Activatable antibodies having non-binding steric moieties and methods of using the same

Abstract

The invention relates generally to activatable antibodies and methods of making and using these activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

1 .- 37 . (canceled) 
     
     
         38 . An isolated nucleic acid molecule encoding an activatable antibody selected from the group consisting of:
 a) an activatable antibody comprising:
 an antibody or antibody fragment (AB) that binds specifically to a target a cleavable linker (CL) covalently linked to the AB, wherein the CL comprises a substrate (S) for an enzyme; and 
 a non-binding steric moiety (NB) covalently linked to the CL; 
 wherein 
 the encoded activatable antibody in an uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: NB-CL-AB or AB-CL-NB, 
 when the encoded activatable antibody is in an uncleaved state, the NB does not bind specifically to the AB and the NB interferes with binding of the AB to the target, 
 when the encoded activatable antibody is in a cleaved state, the NB does not interfere with binding of the AB to the target and 
   b) an activatable antibody comprising:
 an antibody or antibody fragment (AB) that binds specifically to a target; 
 a cleavable linker (CL) covalently linked to the AB, wherein the CL comprises a substrate (S) for an enzyme; and 
 a binding partner (BP) for a non-binding steric moiety (NB), wherein the BP is covalently linked to the CL; and 
 wherein 
 the encoded activatable antibody in an uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: BP-CL-AB or AB-CL-BP, 
 when the encoded activatable antibody is in an uncleaved state and the NB is bound to the BP, the NB does not bind specifically to the AB and the NB interferes with binding of the AB to the target, 
 when the encoded activatable antibody is in a cleaved state, the NB does not interfere with binding of the AB to the target. 
   
     
     
         39 . A vector comprising the isolated nucleic acid molecule of  claim 38 . 
     
     
         40 . A method of producing an activatable antibody by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises the nucleic acid of  claim 38 . 
     
     
         41 .- 44 . (canceled) 
     
     
         45 . The isolated nucleic acid of  claim 38 , wherein the NB is a soluble, globular protein. 
     
     
         46 . The isolated nucleic acid of  claim 38 , wherein the NB is a protein that circulates in the bloodstream. 
     
     
         47 . The isolated nucleic acid of  claim 38 , wherein the NB is selected from the group consisting of albumin, fibrinogen, fibronectin, hemoglobin, transferrin, an immunoglobulin domain, and other serum proteins. 
     
     
         48 . The isolated nucleic acid of  claim 38 , wherein the BP is selected from the group consisting of an albumin binding peptide, a fibrinogen binding peptide, a fibronectin binding peptide, a hemoglobin binding peptide, a transferrin binding peptide, an immunoglobulin domain binding peptide, and other serum protein binding peptides. 
     
     
         49 . The isolated nucleic acid of  claim 38 , wherein the substrate (S) is a protease, and wherein the protease is co-localized with the target of the AB in a tissue. 
     
     
         50 . The isolated nucleic acid of  claim 38 , wherein the CL comprises a first flexible portion (FP1) and a second flexible portion (FP2), wherein the encoded activatable antibody in an uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: NB-FP1-S-FP2-AB, AB-FP2-S-FP1-NB, BP-FP1-S-FP2-AB, and AB-FP2-S-FP1-BP. 
     
     
         51 . The isolated nucleic acid of  claim 50 , wherein the two flexible portions of the CL are not identical to each other. 
     
     
         52 . The isolated nucleic acid of  claim 50 , wherein each of FP1 and FP2 is a peptide of about 1 to 20 amino acids in length. 
     
     
         53 . The isolated nucleic acid of  claim 50 , wherein at least one of FP1 or FP2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GGS) n , (GSGGS) n  (SEQ ID NO: 5), (GGGS) n  (SEQ ID NO: 6), GGSG (SEQ ID NO: 7), GGSGG (SEQ ID NO: 8), GSGSG (SEQ ID NO: 9), GSGGG (SEQ ID NO: 10), GGGSG (SEQ ID NO: 11), and GSSSG (SEQ ID NO: 12), where n is an integer of at least one. 
     
     
         54 . The isolated nucleic acid of  claim 38 , wherein the CL is a polypeptide of up to 50 amino acids in length or wherein the substrate (S) is a polypeptide of up to 15 amino acids in length. 
     
     
         55 . The isolated nucleic acid of  claim 38 , wherein the AB is an antigen binding fragment selected from the group consisting of a Fab fragment, a F(ab′) 2  fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. 
     
     
         56 . The isolated nucleic acid of  claim 38 , wherein the target for the activatable antibody is selected from the group consisting of the targets listed in Table 1. 
     
     
         57 . The isolated nucleic acid of  claim 38 , wherein the AB is or is derived from cetuximab, panitumumab, zalutumumab, mapatumumab, matuzumab, nimotuzumab, ICR62, mAb 528, CH806, MDX-447 or any antibody listed in Table 2. 
     
     
         58 . The isolated nucleic acid of  claim 38 , wherein the AB is or is derived from cetuximab. 
     
     
         59 . The isolated nucleic acid of  claim 38 , wherein the activatable antibody comprises a second AB wherein the target for the second AB is a target selected from the group consisting of the targets listed in Table 1. 
     
     
         60 . The isolated nucleic acid of  claim 38 , wherein the enzyme is selected from the group consisting of the enzymes listed in Table 3. 
     
     
         61 . The isolated nucleic acid of  claim 38 , wherein the enzyme is selected from the group consisting of uPA, legumain, MT-SP1, MMP-9, MMP-14 and TMPRSS4. 
     
     
         62 . The isolated nucleic acid of  claim 38 , wherein the NB in the uncleaved encoded activatable antibody reduces the ability of the AB to bind the target by at least 50%, as compared to the ability of the cleaved encoded activatable antibody to bind the target. 
     
     
         63 . The method of  claim 40  comprising a step of conjugating an agent to the expressed activatable antibody. 
     
     
         64 . The method of  claim 63 , wherein the agent has one or more of the properties selected from the group consisting of:
 (i) the agent is a therapeutic agent,   (ii) the agent is an antineoplastic agent,   (iii) the agent is a toxin or fragment thereof,   (iv) the agent is a detectable marker,   (v) the agent is an agent selected from the group consisting of the agents listed in Table 4,   (vi) the agent is conjugated to the AB via a linker, and   (vii) the agent is conjugated to the AB via a cleavable linker.