CTLA-4 Variants

Abstract

Variants of cytotoxic T-lymphocyte antigen 4 (CTLA-4) with high affinity, potency and stability. Formulations of CTLA-4 variants at high concentration for subcutaneous or intravenous administration, e.g. at monthly or less frequent dosage intervals. Use of CTLA-4 variants for treating rheumatoid arthritis and other inflammatory disorders. Fusion of CTLA-4 with IgG Fc having improved stability and longer in vivo half-life.

1 . An isolated CTLA-4 polypeptide having greater affinity for binding human CD80, greater potency and/or greater stability compared with wild type CTLA-4 SEQ ID NO: 35, the polypeptide comprising an amino acid sequence that is a variant of SEQ ID NO: 35, wherein the variant comprises five or more of the following amino acid mutations in SEQ ID NO: 35:
 R, S, V or T at I16;   T at A24;   N or P at S25;   S at G27;   I at V 32;   G at D41;   G at S42;   E at V44;   K at M54;   S or G at N56;   A, G, S or P at L58;   S or A at T59;   T at F 60;   Q or P at L61;   G at D 62;   Y at D63;   P at S 64;   N, D, V or T at I65;   A, T, M or H at S70;   R at Q80;   Q, S, V, R, K or L at M85;   S at T87;   Q, H, T, E or M at K93;   R, Q or E at L104;   V at I106;   D or S at N108;   V or F at I115;   S at C120;   deletion at T51.   
     
     
         2 .- 49 . (canceled) 
     
     
         50 . A method of producing a further CTLA-4 polypeptide by mutation of a CTLA-4 polypeptide amino acid sequence selected from SEQ ID NOS 36-55 or the CTLA-4 amino acid sequence encoded by nucleic acid deposited under NCIMB accession no. 41948, the method comprising:
 providing a CTLA-4 polypeptide comprising or consisting of amino acid sequence SEQ ID NOS 36-55 or the CTLA-4 amino acid sequence encoded by nucleic acid deposited under NCIMB accession no. 41948;   introducing one or more mutations in the amino acid sequence to provide a further CTLA-4 polypeptide;   testing stability, affinity and/or potency of the further CTLA-4 polypeptide; and   formulating the further CTLA-4 polypeptide into a composition comprising one or more pharmaceutical excipients.   
     
     
         51 . (canceled) 
     
     
         52 . A method of inhibiting T lymphocyte activation comprising providing a CTLA-4 polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 43, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 36, SEQ ID NO: 42, or SEQ ID NO: 47. 
     
     
         53 . A method of reducing or inhibiting IL-2 secretion comprising providing a CTLA-4 polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 43, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 36, SEQ ID NO: 42, or SEQ ID NO: 47. 
     
     
         54 . A method of inhibiting CD4+ lymphocyte proliferation comprising providing a CTLA-4 polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 43, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 36, SEQ ID NO: 42, or SEQ ID NO: 47. 
     
     
         55 . A method of binding CD80 and CD86 comprising providing a CTLA-4 polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 43, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 36, SEQ ID NO: 42, or SEQ ID NO: 47. 
     
     
         56 . The method of  claim 52 , wherein the CTLA-4 polypeptide has greater affinity for binding CD86 than wild type CTLA-4 (SEQ ID NO: 35). 
     
     
         57 . The method of  claim 52 , wherein the CTLA-4 polypeptide is conjugated to an IgG Fc amino acid sequence. 
     
     
         58 . The method of  claim 52 , wherein the IgG Fc is a human IgG1 Fc modified to reduce Fc effector function, and comprises a native human IgG1 Fc hinge region. 
     
     
         59 . The method of  claim 52 , wherein the IgG Fc amino acid sequence comprises SEQ ID NO: 59. 
     
     
         60 . The method of  claim 52 , wherein the IgG Fc amino acid sequence comprises a human IgG1 Fc region in which one or both of the following groups of residues are substituted as follows:
 F at residue 20, E at residue 21, S at residue 117; and   Y at residue 38, T at residue 40, E at residue 42,   
       the residue numbering being defined with reference to SEQ ID NO: 56. 
     
     
         61 . The method of  claim 52 , wherein the CTLA-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 68. 
     
     
         62 . The method of  claim 52 , wherein the CTLA-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 43. 
     
     
         63 . The method of  claim 52 , wherein the CTLA-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 37. 
     
     
         64 . The method of  claim 52 , wherein the CTLA-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 38. 
     
     
         65 . The method of  claim 52 , wherein the CTLA-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 36. 
     
     
         66 . The method of  claim 52 , wherein the CTLA-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 42. 
     
     
         67 . The method of  claim 52 , wherein the CTLA-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 47. 
     
     
         68 . The method of  claim 55 , wherein the CTLA-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 13.