Polypeptides comprising immunoglobulin single variable domains targeting il-13 and tslp

Abstract

The present technology aims at providing a novel type of drug for treating a subject suffering from an inflammatory disease. Specifically, the present technology provides polypeptides comprising at least four immunoglobulin single variable domains (ISVDs), characterized in that at least two ISVDs bind to IL-13 and at least two ISVDs binds to TSLP. The present technology also provides nucleic acids, vectors and compositions.

1 . - 26 . (canceled) 
     
     
         27 . A polypeptide that comprises or consists of at least one immunoglobulin single variable domain (ISVD) that specifically binds to IL13 or TSLP, wherein said ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively); and wherein the at least one ISVD comprises:
 a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 7;
 a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 12; and 
 a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 17, 
   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 8;
 a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and 
 a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 18, 
   c) a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 9;
 a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 14; and 
 a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 19, or 
   d) a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11;
 a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 16; and 
 a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 21. 
   
     
     
         28 . The polypeptide according to  claim 27 , wherein the at least one ISVD that specifically binds to IL-13 or TSLP comprises:
 a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of SEQ ID NO: 17,   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of SEQ ID NO: 18,   c) a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of SEQ ID NO: 19, or   d) a CDR1 that is the amino acid sequence of SEQ ID NO: 11, a CDR2 that is the amino acid sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid sequence of SEQ ID NO: 21.   
     
     
         29 . The polypeptide according to  claim 27 , wherein the amino acid sequence of the at least one ISVD that specifically binds to IL-13 or TSLP comprises:
 a) a sequence identity of more than 90% with SEQ ID NO: 2,   b) a sequence identity of more than 90% with SEQ ID NO: 3,   c) a sequence identity of more than 90% identity with SEQ ID NO: 4, or   d) a sequence identity of more than 90% identity with SEQ ID NO: 6.   
     
     
         30 . The polypeptide according to  claim 27 , wherein said at least one ISVD that specifically binds to IL-13 or TSLP comprises:
 a) the amino acid sequence of SEQ ID NO: 2,   b) the amino acid sequence of SEQ ID NO: 3,   c) the amino acid sequence of SEQ ID NO: 4, or   d) the amino acid sequence of SEQ ID NO: 6.   
     
     
         31 . The polypeptide according to  claim 27 , wherein the polypeptide comprises or consists of at least two ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptidic linkers, and wherein:
 a) a first and a second ISVD specifically binds to IL-13 and comprises
 i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 7; 
 ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 12; and 
 iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 17, 
   b) a first and a second ISVD specifically binds to IL-13 and comprises
 i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 8; 
 ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and 
 iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 18, 
   c) a first ISVD specifically binds to IL-13 and comprises
 i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 7; 
 ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 12; and 
 iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 17, and 
   a second ISVD specifically binds to IL-13 and comprises
 iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 8; 
 v. a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and 
 vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 18, 
   d) a first ISVD specifically binds to IL-13 and comprises
 i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 8; 
 ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and 
 iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 18, and 
   a second ISVD specifically binds to IL-13 and comprises
 iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 7; 
 v. a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 12; and 
 vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 17, 
   e) a first ISVD specifically binds to TSLP and comprises
 i. a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11; 
 ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 16; and 
 iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 21, and 
   a second ISVD specifically binds to TSLP and comprises
 iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 9; 
 v. a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 14; and 
 vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 19, or 
   f) a first ISVD specifically binds to TSLP and comprises
 i. a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 9; 
 ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 14; and 
 iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 19, and 
   a second ISVD specifically binds to TSLP and comprises
 iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11; 
 v. a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 16; and 
 vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 21, 
   wherein the order of the ISVDs indicates their relative position to each other considered from the N-terminus to the C-terminus of said polypeptide.   
     
     
         32 .- 34 . (canceled) 
     
     
         35 . The polypeptide according to  claim 31 , wherein the polypeptide comprises or consists of:
 a) the amino acid sequence of SEQ ID NO: 148,   b) the amino acid sequence of SEQ ID NO: 149,   c) the amino acid sequence of SEQ ID NO: 150,   d) the amino acid sequence of SEQ ID NO: 151,   e) the amino acid sequence of SEQ ID NO: 152,   f) the amino acid sequence of SEQ ID NO: 153,   g) the amino acid sequence of SEQ ID NO: 154,   h) the amino acid sequence of SEQ ID NO: 155,   i) the amino acid sequence of SEQ ID NO: 156,   j) the amino acid sequence of SEQ ID NO: 157,   k) the amino acid sequence of SEQ ID NO: 158, or   l) the amino acid sequence of SEQ ID NO: 159.   
     
     
         36 . The polypeptide according to  claim 27 , wherein the polypeptide comprises or consists of at least four ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptidic linkers, and wherein:
 a) a first ISVD specifically binds to IL-13 and comprises
 i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 7; 
 ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 12; and 
 iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 17; 
   b) a second ISVD specifically binds to IL-13 and comprises
 iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 8; 
 v. a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and 
 vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 18; 
   c) a third ISVD specifically binds to TSLP and comprises
 vii. a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 9; 
 viii. a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 14; and 
 ix. a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 19; and 
   d) a fourth ISVD specifically binds to TSLP and comprises
 x. a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 11; 
 xi. a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 16; and 
 xii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 21, 
   wherein the order of the ISVDs indicates their relative position to each other considered from the N-terminus to the C-terminus of said polypeptide.   
     
     
         37 . The polypeptide according to  claim 36 , wherein:
 a) said first ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of SEQ ID NO: 17;   b) said second ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of SEQ ID NO: 18;   c) said third ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of SEQ ID NO: 19; and   d) said fourth ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 11, a CDR2 that is the amino acid sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid sequence of SEQ ID NO: 21.   
     
     
         38 . (canceled) 
     
     
         39 . The polypeptide according to  claim 36  any of  claims 36  to  38 , wherein:
 a) said first ISVD comprises the amino acid sequence of SEQ ID NO: 2; 
 b) said second ISVD comprises the amino acid sequence of SEQ ID NO: 3; 
 c) said third ISVD comprises the amino acid sequence of SEQ ID NO: 4; and 
 d) said fourth ISVD comprises the amino acid sequence of SEQ ID NO: 6. 
 
     
     
         40 . The polypeptide according to  claim 27 , wherein said polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units. 
     
     
         41 . (canceled) 
     
     
         42 . The polypeptide according to  claim 40 , in which said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG). 
     
     
         43 . The polypeptide according to  claim 42 , in which said binding unit that provides the polypeptide with increased half-life is an ISVD that can bind to human serum albumin. 
     
     
         44 .- 46 . (canceled) 
     
     
         47 . The polypeptide according to  claim 43 , wherein said ISVD binding to human serum albumin comprises the amino acid sequence of SEQ ID NO: 5. 
     
     
         48 . (canceled) 
     
     
         49 . The polypeptide according to  claim 36 , wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1. 
     
     
         50 . A nucleic acid comprising a nucleotide sequence that encodes a polypeptide according to  claim 27 . 
     
     
         51 . A host or host cell comprising a nucleic acid according to  claim 50 . 
     
     
         52 . A method for producing a polypeptide according to, said method at least comprising the steps of:
 a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid according to  claim 50 ; optionally followed by:   b) isolating and/or purifying the polypeptide according to.   
     
     
         53 . A composition comprising at least one polypeptide according to  claim 27 . 
     
     
         54 . (canceled) 
     
     
         55 . A method of treating an inflammatory disease, preferably a type 2 inflammatory disease, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of a polypeptide according to  claim 27 . 
     
     
         56 . The method according to  claim 55 , wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis. 
     
     
         57 .- 58 . (canceled)