Modified variable domain molecules and methods for producing them

Abstract

The present disclosure provides an isolated, engineered or non-naturally occurring protein comprising an antibody light chain variable domain (V L ) which may comprise at least one negatively charged amino acid positioned between residues 49 to 56 according to the numbering system of Kabat, the protein capable of binding specifically to an antigen.

1 . An isolated, engineered or non-naturally occurring protein comprising:
 a) an antibody light chain variable domain (V L ) comprising a negatively charged amino acid at two or more positions between residues 49 and 56 according to the numbering system of Kabat, the protein capable of specifically binding to an antigen; or   b) (i) an antibody light chain variable domain (V L ) comprising a negatively charged amino acid at one or more positions between residues 49 and 56 according to the numbering system of Kabat; and
 (ii) an antibody heavy chain variable domain (V H ) comprising a negatively charged amino acid at one or more positions selected from the group consisting of residues 28, 30, 31, 32, 33 and 35 according to the numbering system of Kabat, wherein the protein is capable of specifically binding to an antigen; or 
   c) a modified antibody light chain variable domain (V L ) capable of specifically binding to an antigen, wherein the V L  comprises a negatively charged amino acid at one or more positions selected from the group consisting of residues 49, 51, 52, 53 and 56 according to the numbering system of Kabat, and wherein the unmodified form of the V L  does not comprise the negatively charged amino acid(s); or   d) a modified antibody light chain variable domain (V L ) capable of specifically binding to an antigen, wherein the V L  comprises a negatively charged amino acid at two or more positions between residues 49 and 56 according to the numbering system of Kabat, and wherein the unmodified form of the V L  does not comprise two or more negatively charged amino acid(s) at the positions; or   e) (i) a modified antibody light chain variable domain (V L ) comprising a negatively charged amino acid at a position between residues 49 and 56 according to the numbering system of Kabat, wherein the unmodified form of the V L  does not comprise a negatively charged amino acid at the position; and
 (ii) a modified antibody heavy chain variable domain (V H ) comprising a negatively charged amino acid at one or more positions selected from the group consisting of residues 28, 30, 31, 32, 33 and 35 according to the numbering system of Kabat, wherein the unmodified form of the V H  does not comprise a negatively charged amino acid at the position, wherein the modified protein is capable of specifically binding to an antigen. 
   
     
     
         2 . The protein of  claim 1  part a), comprising two or more negatively charged amino acids at positions selected from the group consisting of residues 49, 50, 51, 52, 53 and 56 according to the numbering system of Kabat. 
     
     
         3 . (canceled) 
     
     
         4 . The protein of  claim 1  part b) comprising a negatively charged amino acid at two or more positions between residues 49 and 56 of the V L  or comprising a negatively charged amino acid at two or more positions selected from the group consisting of residues 28, 30, 31, 32, 33, 35 of the V H  according to the numbering system of Kabat. 
     
     
         5 . The protein of  claim 1  part b), comprising:
 (i) an antibody light chain variable domain (V L ) comprising a negatively charged amino acid at two or more positions between residues 49 and 56 according to the numbering system of Kabat; and 
 (ii) an antibody heavy chain variable domain (V H ) comprising a negatively charged amino acid at two or more positions selected from the group consisting of residues 28, 30, 31, 32, 33 and 35 according to the numbering system of Kabat, 
 wherein the protein is capable of specifically binding to an antigen. 
 
     
     
         6 . The protein of  claim 1  part b), wherein the V L  comprises negatively charged amino acid at one or more positions selected from the group consisting of residues 49, 50, 51, 52, 53 and 56 according to the numbering system of Kabat. 
     
     
         7 . The protein of  claim 1  part b), wherein the V L  additionally comprises one or more negatively charged amino acids in CDR1 and/or the V H  additionally comprises a negatively charged amino acid at one or more residues selected from the group consisting of residues 26, 39, 40, 50, 52, 52a and 53 according to the numbering system of Kabat. 
     
     
         8 . The protein of  claim 1 , wherein the protein is capable of specifically binding to an antigen with an affinity of more than 10 μM. 
     
     
         9 . The protein of  claim 1 , having a reduced tendency to aggregate compared to the protein without the negatively charged amino acid(s). 
     
     
         10 . The protein of  claim 1 , having a reduced tendency to aggregate after heating to at least about 60° C. compared to the protein without the negatively charged amino acid(s). 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The protein of  claim 1 , wherein the negatively charged amino acids are aspartic acid. 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . The protein of  claim 1  part c), additionally comprising a negatively charged amino acid at position 55. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The protein of  claim 1  part d), comprising a negatively charged amino acid at one or more positions selected from the group consisting of residues 49, 50, 51, 52, 53 and 56 of V L  according to the numbering system of Kabat. 
     
     
         22 . The protein of  claim 1 , wherein the protein is selected from the group consisting of:
 (i) an antibody;   (ii) a single domain antibody;   (iii) a single chain Fv (scFv) containing protein;   (iv) a diabody, a triabody or a tetra body;   (v) a fusion protein comprising any one of (ii)-(iv) and a Fc domain of an antibody or a domain thereof; and   (vi) a fusion protein comprising any one of (ii)-(iv) and a protein capable of binding to an immune effector cell.   
     
     
         23 . The protein according to  claim 1  conjugated to a compound. 
     
     
         24 . A composition comprising the protein of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         25 . A library comprising a plurality of proteins according to  claim 1 . 
     
     
         26 . A library including proteins comprising antibody light chain variable domains (V L s), the V L s comprising negatively charged amino acids at one or more positions between residues 49 and 56 according to the numbering system of Kabat. 
     
     
         27 . The library of  claim 26 , wherein the V L s comprise negatively charged amino acids at two or more of the positions. 
     
     
         28 . The library of  claim 26 , comprising proteins comprising antibody light chain variable domains (V L s) and antibody heavy chain variable domains (V H s), wherein the proteins comprise:
 (a) a V L  comprising at least one negatively charged amino acid at one or more positions between residues 49 and 56 according to the numbering system of Kabat; and   (b) a V H  comprising a negatively charged amino acid at one or more positions selected from the group consisting of residues 28, 30, 31, 32, 33 and 35 according to the numbering system of Kabat.   
     
     
         29 . The library of  claim 26 , wherein the proteins constitute at least 30% of the library. 
     
     
         30 . A method for isolating the protein of  claim 1 , the method comprising contacting the library of  claim 26  with the antigen and isolating a protein that binds thereto. 
     
     
         31 . A method for increasing the aggregation-resistance of a protein comprising an antibody light chain variable domain (V L ), the method comprising:
 a) modifying the V L  by substituting an amino acid at one or more positions selected from the group consisting of residues 49, 50, 51, 52, 53 and 56 according to the numbering system of Kabat with a negatively charged amino acid; or   b) modifying the VL such that it comprises negatively charged amino acids at two or more positions between residues 49 and 56 according to the numbering system of Kabat, wherein the unmodified protein does not comprise the two or more negatively charged amino acids; or   c) when the protein further comprises an antibody heavy chain variable domain (VH), modifying the protein such that it comprises:
 (i) a negatively charged amino acid at one or more positions between residues 49 and 56 of the VL according to the numbering system of Kabat; and 
 (ii) a negatively charged amino acid at one or more positions selected from the group consisting of residues 28, 30, 31, 32, 33 and 35 of the VH according to the numbering system of Kabat, wherein the protein prior to modification does not comprise a negatively charged amino acid at the positions in the VL and the VH. 
   
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 31  part c), comprising:
 (i) modifying the V L  by substituting an amino acid at one or more positions between residues 49 and 56 of the V L  according to the numbering system of Kabat with a negatively charged amino acid; and 
 (ii) modifying the V H  by substituting an amino acid at one or more positions selected from the group consisting of residues 28, 30, 31, 32, 33 and 35 according to the numbering system of Kabat with a negatively charged amino acid. 
 
     
     
         35 . The method of  claim 31  part c), comprising modifying the protein such that it comprises:
 (i) a negatively charged amino acid at two or more positions between residues 49 and 56 of the V L  according to the numbering system of Kabat; and 
 (ii) a negatively charged amino acid at two or more positions selected from the group consisting of residues 28, 30, 31, 32, 33 and 35 of the V H  according to the numbering system of Kabat, 
 wherein the protein prior to modification does not comprise a negatively charged amino acid at the positions in the V L  and the V H . 
 
     
     
         36 . The method of  claim 35 , comprising:
 (i) modifying the V L  by substituting an amino acid at two or more positions between residues 49 and 56 of the V L  according to the numbering system of Kabat with a negatively charged amino acid; and   (ii) modifying the V H  by substituting an amino acid at two or more positions selected from the group consisting of residues 28, 30, 31, 32, 33 and 35 according to the numbering system of Kabat with a negatively charged amino acid.   
     
     
         37 . The method of  claim 31  part c), additionally comprising modifying the protein such that the V L  additionally comprises one or more negatively charged amino acids in CDR1 and/or the V H  additionally comprises a negatively charged amino acid at one or more residues selected from the group consisting of residues 26, 39, 40, 50, 52, 52a and 53 according to the numbering system of Kabat. 
     
     
         38 . A method of treating or preventing a condition in a subject, the method comprising administering the protein of  claim 1  to a subject in need thereof. 
     
     
         39 . A method for delivering a compound to a cell, the method comprising contacting the cell with the protein of  claim 23 , wherein the protein is conjugated to the compound. 
     
     
         40 . A method for diagnosing or prognosing a condition in a subject, the method comprising contacting a sample from the subject with the protein of  claim 1  such that the protein binds to an antigen and forms a complex and detecting the complex, wherein detection of the complex is diagnostic or prognostic of the condition in the subject. 
     
     
         41 . (canceled) 
     
     
         42 . A method for localising or detecting an antigen in a subject, said method comprising detecting or localising the protein of  claim 23  in the subject, wherein the protein binds to the antigen, and wherein the protein is conjugated to a detectable label.