Anti-serum albumin binding variants

Abstract

The invention relates to improved variants of the anti-serum albumin immunoglobulin single variable domain DOM7h-11, as well as ligands and drug conjugates comprising such variants, compositions, nucleic acids, vectors and hosts.

1 . An anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11 (SEQ ID NO: 438), said variant having a Tm of at least 54° C. 
     
     
         2 . An anti-SA immunoglobulin as claimed in  claim 1 , wherein said variant comprises at least one mutation at position 22, 42 or 91 (numbering according to Kabat) compared to DOM7h-11, and, wherein said variant is a variant of DOM7h-11-15 (SEQ ID NO: 2) and comprises at least one mutation at position 22, 42, or 91 (numbering according to Kabat) compared to DOM7h-11-15. 
     
     
         3 . The variant of any of  claim 1 , wherein the variant comprises at least one mutation selected from the following:
 Position 22=Ser, Phe, Thr, Ala or Cys;   Position 42=Glu or Asp;   Position 91=Thr or Ser.   
     
     
         4 . An anti-SA immunoglobulin single variable domain variant as claimed in  claim 3  wherein position 22 is Ser or Phe; or wherein position 42 is Glu and position 91 is Thr; or wherein position 91 is Thr; or wherein position 22 is Phe. 
     
     
         5 . The variant of  claim 1 , wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-56 (SEQ ID NO: 412), DOM7h-11-68 (SEQ ID NO: 416), DOM7h-11-79 (SEQ ID NO:418), DOM7h-11-80 (SEQ ID NO: 419), DOM 7h-11-90 (SEQ ID NO: 420), DOM 7h-11-86 (SEQ ID NO: 421), DOM 7h-11-87 (SEQ ID NO: 422), and DOM 7h-11-88 (SEQ ID NO: 423) or has up to 4 changes compared to the selected amino acid sequence. 
     
     
         6 . An anti-SA immunoglobulin single variable domain variant as claimed in  claim 1  wherein the variant comprises at least one mutation in the FW3 region (positions 57 to 88, numbering according to Kabat) or in the CDR3 region (positions 89 to 97, numbering according to Kabat) compared to DOM7h-11, preferably wherein said variant is a variant of DOM7h-11-15 (SEQ ID NO: 2) and comprises at least one mutation in the FW3 region (positions 57 to 88, numbering according to Kabat) or in the CDR3 region (positions 89 to 97, numbering according to Kabat) compared to DOM7h-11-15. 
     
     
         7 . An anti-SA immunoglobulin single variable domain variant as claimed in  claim 6  wherein said variant comprises at least one mutation at any of positions 77, 83, 93 or 95 (numbering according to Kabat), and, preferably, wherein the variant comprises at least one mutation selected from the following:
 Position 77=Asn, Gln 
 Position 83=Val, Ile, Met, Leu, Phe, Ala or Norleucine. 
 Position 93=Val, Ile, Met, Leu, Phe, Ala or Norleucine. 
 Position 95=His, Asn, Gln, Lys or Arg. 
 
     
     
         8 . An anti-SA immunoglobulin single variable domain as claimed in  claim 1  further comprising a mutation at position 106 or 108 (numbering according to Kabat); preferably wherein position 106 is Asn or Gln; or wherein position 108 is Trp, Tyr or Phe. 
     
     
         9 . An anti-SA immunoglobulin single variable domain variant as claimed in  claim 1  wherein position 77 is Asn; or wherein position 83 is Val; or wherein position 95 is His; or wherein position 93 is Val. 
     
     
         10 . An anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11 (SEQ ID NO: 438), said variant having a Tm of at least 54° C., wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-57 (SEQ ID NO: 413), DOM7h-11-65 (SEQ ID NO: 414), DOM7h-11-67 (SEQ ID NO:415) and DOM7h-11-69 (SEQ ID NO: 417) or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has at least one mutation in the FW3 or CDR3 region as defined in of  claim 1 . 
     
     
         11 . A variant as claimed in  claim 6  said variant having a Tm of at least 57° C. 
     
     
         12 . A variant as claimed in  claim 1 , wherein said variant has an increased Tm value compared to DOM7h-11, or an increased Tm value compared to DOM7h-11-15. 
     
     
         13 . The variant of  claim 1 , wherein
 a) the variant comprises a binding site that specifically binds human SA with a dissociation constant (KD) of from about 0.1 to about 10000 nM, optionally from about 1 to about 6000 nM, as determined by surface plasmon resonance; or   b) the variant comprises a binding site that specifically binds human SA with an off-rate constant (K d ) of from about 1.5×10 −4  to about 0.1 sec −1 , optionally from about 3×10 −4  to about 0.1 sec −1  as determined by surface plasmon resonance; or   c) the variant comprises a binding site that specifically binds human SA with an on-rate constant (K a ) of from about 2×10 6  to about 1×10 4 M −1  sec −1 , optionally from about 1×10 6  to about 2×10 4  M −1  sec −1  as determined by surface plasmon resonance; or   d) the variant comprises a binding site that specifically binds Cynomolgus monkey SA with a dissociation constant (KD) of from about 0.1 to about 10000 nM, optionally from about 1 to about 6000 nM, as determined by surface plasmon resonance; or   e) the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an off-rate constant (K d ) of from about 1.5×10 −4  to about 0.1 sec −1 , optionally from about 3×10 −4  to about 0.1 sec −1  as determined by surface plasmon resonance; or   f) the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an on-rate constant (K a ) of from about 2×10 6  to about 1×10 4 M −1  sec −1 , optionally from about 1×10 6  to about 5×10 3 M −1  sec −1  as determined by surface plasmon resonance.   
     
     
         14 . A multispecific ligand comprising an anti-SA variant of  claim 1  and a binding moiety that specifically binds a target antigen other than SA. 
     
     
         15 . An anti-SA variant single variable domain of  claim 1 , wherein the variable domain is conjugated to a drug (optionally an NCE drug), optionally wherein the selected variant is DOM7h-11-56 (SEQ ID NO: 412), DOM7h-11-57 (SEQ ID NO: 413), DOM7h-11-65 (SEQ ID NO: 414), DOM7h-11-67 (SEQ ID NO:415), DOM7h-11-68 (SEQ ID NO:416), DOM7h-11-69 (SEQ ID NO: 417), DOM7h-11-79 (SEQ ID NO:418), DOM7h-11-80 (SEQ ID NO:419), DOM7h-11-90 (SEQ ID NO:420), DOM7h-11-86 (SEQ ID NO:421), DOM7h-11-87 (SEQ ID NO:422) or DOM7h-11-88 (SEQ ID NO:423). 
     
     
         16 . A fusion protein comprising a polypeptide or peptide drug fused to a variant according to  claim 1 , optionally wherein the selected variant is DOM7h-11-56 (SEQ ID NO: 412), DOM7h-11-57 (SEQ ID NO: 413), DOM7h-11-65 (SEQ ID NO: 414), DOM7h-11-67 (SEQ ID NO:415), DOM7h-11-68 (SEQ ID NO:416), DOM7h-11-69 (SEQ ID NO: 417), DOM7h-11-79 (SEQ ID NO:418), DOM7h-11-80 (SEQ ID NO:419), DOM7h-11-90 (SEQ ID NO:420), DOM7h-11-86 (SEQ ID NO:421), DOM7h-11-87 (SEQ ID NO:422) or DOM7h-11-88 (SEQ ID NO:423), preferably wherein the fusion protein comprises a linker (e.g., a linker comprising the amino acid sequence TVA, optionally TVAAPS) between the variant and the drug. 
     
     
         17 . A composition comprising a variant, fusion protein or ligand of  claim 1  and a pharmaceutically acceptable diluent, carrier, excipient or vehicle. 
     
     
         18 . A method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of a variant, multispecific ligand or fusion protein according to  claim 1  to said patient.