sequence of SEQ ID NO:6, said VH2 comprises the amino acid sequence of SEQ ID NO:5, said VL1 comprises the amino acid sequence of SEQ ID NO:8 and said VH1 comprises the amino acid sequence of SEQ ID NO:7. 
     
     
         46 . The method of  claim 41 , wherein in said first polypeptide further comprises a first linker between said VL1 and said VH2 and said second polypeptide further comprises a second linker between said VL2 and said VH1, wherein optionally said first linker and/or said second linker comprises the amino acid sequence of GGGSGGG (SEQ ID NO:9). 
     
     
         47 . The method of  claim 41 , wherein said first polypeptide further comprises a heavy chain constant region 1 domain (CH1) and said second polypeptide further comprises a light chain constant region domain (CL), wherein said CL and said CH1 are associated together via a disulfide bond to form a C1 domain. 
     
     
         48 . The method of  claim 46 , wherein said first polypeptide further comprises a third linker between said VH2 and said CH1 and said second polypeptide further comprises a fourth linker between said VH1 and said CL, wherein optionally said third linker comprises the amino acid sequence of FNRGES (SEQ ID NO:11) and/or said fourth linker comprises the amino acid sequence of VEPKSS (SEQ ID NO:12) or said third linker and/or said fourth linker comprises the amino acid sequence of LGGGSG (SEQ ID NO:10). 
     
     
         49 . The method of  claim 41 , wherein said heavy chain constant region 2 (CH2) of (C) (iii) comprises an alanine at positions 234 and an alanine at position 235, numbered according to the EU index as in Kabat for the CH2 of a conventional antibody. 
     
     
         50 . The method of  claim 41 , wherein said the amino acid sequence of said hinge region, said heavy chain constant region 2 (CH2) or said heavy chain constant region 3 (CH3) is derived from a IgG1 or from a IgG4, optionally wherein said hinge region comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO:40). 
     
     
         51 . The method of  claim 41 , wherein said compound comprises two said first polypeptides and two said second polypeptides, wherein said two first polypeptides are associated together via at least one disulfide bond. 
     
     
         52 . The method of  claim 41 , wherein:
 (i) said first polypeptide comprises the amino acid sequence of SEQ ID NO:13 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:14;   (ii) said first polypeptide comprises the amino acid sequence of SEQ ID NO:15 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:16;   (iii) said first polypeptide comprises the amino acid sequence of SEQ ID NO:17 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:18;   (iv) said first polypeptide comprises the amino acid sequence of SEQ ID NO:19 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:20;   (v) said first polypeptide comprises the amino acid sequence of SEQ ID NO:21 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:22;   (vi) said first polypeptide comprises the amino acid sequence of SEQ ID NO:23 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:24;   (vii) said first polypeptide comprises the amino acid sequence of SEQ ID NO:25 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:26;   (viii) said first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:28;   (ix) said first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:30;   (x) said first polypeptide comprises the amino acid sequence of SEQ ID NO:31 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:32;   (xi) said first polypeptide comprises the amino acid sequence of SEQ ID NO:33 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:34; or   (xii) said first polypeptide comprises the amino acid sequence of SEQ ID NO:35 and said second polypeptide comprises the amino acid sequence of SEQ ID NO:36.   
     
     
         53 . A composition comprising a purified bispecific compound obtained by the method of  claim 36 . 
     
     
         54 . A composition comprising a purified bispecific compound obtained by the method of  claim 41 .