Modified antibody compositions, methods of making and using thereof

Abstract

The present disclosure provides modified antibodies which contain an antibody or antibody fragment (AB) modified with a masking moiety (MM). Such modified antibodies can be further coupled to a cleavable moiety (CM), resulting in activatable antibodies (AAs), wherein the CM is capable of being cleaved, reduced, photolysed, or otherwise modified. AAs can exhibit an activatable conformation such that the AB is more accessible to a target after, for example, removal of the MM by cleavage, reduction, or photolysis of the CM in the presence of an agent capable of cleaving, reducing, or photolysing the CM. The disclosure further provides methods of making and using such modified antibodies and activatable antibodies.

1 . A modified antibody comprising: an antibody or antibody fragment (AB), capable of specifically binding its target, coupled to a masking moiety (MM), wherein the coupling of the MM reduces the ability of the AB to bind its target such that that the dissociation constant (K d ) of the AB when coupled to the MM towards the target is at least 100 times greater than the K d  of the AB when not coupled to the MM towards the target. 
     
     
         2 - 3 . (canceled) 
     
     
         4 . The modified antibody of  claim 1  wherein in the presence of a target, the coupling of the MM to the AB reduces the ability of the AB to bind its target by at least 90%, as compared to the ability of the AB to bind its target when not coupled to the MM, when assayed in vitro using a target displacement assay. 
     
     
         5 . The modified antibody of  claim 4  wherein the coupling of the MM to the AB reduces the ability of the AB to bind its target for at least 12 hours. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The modified antibody of  claim 1  wherein the dissociation constant (K d ) of the MM towards the AB is at least 100 times greater than the K d  of the AB towards the target. 
     
     
         9 . The modified antibody of  claim 8  wherein the K d  of the MM towards the AB is lower than 10 nM. 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The modified antibody of  claim 1  further coupled to a cleavable moiety (CM). 
     
     
         13 . The modified antibody of  claim 12  wherein the CM is capable of being cleaved by an enzyme, reduced by a reducing agent, or photolysed. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . The modified antibody of  claim 1  wherein the MM is capable of specifically binding to the antigen-binding domain of the AB. 
     
     
         17 . The modified antibody of  claim 16  wherein the binding of the MM to the antigen-binding domain is non-covalent. 
     
     
         18 . The modified antibody of  claim 1  wherein the MM reduces the AB's ability to bind its target allosterically. 
     
     
         19 . The modified antibody of  claim 1  wherein the MM reduces the AB's ability to bind its target sterically. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . The modified antibody of  claim 1  wherein the antibody fragment is 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. 
     
     
         24 . The modified antibody of  claim 1  wherein the AB is selected from the group consisting of the antibodies in Table 2. 
     
     
         25 . (canceled) 
     
     
         26 . The modified antibody of  claim 24  wherein the AB is cetuximab, panitumumab, infliximab, adalimumab, efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8, alemtuzumab, ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab, infliximab, bevacizumab, or figitumumab. 
     
     
         27 . The modified antibody of  claim 1  wherein the target is selected from the group consisting of the targets in Table 1. 
     
     
         28 . (canceled) 
     
     
         29 . The modified antibody of  claim 27  wherein the target is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4. 
     
     
         30 . The modified antibody of  claim 1  further comprising a second AB wherein the target for the second AB is selected from the group consisting of the targets in Table 1. 
     
     
         31 . The modified antibody of  claim 12  wherein the CM is located within the MM. 
     
     
         32 . The modified antibody of  claim 12  wherein the CM is a substrate for an enzyme selected from the group consisting of the enzymes in Table 3. 
     
     
         33 . The modified antibody of  claim 32  wherein the CM is a substrate for legumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, or PSA. 
     
     
         34 . The modified antibody of  claim 32  wherein the AB is selected from the group consisting of the antibodies in Table 2. 
     
     
         35 . (canceled) 
     
     
         36 . The modified antibody of  claim 34  wherein the AB is cetuximab, panitumumab, infliximab, adalimumab, efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8, alemtuzumab, ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab, infliximab, bevacizumab, or figitumumab. 
     
     
         37 . The modified antibody of  claim 32  wherein the target is selected from the group consisting of the targets in Table 1. 
     
     
         38 . (canceled) 
     
     
         39 . The modified antibody of  claim 37  wherein the target is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4. 
     
     
         40 - 41 . (canceled) 
     
     
         42 . The modified antibody of  claim 1  wherein the MM does not comprise more than 50% amino acid sequence similarity to a natural binding partner of the AB. 
     
     
         43 - 45 . (canceled) 
     
     
         46 . The modified antibody of  claim 12  further comprising two linker peptides, wherein the first linker peptide is between the AB and the CM and the second linker peptide is positioned between the MM and the CM. 
     
     
         47 - 49 . (canceled) 
     
     
         50 . The modified antibody of  claim 46  wherein the linkers are selected from the group consisting of a cleavable linker, a non-cleavable linker, and a branched linker. 
     
     
         51 . The modified antibody of  claim 4 - 12  further comprising a detectable moiety. 
     
     
         52 . (canceled) 
     
     
         53 . The modified antibody of  claim 51  wherein the detectable moiety is a diagnostic agent. 
     
     
         54 . The modified antibody of  claim 1  further comprising an agent conjugated to the AB. 
     
     
         55 . The modified antibody of  claim 54  wherein the agent is a therapeutic agent. 
     
     
         56 . The modified antibody of  claim 54  further comprising a cleavable moiety (CM) coupled to the AB, capable of being specifically cleaved. 
     
     
         57 . The modified antibody of  claim 56  wherein the agent is an antineoplastic agent. 
     
     
         58 - 69 . (canceled) 
     
     
         70 . The modified antibody of  claim 12  wherein the serum half-life of the composition is at least 5 days when administered to an organism. 
     
     
         71 . The modified antibody of  claim 1  wherein the consensus sequence of the MM is CISPRGC, C(N/P)H(HVF)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG, xCxxYQCLxxxxxx, XXQPxPPRVXX, PxPGFPYCxxxx, xxxxQxxPWPP, GxGxCYTILExxCxxxR, GxxxCYxlxExxCxxxx, GxxxCYxIxExWCxxxx, xxxCCxxYxIxxCCxxx, or xxxxxYxILExxxxx. 
     
     
         72 . The modified antibody of  claim 57  wherein the consensus sequence is specific for binding to an anti-VEGF antibody, an anti-EFGR antibody, or an anti-CTLA-4 antibody. 
     
     
         73 - 133 . (canceled) 
     
     
         134 . An activatable antibody complex (AAC) comprising:
 (a) two antibodies or antibody fragments (AB1 and AB2), each capable of specifically binding its target;   (b) at least one masking moiety (MM) coupled to either AB1 or AB2, capable of inhibiting the specific binding of AB1 and AB2 to their targets; and   (c) at least one cleavable moiety (CM) coupled to either AB1 or AB2, capable of being specifically cleaved by an enzyme whereby activating the AAC composition;   wherein when the AAC is in an uncleaved state, the MM inhibits the specific binding of AB1 and AB2 to their targets and when the AAC is in a cleaved state, the MM does not inhibit the specific binding of AB1 and AB2 to their targets.   
     
     
         135 - 152 . (canceled) 
     
     
         153 . A method of treating or diagnosing a condition in a subject including administering to the subject a composition comprising:
 (a) an antibody or antibody fragment (AB), capable of specifically binding its target;   (b) a masking moiety (MM) coupled to the AB, capable of inhibiting the specific binding of the AB to its target; and   (c) a cleavable moiety (CM) coupled to the AB, capable of being specifically cleaved by an enzyme;   wherein upon administration to the subject, when the AA is not in the presence of sufficient enzyme activity to cleave the CM, the MM reduces the specific binding of the AB to its target by at least 90% when compared to when the AA is in the presence of sufficient enzyme activity to cleave the CM and the MM does not inhibit the specific binding of the AB to its target.   
     
     
         154 - 164 . (canceled) 
     
     
         165 . A method of inhibiting angiogenesis in a mammalian subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of  claim 12 . 
     
     
         166 - 182 . (canceled) 
     
     
         183 . The antibody of  claim 1  wherein the affinity of binding of the antibody to its target is lower in a first tissue when compared to the binding of the antibody to its target in a second tissue. 
     
     
         184 . The antibody therapeutic of  claim 183  wherein the target is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4. 
     
     
         185 . The antibody of  claim 183  wherein the first tissue is a healthy tissue and the second tissue is a diseased tissue; the first tissue is an early stage tumor and the second tissue is a late stage tumor; the first tissue is a benign tumor and the second tissue is a malignant tumor; the first tissue and second tissue are spatially separated; or the first tissue is epithelial tissue and the second tissue is breast, head, neck, lung, pancreatic, nervous system, liver, prostate, urogenital, or cervical tissue. 
     
     
         186 - 234 . (canceled)