Clostridium difficile-specific antibodies and uses thereof

Abstract

The present invention is directed to Clostridium difficile toxin-specific antibodies, compositions, and uses thereof. The anti-toxin antibodies may be specific for either TcdA or TcdB. The invention also includes methods of treating a Clostridium difficile infection, methods of capturing Clostridium difficile toxins, and methods of detecting Clostridium difficile toxins.

1 . An isolated or purified antibody or fragment thereof, comprising
 a sequence of complementarity determining region (CDR) 1 selected from GRTFNTLS (SEQ ID NO:1); GRTFSMYR (SEQ ID NO:2); GRTLSSYI (SEQ ID NO:3); GRTFSMDP (SEQ ID NO:4); IRSFSNRN (SEQ ID NO:5); and ERTFSRYP (SEQ ID NO:6);   a sequence of CDR2 selected from VSRSGGST (SEQ ID NO:7); ITRNGSST (SEQ ID NO:8); ISRRGGNS (SEQ ID NO:9); GSSTGRTT (SEQ ID NO:10); ISWGGGST (SEQ ID NO:11); and ISSTGTST (SEQ ID NO:12); and   a sequence of CDR3 selected from AAAATKSNTTAYRLSFDY (SEQ ID NO:13); AATSGSSYLDAAHVYDY (SEQ ID NO:14); AADGSVAGWGRRSVSVSSYDY (SEQ ID NO:15); AAAPYGANWYRDEYAY (SEQ ID NO:16); AAEFGHNIATSSDEYDY (SEQ ID NO:17); and AVNSQRTRLQDPNEYDY (SEQ ID NO:18),   
       wherein the antibody or fragment thereof is specific for TcdA. 
     
     
         2 . The isolated or purified antibody or fragment thereof of  claim 1 , selected from the group consisting of:
 an isolated or purified antibody or fragment thereof comprising a sequence of CDR 1 of GRTFNTLS (SEQ ID NO:1); CDR2 of VSRSGGST (SEQ ID NO:7); and CDR3 of AAAATKSNTTAYRLSFDY (SEQ ID NO:13);   an isolated or purified antibody or fragment thereof comprising a sequence of CDR 1 of GRTFSMYR (SEQ ID NO:2); CDR2 of ITRNGSST (SEQ ID NO:8); and CDR3 of AATSGSSYLDAAHVYDY (SEQ ID NO:14);   an isolated or purified antibody or fragment thereof comprising a sequence of CDR 1 of GRTLSSYI (SEQ ID NO:3); CDR2 of ISRRGGNS (SEQ ID NO:9); and CDR3 of AADGSVAGWGRRSVSVSSYDY (SEQ ID NO:15);   an isolated or purified antibody or fragment thereof comprising a sequence of CDR 1 of GRTFSMDP (SEQ ID NO:4); CDR2 of GSSTGRTT (SEQ ID NO:10); and CDR3 of AAAPYGANWYRDEYAY (SEQ ID NO:16);   an isolated or purified antibody or fragment thereof comprising a sequence of CDR 1 of IRSFSNRN (SEQ ID NO:5); CDR2 of ISWGGGST (SEQ ID NO:11); and CDR3 of AAEFGHNIATSSDEYDY (SEQ ID NO:17); or   an isolated or purified antibody or fragment thereof comprising a sequence of CDR 1 of ERTFSRYP (SEQ ID NO:6); CDR2 of ISSTGTST (SEQ ID NO:12); and CDR3 of AVNSQRTRLQDPNEYDY (SEQ ID NO:18).   
     
     
         3 . The isolated or purified antibody or fragment thereof of  claim 1 , comprising a sequence selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO: 34) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREF 
                 
                     
                 
                   VAAVSRSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 35) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREF 
                 
                     
                 
                   VGVITRNGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALY 
                 
                     
                 
                   YCAATSGSSYLDAAHVYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 36) 
                 
                   QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREF 
                 
                     
                 
                   VAGISRRGGNSAYVESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 37) 
                 
                   QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREF 
                 
                     
                 
                   VAAGSSTGRTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAAPYGANWYRDEYAYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 38) 
                 
                   QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREF 
                 
                     
                 
                   VAGISWGGGSTRYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAEFGHNIATSSDEYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 39) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREF 
                 
                     
                 
                   VAVISSTGTSTYYADSVKGRFTISRDNAKVTVYLQMNNLKREDTAVY 
                 
                     
                 
                   FCAVNSQRTRLQDPNEYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 45) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREF 
                 
                     
                 
                   VCAVSRSGGSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 46) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREF 
                 
                     
                 
                   VCVITRNGSSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTALY 
                 
                     
                 
                   YCAATSGSSYLDAAHVYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 47) 
                 
                   QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREF 
                 
                     
                 
                   VCGISRRGGNSAYVESVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 48) 
                 
                   QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREF 
                 
                     
                 
                   VCAGSSTGRTTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAAPYGANWYRDEYAYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 49) 
                 
                   QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREF 
                 
                     
                 
                   VCGISWGGGSTRYADSVKGRFTCSRDNAKKTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAEFGHNIATSSDEYDYWGQGTQVTVSS; 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO: 50) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREF 
                 
                     
                 
                   VCVISSTGTSTYYADSVKGRFTCSRDNAKVTVYLQMNNLKREDTAVY 
                 
                     
                 
                   FCAVNSQRTRLQDPNEYDYWGQGTQVTVSS, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       or a sequence substantially identical thereto. 
     
     
         4 . The isolated or purified antibody or fragment thereof of any one of  claims 1  to  3 , wherein the antibody or fragment thereof binds to a conformational or linear epitope. 
     
     
         5 . The isolated or purified antibody or fragment thereof of  claim 1 , wherein the antibody or fragment thereof is in a multivalent display format. 
     
     
         6 . A nucleic acid molecule encoding the isolated or purified antibody or fragment thereof of  claim 1 . 
     
     
         7 . A vector comprising the nucleic acid molecule of  claim 6 . 
     
     
         8 . The isolated or purified antibody or fragment thereof of  claim 1 , wherein the antibody or fragment thereof is immobilized onto a surface. 
     
     
         9 . The isolated or purified antibody or fragment thereof of  claim 1 , wherein the antibody or fragment thereof is linked to a cargo molecule. 
     
     
         10 . The isolated or purified antibody or fragment thereof of  claim 9 , wherein the cargo molecule is a detectable agent, a therapeutic, a drug, a peptide, a carbohydrate moiety, an enzyme, or a cytotoxic agent; one or more liposomes loaded with a detectable agent, a therapeutic, a drug, a peptide, an enzyme, or a cytotoxic agent; or one or more nanoparticle, nanowire, nanotube, or quantum dots. 
     
     
         11 . A composition comprising one or more than one isolated or purified antibody or fragment thereof of  claim 1  and a pharmaceutically-acceptable carrier, diluent, or excipient. 
     
     
         12 . A method of treating a  Clostridium difficile  infection, comprising administering the isolated or purified antibody or fragment thereof of  claim 1  to a subject in need thereof. 
     
     
         13 . A method of capturing  Clostridium difficile  toxins, comprising contacting a sample with one or more than one isolated or purified antibody or fragment thereof of  claim 8  and allowing the toxin(s) to bind to the isolated or purified antibody or fragment thereof. 
     
     
         14 . The method of  claim 13 , further comprising identification of the toxin by a mass spectrometric method. 
     
     
         15 . The method of  claim 13 , further comprising eluting the bound toxin. 
     
     
         16 . The method of  claim 15 , wherein elution of the bound toxin is accomplished by heat elution. 
     
     
         17 . A method of detecting  Clostridium difficile  toxins, comprising contacting a sample with one or more than one isolated or purified antibody or fragment thereof of  claim 9  or  10 , and detecting the bound antibody or fragment thereof using a suitable detection and/or imaging technology. 
     
     
         18 . A method of treating a  Clostridium difficile  infection, comprising administering isolated or purified single domain antibody, comprising a sequence selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO: 34) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREF 
                 
                     
                 
                   VAAVSRSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 35) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREF 
                 
                     
                 
                   VGVITRNGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALY 
                 
                     
                 
                   YCAATSGSSYLDAAHVYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 36) 
                 
                   QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREF 
                 
                     
                 
                   VAGISRRGGNSAYVESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 37) 
                 
                   QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREF 
                 
                     
                 
                   VAAGSSTGRTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAAPYGANWYRDEYAYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 38) 
                 
                   QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREF 
                 
                     
                 
                   VAGISWGGGSTRYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAEFGHNIATSSDEYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 39) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREF 
                 
                     
                 
                   VAVISSTGTSTYYADSVKGRFTISRDNAKVTVYLQMNNLKREDTAVY 
                 
                     
                 
                   FCAVNSQRTRLQDPNEYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 45) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASGRTFNTLSMGWFRQAPGKEREF 
                 
                     
                 
                   VCAVSRSGGSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAAATKSNTTAYRLSFDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 46) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASGRTFSMYRMGWFRQAPGKEREF 
                 
                     
                 
                   VCVITRNGSSTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTALY 
                 
                     
                 
                   YCAATSGSSYLDAAHVYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 47) 
                 
                   QVKLEESGGGLVQPGGSLRLSCAASGRTLSSYIVAWFRQAPGKEREF 
                 
                     
                 
                   VCGISRRGGNSAYVESVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAADGSVAGWGRRSVSVSSYDYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 48) 
                 
                   QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAWFRQPPGKEREF 
                 
                     
                 
                   VCAGSSTGRTTYYADSVKGRFTCSRDNAKNTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAAPYGANWYRDEYAYWGQGTQVTVSS; 
                 
                     
                 
                   (SEQ ID NO: 49) 
                 
                   QVQLVESGGGLVQAGGSLRLSCAASIRSFSNRNMGWFRQPPGKEREF 
                 
                     
                 
                   VCGISWGGGSTRYADSVKGRFTCSRDNAKKTVYLQMNSLKPEDTAVY 
                 
                     
                 
                   YCAAEFGHNIATSSDEYDYWGQGTQVTVSS; 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO: 50) 
                 
                   QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWFRQAPGAEREF 
                 
                     
                 
                   VCVISSTGTSTYYADSVKGRFTCSRDNAKVTVYLQMNNLKREDTAVY 
                 
                     
                 
                   FCAVNSQRTRLQDPNEYDYWGQGTQVTVSS, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       allowing the toxin to bind the isolated or purified single domain antibody or fragment thereof and detecting the bound single domain antibody or fragment thereof using a suitable detection and/or imaging technology.