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.

What is claimed is: 
     
         1 . An isolated polypeptide comprising an antibody or antigen binding fragment thereof (AB) that binds a target and a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of PTNL (SEQ ID NO: 362), AANL (SEQ ID NO: 363), and PTN (SEQ ID NO: 364), wherein the cleavable moiety is a substrate for a protease. 
     
     
         2 . The isolated polypeptide of  claim 1 , wherein the CM is cleaved by at least legumain. 
     
     
         3 . The isolated polypeptide of  claim 1 , wherein the CM is a substrate for a protease that is co-localized in a tissue with the target. 
     
     
         4 . The isolated polypeptide of  claim 1 , wherein the antigen binding fragment thereof 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. 
     
     
         5 . The isolated polypeptide of  claim 1 , wherein the AB is linked directly to the CM. 
     
     
         6 . The isolated polypeptide of  claim 1 , wherein the AB is linked to the CM via a linking peptide. 
     
     
         7 . The isolated polypeptide of  claim 1 , wherein the isolated polypeptide comprises a masking moiety (MM), wherein the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB for binding to the target. 
     
     
         8 . The isolated polypeptide of  claim 7 , wherein the MM is a polypeptide of about 2-40 amino acids in length. 
     
     
         9 . The isolated polypeptide of  claim 7 , wherein the MM is linked to the CM such that the isolated polypeptide in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM. 
     
     
         10 . The isolated polypeptide of  claim 9 , wherein the isolated polypeptide comprises a linking peptide between the MM and the CM. 
     
     
         11 . The isolated polypeptide of  claim 9 , wherein the isolated polypeptide comprises a linking peptide between the CM and the AB. 
     
     
         12 . The isolated polypeptide of  claim 9 , wherein the isolated polypeptide comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the isolated polypeptide has the structural arrangement from N-terminus to C-terminus as follows in the uncleaved state: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. 
     
     
         13 . The isolated polypeptide of  claim 12 , wherein the two linking peptides need not be identical to each other. 
     
     
         14 . The isolated polypeptide of  claim 12 , wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length. 
     
     
         15 . The isolated polypeptide of  claim 7 , wherein the amino acid sequence of the MM is different from that of the target and is no more than 50% identical to the amino acid sequence of a natural binding partner of the AB. 
     
     
         16 . The isolated polypeptide of  claim 7 , wherein the MM does not interfere or compete with the AB for binding to the target in a cleaved state. 
     
     
         17 . The polypeptide of  claim 1 , wherein the CM is resistant to cleavage by one or more proteases selected from the group consisting of KLK5, KLK7 and plasmin. 
     
     
         18 . An isolated polypeptide comprising a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of:
 a) X 1 X 2 PX 3 VKX 4 X 5 X 6 X 7  (SEQ ID NO: 366), where X 1  is Ala, Arg, Asp, Glu, Lys, Pro, or Tyr; X 2  is Ala, Arg, Gln, Gly, His, Leu, Pro, Ser, Thr or Val; X 3  is Arg, Glu, Gly, Leu, Lys, Phe, Pro, or Ser; X 4  is Ala, Met, Ser, Thr, or Val; X 5  is Leu, Met or Val; X 6  is Asp, Glu, Gly, Leu, Lys, Pro, Ser, Thr, or Val; and X 7  is Arg, Asp, Gln, Glu, Gly, Leu, Lys, Met, Phe, Ser, or Val;   b) X 1 X 2 PX 3 VKX 4 VX 5 X 6  (SEQ ID NO: 367), where X 1  is Ala, Glu, Lys, or Pro; X 2  is Arg, Gln, Gly, His, or Leu; X 3  is Arg, Glu, Lys, Phe, or Ser; X 4  is Met, Ser, or Val; X 5  is Asp, Lys, Ser, or Thr; and X 6  is Gln, Glu, Gly, or Leu;   c) X 1 X 2 PX 3 VKX 4 LX 5 X 6  (SEQ ID NO: 368), where X 1  is Asp, Glu, or Tyr; X 2  is Ala, Arg, Leu, or Val; X 3  is Arg, Gly, Leu, or Lys; X 4  is Ala or Ser; X 5  is Glu, Pro, or Val; and X 6  is Leu, Lys, Met, or Ser;   d) X 1 X 2 PX 3 VKX 4 MX 5 X 6  (SEQ ID NO: 369), where X 1  is Ala, Arg, Asp, Glu, or Pro; X 2  is Arg, Pro, Ser, Thr, or Val; X 3  is Leu, Lys, Pro, or Ser; X 4  is Met, Ser, Thr, or Val; X 5  is Asp, Gly, Leu, Pro, or Val; and X 6  is Arg, Asp, Glu, Phe, or Val;   e) X 1 X 2 PX 3 MKX 4 X 5 X 6 X 7  (SEQ ID NO: 370), where X 1  is Ala, Asp, Arg, Glu, Gly, or Pro; X 2  is Arg, Glu, Lys, Phe, Pro, Ser, or Val; X 3  is Arg, Asp, Glu, His, Phe, Pro, Thr, or Val; X 4  is Leu, Ser, or Thr; X 5  is Leu, Met, Phe, or Val; X 6  is Ala, Arg, Gln, Gly, Pro, or Ser; and X 7  is Gly, His, Leu, Pro, or Trp;   f) X 1 X 2 PX 3 MKLFX 5 G (SEQ ID NO: 371), where X 1  is Glu or Pro; X 2  is Lys or Pro; X 3  is Arg or Asp; and X 5  is Gln or Pro;   g) X 1 X 2 PX 3 MKSX 4 X 5 X 6  (SEQ ID NO: 372), where X 1  is Arg, Asp, or Glu; X 2  is Arg, Glu, or Ser; X 3  is Glu, Phe, or Val; X 4  is Leu or Met; X 5  is Ala, Pro, or Ser; and X 6  is Gly, Leu, or Trp;   h) X 1 X 2 PX 3 MKTX 4 X 5 X 6  (SEQ ID NO: 373), where X 1  is Ala, Asp, or Gly; X 2  is Phe, Ser, or Val; X 3  is His, Pro, or Thr; X 4  is Leu, Phe, or Val; X 5  is Arg, Gln, or Gly; and X 6  is Leu, His, or Pro;   i) X 1 X 2 PSFKLVX 3 X 4  (SEQ ID NO: 374), where X 1  is Ala or Asn; X 2  is Met or Pro; X 3  is Asn or Thr; and X 4  is Ala or Gly;   j) X 1 X 2 PX 3 LKX 4 X 5 X 6 X 7  (SEQ ID NO: 375), where X 1  is Arg, Pro or Val; X 2  is Ala, Phe, Pro, or Val; X 3  is Arg, Gln, Ser, or Val; X 4  is Leu or Ser; X 5  is Ile, Leu, or Phe; X 6  is Glu, Pro, Lys, or Val; and X 7  is Asp, Leu, Pro, or Trp;   k) X 1 X 2 PX 3 X 4 RX 5 X 6 X 7 X 8  (SEQ ID NO: 376), where X 1  is Ala, Pro, Ser, or Val; X 2  is Ala, Ile, Leu, Phe, Pro, or Val; X 3  is Arg, Asp, Glu, Lys, or Tyr; X 4  is Leu, Met, Phe, or Val; X 5  is Leu, Met, Ser, or Val; X 6  is Met, Phe, or Val; X 7  is Arg, Asp, Phe, Pro, or Ser; and X 8  is Glu, Met, Ser, or Val; and   l) GLGTPRGLFA (SEQ ID NO: 290),   wherein the cleavable moiety is a substrate for a protease.   
     
     
         19 . The isolated polypeptide of  claim 18 , wherein the CM comprises the amino acid sequence of X 1 X 2 PX 3 VKX 4 X 5 X 6 X 7  (SEQ ID NO: 366), where X 1  is Ala, Asp, or Glu; X 2  is Ala, Arg, Gln, Gly, His, Leu, Pro, Ser, Thr or Val; X 3  is Glu or Lys; X 4  is Ala, Met, Ser, Thr, or Val; X 5  is Leu, Met or Val; X 6  is Asp or Pro; and X 7  is Glu; or the amino acid sequence of X 1 X 2 PX 3 X 4 RX 5 X 6 X 7 X 8  (SEQ ID NO: 376), where X 1  is Ala; X 2  is Ala, Ile, Leu, Phe, Pro, or Val; X 3  is Glu or Lys; X 4  is Leu, Met, Phe, or Val; X 5  is Leu, Met, Ser, or Val; X 6  is Met, Phe, or Val; X 7  is Asp or Pro; and X 8  is Glu. 
     
     
         20 . The isolated polypeptide of  claim 18 , wherein the CM is cleaved by at least plasmin. 
     
     
         21 . The isolated polypeptide of  claim 18 , wherein the CM comprises an amino acid sequence selected from the group consisting of:
 a) the amino acid sequence of X 1 X 2 PX 3 VKX 4 X 5 X 6 X 7  (SEQ ID NO: 366) or X 1 X 2 PX 3 VKX 4 VX 5 X 6  (SEQ ID NO: 367) and comprises an amino acid sequence selected from the group consisting of EHPRVKVVSE (SEQ ID NO: 281), ALPSVKMVSE (SEQ ID NO: 287), KGPKVKVVTL (SEQ ID NO: 296), PRPFVKSVDQ (SEQ ID NO: 299), and EQPEVKMVKG (SEQ ID NO: 306);   b) the amino acid sequence of X 1 X 2 PX 3 VKX 4 X 5 X 6 X 7  (SEQ ID NO: 366) or X 1 X 2 PX 3 VKX 4 LX 5 X 6  (SEQ ID NO: 368) and comprises an amino acid sequence selected from the group consisting of ERPGVKSLVL (SEQ ID NO: 297), DLPLVKSLPS (SEQ ID NO: 308), EAPKVKALPK (SEQ ID NO: 309), and YVPRVKALEM (SEQ ID NO: 317);   c) the amino acid sequence of X 1 X 2 PX 3 VKX 4 X 5 X 6 X 7  (SEQ ID NO: 366) or X 1 X 2 PX 3 VKX 4 MX 5 X 6  (SEQ ID NO: 369) and comprises an amino acid sequence selected from the group consisting of ETPSVKTMGR (SEQ ID NO: 288), DRPKVKTMDF (SEQ ID NO: 291), RVPKVKVMLD (SEQ ID NO: 292), APPLVKSMVV (SEQ ID NO: 293), and PSPPVKMMPE (SEQ ID NO: 365);   d) the amino acid sequence of X 1 X 2 PX 3 MKX 4 X 5 X 6 X 7  (SEQ ID NO: 370) or X 1 X 2 PX 3 MKLX 4 X 5 X 6  (SEQ ID NO: 371) and comprises an amino acid sequence selected from the group consisting of PPPDMKLFPG (SEQ ID NO: 282) and EKPRMKLFQG (SEQ ID NO: 316);   e) the amino acid sequence of X 1 X 2 PX 3 MKX 4 X 5 X 6 X 7  (SEQ ID NO: 370) or X 1 X 2 PX 3 MKSX 4 X 5 X 6  (SEQ ID NO: 372) and comprises an amino acid sequence selected from the group consisting of REPFMKSLPW (SEQ ID NO: 294), ESPVMKSMAL (SEQ ID NO: 301), and DRPEMKSLSG (SEQ ID NO: 305);   f) the amino acid sequence of X 1 X 2 PX 3 MKX 4 X 5 X 6 X 7  (SEQ ID NO: 370) or X 1 X 2 PX 3 MKTX 4 X 5 X 6  (SEQ ID NO: 373) and comprises an amino acid sequence selected from the group consisting of GFPHMKTFQH (SEQ ID NO: 310), ASPTMKTVGL (SEQ ID NO: 312), and DVPPMKTLRP (SEQ ID NO: 313);   g) the amino acid sequence of X 1 X 2 PSFKLVX 3 X 4  (SEQ ID NO: 374) and comprises an amino acid sequence selected from the group consisting of APPSFKLVNA (SEQ ID NO: 285) and NMPSFKLVTG (SEQ ID NO: 304);   h) the amino acid sequence of X 1 X 2 PX 3 LKX 4 X 5 X 6 X 7  (SEQ ID NO: 375) and comprises an amino acid sequence selected from the group consisting of PPPVLKLLEW (SEQ ID NO: 283), PVPRLKLIKD (SEQ ID NO: 295), RFPSLKSFPL (SEQ ID NO: 300), and VAPQLKSLVP (SEQ ID NO: 302); and   i) the amino acid sequence of X 1 X 2 PX 3 X 4 RX 5 X 6 X 7 X 8  (SEQ ID NO: 376) and comprises an amino acid sequence selected from the group consisting of VLPELRSVFS (SEQ ID NO: 284), PPPEVRSFSV (SEQ ID NO: 286), AIPRVRLFDV (SEQ ID NO: 289), AVPKVRVVPE (SEQ ID NO: 307), AFPDMRSVRS (SEQ ID NO: 314), and SAPYFRMMDM (SEQ ID NO: 315).   
     
     
         22 . The isolated polypeptide of  claim 18 , wherein the CM comprises an amino acid sequence selected from the group consisting of APPLVKSMVV (SEQ ID NO: 293), PRPFVKSVDQ (SEQ ID NO: 299), NMPSFKLVTG (SEQ ID NO: 304), GFPHMKTFQH (SEQ ID NO: 310), and ASPTMKTVGL (SEQ ID NO: 312); and wherein the cleavable moiety is a substrate for a protease. 
     
     
         23 . The isolated polypeptide of  claim 18 , wherein the isolated polypeptide comprises an antibody or antigen binding fragment thereof (AB) that binds a target. 
     
     
         24 . The isolated polypeptide of  claim 23 , wherein the isolated polypeptide comprises a masking moiety (MM), wherein the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB for binding to the target. 
     
     
         25 . The isolated polypeptide of  claim 18 , wherein the CM is resistant to cleavage by one or more proteases selected from the group consisting of MMP-9, KLK5 and KLK7. 
     
     
         26 . An isolated polypeptide comprising an antibody or antigen binding fragment thereof (AB) that binds a target and a cleavable moiety (CM) comprising the amino acid sequence DEVD (SEQ ID NO: 379), wherein the cleavable moiety is a substrate for a protease. 
     
     
         27 . The isolated polypeptide of  claim 26 , wherein the CM is cleaved by at least caspase. 
     
     
         28 . The isolated polypeptide of  claim 26 , wherein the isolated polypeptide comprises a masking moiety (MM), wherein the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB for binding to the target. 
     
     
         29 . The isolated polypeptide of  claim 26 , wherein the CM is resistant to cleavage by one or more proteases selected from the group consisting of KLK5, KLK7, and plasmin. 
     
     
         30 . A method of manufacturing the isolated polypeptide of  claim 1 , the method comprising culturing a cell comprising a nucleic acid construct that encodes the isolated polypeptide under conditions that lead to expression of the isolated polypeptide. 
     
     
         31 . A method of manufacturing the isolated polypeptide of  claim 18 , the method comprising culturing a cell comprising a nucleic acid construct that encodes the isolated polypeptide under conditions that lead to expression of the isolated polypeptide. 
     
     
         32 . A method of manufacturing the isolated polypeptide of  claim 26 , the method comprising culturing a cell comprising a nucleic acid construct that encodes the isolated polypeptide under conditions that lead to expression of the isolated polypeptide. 
     
     
         33 . A method of treating a neoplasm, an autoimmune disease or an inflammatory disease or inhibiting angiogenesis in a subject, the method comprising administering a therapeutically effective amount of the isolated polypeptide of  claim 1  to a subject in need thereof. 
     
     
         34 . A method of treating a neoplasm, an autoimmune disease or an inflammatory disease or inhibiting angiogenesis in a subject, the method comprising administering a therapeutically effective amount of the isolated polypeptide of  claim 18  to a subject in need thereof. 
     
     
         35 . A method of treating a neoplasm, an autoimmune disease or an inflammatory disease or inhibiting angiogenesis in a subject, the method comprising administering a therapeutically effective amount of the isolated polypeptide of  claim 26  to a subject in need thereof.