Anti-rho gtpase conformational single domain antibodies and uses thereof

Abstract

Active forms of specific anti Rho GTPase conformational single domain antibodies are useful in the therapeutic and diagnostic fields. In particular, single domain antibodies wherein the amino acid sequences of CDR1-IMGT, CDR2-IMGT and CDR3-IMGT have at least 90% of identity with the amino acid sequences of the CDR1-IMGT, CDR2-IMGT and CDR3-IMGT of the H12, B6, 4P75, 4SP1, 4SNP36, 4SNP61, 5SP10, 5SP11, 5SP58, 5SNP47, 5SNP48, 5SNP65, B20, B15, B5, B71, E3, A6, G12, NB61, 212B, 111B or 404F (hs2dAb) are defined in Table B.

1 . A single domain antibody wherein the amino acid sequences of CDR1-IMGT, CDR2-IMGT and CDR3-IMGT of the single domain antibody have at least 90% identity, respectively, with each sequence of each group selected from
 SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7;   SEQ ID NO. 8, SEQ ID NO 9, and SEQ ID NO. 10;   SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13;   SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 16;   SEQ ID NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19;   SEQ ID NO. 20, SEQ ID NO. 21, and SEQ ID NO. 22;   SEQ ID NO. 23, SEQ ID NO. 24, and SEQ ID NO. 25;   SEQ ID NO. 26, SEQ ID NO. 27, and SEQ ID NO. 28;   SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 31;   SEQ ID NO. 32, SEQ ID NO. 33, and SEQ ID NO. 34;   SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37;   SEQ ID NO. 38, SEQ ID NO. 39, and SEQ ID NO. 40;   SEQ ID NO. 41, SEQ ID NO. 42, and SEQ ID NO. 43;   SEQ ID NO. 44, SEQ ID NO. 45, and SEQ ID NO. 46;   SEQ ID NO. 47, SEQ ID NO. 48, and SEQ ID NO. 49;   SEQ ID NO. 50, SEQ ID NO. 51, and SEQ ID NO. 52;   SEQ ID NO. 53, SEQ ID NO. 54, and SEQ ID NO. 55;   SEQ ID NO. 56, SEQ ID NO. 57, and SEQ ID NO. 58;   SEQ ID NO. 59, SEQ ID NO. 60, and SEQ ID NO. 61;   SEQ ID NO. 80, SEQ ID NO. 81, and SEQ ID NO. 83;   SEQ ID NO. 84, SEQ ID NO. 85, and SEQ ID NO. 86;   SEQ ID NO. 87, SEQ ID NO. 88, and SEQ ID NO. 89; and   SEQ ID NO. 90, SEQ ID NO. 91, and SEQ ID NO. 92.   
     
     
         2 . The single domain antibody of  claim 1  which comprises a framework region FR1 having at least 90% identity with SEQ ID NO:1. 
     
     
         3 . The single domain antibody of  claim 1  which comprises a framework region FR2 having at least 90% identity with SEQ ID NO:2. 
     
     
         4 . The single domain antibody of  claim 1  which comprises a framework region FR3 having at least 90% identity with SEQ ID NO:3. 
     
     
         5 . The single domain antibody of  claim 1  which comprises a framework region FR4 having at least 90% identity with SEQ ID NO:4. 
     
     
         6 . The single domain antibody of  claim 1  which comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NO:62-80 and SEQ ID NO: 93-96. 
     
     
         7 . The single domain antibody of  claim 1  which comprises an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NO:62-80 and SEQ ID NO: 93-96. 
     
     
         8 . The single domain antibody of  claim 1  which is fused to a heterologous polypeptide to form a fusion protein. 
     
     
         9 . The single domain antibody of  claim 1  which is fused to an immunoglobulin domain, a single domain antibody, a carrier polypeptide, a fluorescent polypeptide, an enzyme, a polypeptide that facilitates purification or isolation of the fusion protein, a cell-penetrating peptide or an ubiquitin ligase domain. 
     
     
         10 . The single domain antibody of  claim 1  which is fused to a F-box domain. 
     
     
         11 . A nucleic acid molecule which encodes for the single domain antibody of  claim 1 ; or a vector which comprises the nucleic acid molecule; or a host cell transformed with the nucleic acid molecule. 
     
     
         12 . A method of detecting the presence of at least one activated form of a Rho GTPase, comprising the steps of i) obtaining a sample from a subject, ii) contacting, in vitro, the sample with a single domain antibody of  claim 1 , iii) detecting the binding of said single domain antibody to said sample, and iv) comparing the binding detected in step (iii) with a standard, wherein a difference in binding relative to said standard indicates the presence of the at least one activated form of the Rho GTPase in the sample. 
     
     
         13 . A method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the single domain antibody of  claim 1  or a nucleic acid molecule encoding the single domain antibody.