Polypeptides

Abstract

The present disclosure relates to a class of engineered polypeptides having a binding affinity for carbonic anhydrase IX (CAIX), and provides a CAIX binding polypeptide comprising the sequence EX2X3X4AX6X7EIX10X11LPN LX16X17X18QX20X21AFIX25X26LWD (SEQ ID NO. 369). The present disclosure also relates to the use of such a CAIX binding polypeptide as a diagnostic, prognostic agent and/or therapeutic agent.

What is claimed is: 
     
         1 . CAIX binding polypeptide, comprising a CAIX binding motif BM, which motif consists of the amino acid sequence selected from:
 iii)   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 369) 
                 
                     
                   EX 2 X 3 X 4 AX 6 X 7 EIX 10  X 11 LPNLX 16 X 17 X 18 QX 20  X 21 AFIX 25 X 26 LWD 
                 
             
                
                
               
            
           
         
         
           wherein, independently from each other, 
           X 2  is selected from D, H, N, Q and W; 
           X 3  is selected from E, F, I, K, L, Q, R, S, T, V, W and Y; 
           X 4  is selected from A, F, N, W and Y; 
           X 6  is selected from A, G, S and W; 
           X 7  is selected from A, E, G, I, Q, T, V and W; 
           X 10  is selected from A, D, E, G, H, I, N, Q, R, S, T and V; 
           X 11  is selected from D, E, K, R and S; 
           X 16  is selected from N and T; 
           X 17  is selected from A, D, E, G, H, I, Q, S, T, V and W; 
           X 18  is selected from A, D, E, F, G, H, I, L, N, Q, R, S, T, V, W and Y; 
           X 20  is selected from K, Q and R; 
           X 21  is selected from D, H, N and Q; 
           X 25  is selected from F and Y; 
           X 26  is selected from K and S; and 
         
         iv) the amino acid sequence which has at least 89% identity to the sequence defined in i). 
       
     
     
         2 . CAIX binding polypeptide according to  claim 1 , wherein sequence i) fulfills at least seven of the twelve conditions I-XII:
 III. X 2  is N;   IV. X 3  is selected from I and L;   III. X 4  is F;   IV. X 6  is G;   V. X 7  is W;   VI. X 10  is selected from D and S;   VII. X 11  is D;   VIII. X 16  is T;   IX. X 17  is selected from E and D;   X. X 18  is selected from D, Y, E and S;   XI. X 21  is N; and   XII. X 26  is K.   
     
     
         3 . CAIX binding polypeptide according to  claim 1 , wherein sequence i) is selected from SEQ ID NO:1-113 and SEQ ID NO:340-344. 
     
     
         4 . CAIX binding polypeptide according to  claim 3 , wherein sequence i) is selected from SEQ ID NO:1-14. 
     
     
         5 . CAIX binding polypeptide according to  claim 4 , wherein sequence i) is selected from SEQ ID NO:1-5. 
     
     
         6 . CAIX binding polypeptide according to  claim 1 , which comprises the amino acid sequence selected from:
 iii)   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 371) 
                 
                     
                   K-[ BM ]-DPSQS X a X b LLX c  EAKKL NDX d Q; 
                 
             
                
                
               
            
           
         
         
           wherein 
           [BM] is an CAIX binding motif as defined in  claim 1 ; 
           X a  is selected from A and S; 
           X b  is selected from N and E; 
           X c  is selected from A, S and C; 
           X d  is selected from A and S; and 
         
         iv) the amino acid sequence which has at least 81% identity to a sequence defined by iii). 
       
     
     
         7 . CAIX binding polypeptide according to  claim 1 , which comprises the amino acid sequence selected from:
 ix)   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 384) 
                 
                     
                   VDAKYAK-[ BM ]-DPSQSSELLSEAKKLNDSQAPK; 
                 
             
                
                
               
            
           
         
       
       wherein [BM] is an CAIX binding motif as defined in  claim 1 ; and
 x) the amino acid sequence which has at least 83% identity to the sequence defined in ix). 
 
     
     
         8 . CAIX binding polypeptide according to  claim 7 , in which sequence ix) is selected from SEQ ID NO:227-339 and SEQ ID NO:350-354. 
     
     
         9 . CAIX binding polypeptide according to  claim 8 , in which sequence ix) is selected from SEQ ID NO:227-240. 
     
     
         10 . CAIX binding polypeptide according to  claim 9 , in which sequence ix) is SEQ ID NO:227-231. 
     
     
         11 . CAIX binding polypeptide according to  claim 1 , which is capable of binding to CAIX such that the K D  value of the interaction is at most 1×10 −6  M. 
     
     
         12 . CAIX binding polypeptide according to  claim 1 , which comprises an additional moiety consisting of a polypeptide having a desired biological activity, such that the polypeptide is a fusion protein or conjugate comprising a first moiety consisting of CAIX binding polypeptide according to any preceding claim and a second moiety consisting of a polypeptide having a desired biological activity. 
     
     
         13 . Composition comprising a CAIX binding polypeptide according to  claim 1  and at least one pharmaceutically acceptable excipient or carrier. 
     
     
         14 . Method of treatment of a CAIX related condition, comprising administering to a subject in need thereof an effective amount of a CAIX binding polypeptide according to  claim 1 . 
     
     
         15 . Method of treatment of a CAIX related condition, comprising administering to a subject in need thereof an effective amount of a composition according to  claim 13 . 
     
     
         16 . Method according to  claim 14  or  15 , wherein said CAIX binding polypeptide, fusion protein or conjugate or composition modulates CAIX function in vivo. 
     
     
         17 . Method according to  claim 14  or  15 , wherein said CAIX related condition is cancer. 
     
     
         18 . Method according to  claim 17 , wherein said cancer is selected from the group consisting of kidney cancer, lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, vulval cancer, brain cancer, head and neck cancer, soft tissue sarcoma, astrocytomas, cancer of the oral cavity and any cancer manifested by solid tumors with hypoxia and CAIX expression. 
     
     
         19 . Method of detecting CAIX, comprising providing a sample suspected to contain CAIX, contacting said sample with a CAIX binding polypeptide according to  claim 1 , and detecting the binding of the CAIX binding polypeptide to indicate the presence of CAIX in the sample. 
     
     
         20 . Method for determining the presence of CAIX in a subject, the method comprising the steps:
 contacting the subject, or a sample isolated from the subject, with a CAIX binding polypeptide according to  claim 1 , and   obtaining a value corresponding to the amount of the CAIX binding polypeptide that has bound in said subject or to said sample.   
     
     
         21 . Method for determining the presence of CAIX in a subject, the method comprising the steps:
 contacting the subject, or a sample isolated from the subject, with a composition according to  claim 13 , and   obtaining a value corresponding to the amount of the composition that has bound in said subject or to said sample.   
     
     
         22 . Method according to  claim 20  or  21 , further comprising a step of comparing said value to a reference. 
     
     
         23 . Method according to  claim 20  or  21 , wherein said subject is a mammalian subject, such as a human subject. 
     
     
         24 . Method according to  claim 20  or  21 , which is performed in vivo. 
     
     
         25 . Method according to  claim 20  or  21 , which is performed in vitro.