High affinity fusion proteins and therapeutic and diagnostic methods for use

Abstract

High affinity fusion proteins capable of binding and inhibiting the activity of soluble, interacting proteins (“SIPs”) are described. In specific embodiments the fusion proteins are multimers, preferably dimers, of SIP-specific fusion polypeptides which comprise SIP binding domains derived from SIP targets and/or anti-SIP immunoglobulin domains, as well as multimerizing components.

1 . An isolated nucleic acid encoding a SIP-specific fusion polypeptide, wherein the fusion polypeptide comprises: 
 (a) one or more components which comprise a SIP binding domain of a SIP target (TBD);    (b) one or more components which comprise a SIP binding domain of an immunoglobulin (IBD); and    (c) a multimerizing component (M) capable of multimerizing with a multimerizing component on another fusion polypeptide to form a multimer of the fusion polypeptides.    
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein the multimerizing component comprises a constant region(s) of an immunoglobulin or derivative thereof.  
     
     
         3 . The isolated nucleic acid of  claim 2 , wherein the multimerizing component is selected from the group consisting of the Fc domain of IgG, the Fc domain of the heavy chain of IgG, the Fc domain of the light chain of IgG, and the heavy chain CH 2  and CH 3  constant regions.  
     
     
         4 . The isolated nucleic acid of  claim 1 , wherein the IBD is selected from the group consisting of a heavy chain variable domain, a light chain variable domain and an ScFv.  
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein the SIP-specific fusion polypeptide component arrangements are selected from the group consisting of (TBD) x -(IBD) y -M; (TBD) x -M-(IBD) y ; (IBD) x -(TBD) y -M; (IBD) y -M-(TBD) x , M-(IBD) y -(TBD) x , and M-(TBD) x -(IBD) y , wherein TBD is a SIP target binding domain, IBD is an immunoglobulin-derived SIP-binding domain, M is a multimerizing component, and x≧1 and y≧1.  
     
     
         6 . A fusion polypeptide encoded by the nucleic acid of any of  claims 1  to  5 .  
     
     
         7 . A trapbody comprising two of the fusion polypeptides of  claim 6 .  
     
     
         8 . An isolated nucleic acid encoding a SIP-specific fusion polypeptide, wherein the fusion polypeptide comprises: 
 (a) two or more components which comprise a SIP binding domain of an immunoglobulin (IBD); and    (b) a multimerizing (M) component.    
     
     
         9 . The isolated nucleic acid of  claim 8 , wherein the fusion polypeptide comprises IBD′-IBD″-M, wherein IBD′ and IBD″ are directed to different epitopes of the same SIP.  
     
     
         10 . The isolated nucleic acid of  claim 8 , wherein the SIP is a cytokine.  
     
     
         11 . The isolated nucleic acid of  claim 10 , wherein the SIP target is a specificity-determining component of a cytokine receptor or a signaling component of a cytokine receptor.  
     
     
         12 . The isolated nucleic acid of  claim 11 , wherein the cytokine is selected from the group consisting of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13, IL-15, IL-18, granulocyte macrophage colony stimulating factor, oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, MIF, interferon gamma, and cardiotrophin-1.  
     
     
         13 . A fusion polypeptide encoded by the nucleic acid molecule of any of  claims 8  to  12 .  
     
     
         14 . A trapbody which is a dimer comprising two of the fusion polypeptides of  claim 13 .  
     
     
         15 . A method of producing a SIP-specific fusion polypeptide, comprising culturing a host cell transfected with a vector comprising the nucleic acid of  claim 1  or  8 , under conditions suitable for expression of the protein from the host cell, and recovering the fusion protein so produced.  
     
     
         16 . An isolated nucleic acid encoding (i) a V L  CDR selected from the group consisting of SEQ ID NOs:27-32, 43-48, 59-64, 75-80, 91-96 and 107-112, and (i) a V H  CDR selected from the group consisting of SEQ ID NOs:19-24, 35-40, 51-56, 67-72, 83-88, and 99-104.  
     
     
         17 . An IL-6-specific antibody or antibody fragment, comprising at least one CDR of  claim 16 .  
     
     
         18 . An IL-6-specific fusion polypeptide comprising two IL-6-specific immunoglobulin domains and a multimerizing component, wherein each immunoglobulin domain comprises at least one CDR of  claim 16 .  
     
     
         19 . The IL-6-specific fusion polypeptide of  claim 18 , wherein at least one immunoglobulin domain is humanized.  
     
     
         20 . A trapbody comprising two IL-6-specific fusion polypeptides of  claim 18  or  claim 19 .  
     
     
         21 . A trapbody, comprising two fusion polypeptides and capable of binding a soluble protein (SIP), wherein each fusion polypeptide comprises IBD′-IBD″-M, wherein IBD′ and IBD″ are specific to different epitopes of the same SIP, and the trapbody exhibits at least a five-fold increase in affinity relative to a dimer composed of two fusion polypeptides each having a single IBD and a multimerizing component.  
     
     
         22 . A human IL-6-specific trapbody, comprising IBD-IBD-M, wherein each IBD comprise at least one CDR sequence selected from the group consisting of SEQ ID NOs:19-24, 27-32, 35-40, 43-48, 51-56, 59-64, 67-72, 75-80, 83-88, 91-96, 99-104 and 107-112.  
     
     
         23 . The IL-6-specific trapbody of  claim 22 , wherein each IBD comprises (i) at least one V L  CDR selected from the group consisting of SEQ ID NOs:27-32, 43-48, 59-64, 75-80, 91-96 and 107-112, and (i) at least one V H  CDR selected from the group consisting of SEQ ID NOs:19-24, 35-40, 51-56, 67-72, 83-88, and 99-104.  
     
     
         24 . An antibody heavy chain or heavy chain fragment, comprising one or more of CDR1, CDR2 and CDR3, wherein (i) CDR1 is selected from the group consisting of SEQ ID NO:19, 22, 35, 38, 51, 54, 67, 70, 83, 86, 99 and 102; (ii) CDR2 is selected from the group consisting of SEQ ID NO: 20, 23, 36, 39, 52, 55, 68, 71, 84, 87, 100 and 103; and (iii) CDR3 is selected from the group consisting of SEQ ID NO: 21, 24, 37, 40, 53, 56, 69, 72, 85, 88, 101 and 104.  
     
     
         25 . The antibody heavy chain or heavy chain fragment of  claim 24  which is humanized.  
     
     
         26 . An antibody light chain or light chain fragment, comprising one or more of CDR1, CDR2 and CDR3, wherein (i) CDR1 is selected from the group consisting of SEQ ID NO: 27, 30, 43, 46, 59, 62, 75, 78, 91, 94, 107 and 110; (ii) CDR2 is selected from the group consisting of SEQ ID NO: 28, 31, 44, 47, 60, 63, 76, 79, 92, 95, 108, and 111; and (iii) CDR3 is selected from the group consisting of SEQ ID NO: 29, 32, 45, 48, 61, 64, 77, 80, 93, 96, 109 and 112.  
     
     
         27 . The antibody light chain or light chain fragment of  claim 26  which is humanized.  
     
     
         28 . An antibody or antibody fragment, comprising the heavy chain or heavy chain fragment of  claim 24  and the light chain or light chain fragment of  claim 26 .  
     
     
         29 . The antibody or antibody fragment of  claim 28  which is humanized.