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 (DOM7h-11 as shown in  FIG. 1 ), wherein the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11, and wherein the variant has from 2 to 8 changes compared to the amino acid sequence of DOM7h-11. 
     
     
         2 . The variant of  claim 1 , wherein position 49 (according to Kabat) is Leu. 
     
     
         3 . The variant of  claim 1 , wherein position 50 (according to Kabat) is Ala or Trp. 
     
     
         4 . The variant of  claim 1 , wherein position 51 (according to Kabat) is Phe or Asn. 
     
     
         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-11-3 (SEQ ID NO: 5), DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-12 (SEQ ID NO: 1) and DOM7h-11-19 (SEQ ID NO: 4) 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 FW2/CDR2 junction. 
     
     
         6 . The variant of  claim 1 , wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of DOM7h-11-15 S12P  (SEQ ID NO: 414) or has up to 4 changes compared to the amino acid sequence of DOM7h-11-15 S12P , provided that the amino acid sequence of the variant has at least one mutation in the FW2/CDR2 junction. 
     
     
         7 . An anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11, wherein the variant comprises a Met at position 32 (numbering according to Kabat) compared to DOM7h-11 (as shown in  FIG. 1 ), and wherein the variant has from 0 to 4 further changes compared to the amino acid sequence of DOM7h-11. 
     
     
         8 . The variant of  claim 7 , wherein the variant comprises at least one mutation in the FW2/CDR2 junction (positions 49 to 51, numbering according to Kabat) compared to DOM7h-11 (as shown in  FIG. 1 ). 
     
     
         9 . The variant of  claim 7 , wherein the variant comprises at least one mutation compared to DOM7h-11 (as shown in  FIG. 1 ) selected from the following
 Position 49=L,   Position 50=A or W,   Position 51=F or N,   Position 87=H, and   Position 91=T.   
     
     
         10 . The variant of  claim 7 , wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of a single variable domain selected from DOM7h-11-12 (SEQ ID NO: 1), DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-18 (SEQ ID NO: 3) and DOM7h-11-19 (SEQ ID NO: 4) or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has Met at position 32. 
     
     
         11 . The variant of  claim 7 , wherein the variant comprises an amino acid sequence that is identical to the amino acid sequence of DOM7h-11-15 S12P  (SEQ ID NO: 414) or has up to 4 changes compared to the selected amino acid sequence, provided that the amino acid sequence of the variant has Met at position 32. 
     
     
         12 . The variant of  claim 1 , wherein 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. 
     
     
         13 . The variant of  claim 1 , wherein 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. 
     
     
         14 . The variant of  claim 1 , wherein 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. 
     
     
         15 . The variant of  claim 1 , wherein 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. 
     
     
         16 . The variant of  claim 1 , wherein 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. 
     
     
         17 . The variant of  claim 1 , wherein 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. 
     
     
         18 . A multispecific ligand comprising an anti-SA variant of  claim 1  and a binding moiety that specifically binds a target antigen other than SA. 
     
     
         19 . 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-11-15 (SEQ ID NO: 2), DOM7h-11-15 S12P  (SEQ ID NO: 414) or DOM7h-11-12 (SEQ ID NO: 1). 
     
     
         20 . A fusion protein comprising a polypeptide or peptide drug fused to a variant according to  claim 1 , optionally wherein the selected variant is DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-15 S12P  (SEQ ID NO: 414) or DOM7h-11-12 (SEQ ID NO: 1). 
     
     
         21 . A fusion protein according to  claim 20 , wherein the fusion protein comprises a linker (eg, a linker comprising the amino acid sequence TVA, optionally TVAAPS) between the variant and the drug. 
     
     
         22 . A composition comprising a variant, fusion protein or ligand of  claim 1  and a pharmaceutically acceptable diluent, carrier, excipient or vehicle. 
     
     
         23 . A nucleic acid comprising a nucleotide sequence encoding a variant according to  claim 1 . 
     
     
         24 . A nucleic acid comprising the nucleotide sequence of a DOM7h-11 variant selected from the nucleotide sequence of DOM7h-11-3 (SEQ ID NO: 5), DOM7h-11-15 (SEQ ID NO: 2), DOM7h-11-12 (SEQ ID NO: 1), DOM7h-11-18 (SEQ ID NO: 3) and DOM7h-11-19 (SEQ ID NO: 4) or a nucleotide sequence that is at least 80% identical to said selected sequence. 
     
     
         25 . A nucleic acid comprising the nucleotide sequence of DOM7h-11-15 S12P  (SEQ ID NO: 414) or a nucleotide sequence that is at least 80% identical to said selected sequence. 
     
     
         26 . A vector comprising the nucleic acid of  claim 23 . 
     
     
         27 . An isolated host cell comprising the vector of  claim 26 . 
     
     
         28 . A method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of a variant according to  claim 1  to said patient.