Serine protease molecules and therapies

Abstract

Cell-targeted serine protease constructs are provided. Such constructs can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer). In some aspects, recombinant serine proteases, such as Granzyme B polypeptides, are provided that exhibit improved stability and cell toxicity. Methods and compositions for treating lapatinib or trastuzumab-resistant cancers are also provided.

What is claimed is: 
     
         1 . A recombinant polypeptide comprising a Granzyme B (GrB) coding sequence at least 80% identical to SEQ ID NO: 1 wherein the GrB coding sequence comprises one or more of the following features:
 (a) an amino acid substitution or deletion at the position corresponding to Asp 37;   (b) an amino acid substitution or deletion at the position corresponding to Asn 51;   (c) an amino acid substitution or deletion at the position corresponding to Asn 84;   (d) an amino acid substitution or deletion at the position corresponding to Arg 96;   (e) an amino acid substitution or deletion at the position corresponding to Arg 100;   (f) an amino acid substitution or deletion at the position corresponding to Arg 102;   (g) an amino acid substitution or deletion at the position corresponding to Asp 150;   (h) an amino acid substitution or deletion at the position corresponding to Arg 201;   (i) an amino acid substitution or deletion at the position corresponding to Cys 210;   (j) an amino acid substitution or deletion at the position corresponding to Lys 221;   (k) an amino acid substitution or deletion at the position corresponding to Lys 222;   (l) an amino acid substitution or deletion at the position corresponding to Lys 225; or   (m) an amino acid substitution or deletion at the position corresponding to Arg 226.   
     
     
         2 . The polypeptide of  claim 1 , wherein the polypeptide further comprises an amino acid sequence comprising a Cys, wherein the amino acid sequence is positioned C-terminally relative to the GrB coding sequence. 
     
     
         3 . The polypeptide of  claim 2 , wherein the polypeptide further comprises the sequence SSCSGSA (SEQ ID NO: 12) positioned C-terminally relative to the GrB coding sequence. 
     
     
         4 . The polypeptide of  claim 1 , wherein the GrB coding sequence comprises two, three, four or five of said features. 
     
     
         5 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar side chain at the position corresponding to Asp 37. 
     
     
         6 . The polypeptide of  claim 5 , wherein the polypeptide comprises an Asn substitution at the position corresponding to Asp 37. 
     
     
         7 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar side chain at the position corresponding to Asp 150. 
     
     
         8 . The polypeptide of  claim 7 , wherein the polypeptide comprises an Asn substitution at the position corresponding to Asp 150. 
     
     
         9 . The polypeptide of  claim 1 , wherein the polypeptide comprises an Ala or Ser substitution at the position corresponding to Asn 51. 
     
     
         10 . The polypeptide of  claim 1 , wherein the polypeptide comprises an Ala or Ser substitution at the position corresponding to Asn 84. 
     
     
         11 . The polypeptide of  claim 1 , wherein the polypeptide comprises an Ala substitution at the position corresponding to Cys 210. 
     
     
         12 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar or positively charged side chain at the position corresponding to Arg 96. 
     
     
         13 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar or positively charged side chain at the position corresponding to Arg 100. 
     
     
         14 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar or positively charged side chain at the position corresponding to Arg 102. 
     
     
         15 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar or positively charged side chain at the position corresponding to Arg 201. 
     
     
         16 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar or positively charged side chain at the position corresponding to Lys 221. 
     
     
         17 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar or positively charged side chain at the position corresponding to Lys 222. 
     
     
         18 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar or positively charged side chain at the position corresponding to Lys 225. 
     
     
         19 . The polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid substitution for a residue having polar or positively charged side chain at the position corresponding to Arg 226. 
     
     
         20 . The polypeptide of  claim 1 , wherein the polypeptide is conjugated to or fused with a cell binding moiety. 
     
     
         21 . The polypeptide of  claim 20 , wherein the polypeptide is conjugated to the cell binding moiety by a thioester linkage. 
     
     
         22 . The polypeptide of  claim 1 , wherein the polypeptide fused with a cell binding moiety positioned C-terminally relative to the GrB coding sequence. 
     
     
         23 . The polypeptide of  claim 20 , wherein the cell binding moiety is a VEGF, BLyS, an antibody or a cell-binding portion of any of the foregoing. 
     
     
         24 . The polypeptide of  claim 23 , wherein the antibody is a monoclonal, chimeric antibody, Fab′, Fab, F(ab′)2, single domain antibody, Fv, or single chain Fv (scFv) antibody. 
     
     
         25 . The polypeptide of  claim 23 , wherein the antibody is a human antibody, a humanized antibody or a deimmunized antibody. 
     
     
         26 . The polypeptide of  claim 23 , wherein the antibody is a 15A8, ZME-018, ScFvMEL, cetuximab or trastuzumab antibody. 
     
     
         27 . The polypeptide of  claim 20 , wherein the cell binding moiety binds to a protein, carbohydrate or lipid expressed on cancer cells. 
     
     
         28 . The polypeptide of  claim 20 , wherein the cell binding moiety binds to GP240, 5T4, HER1, HER2, CD-33, CD-38, flt1, Flk-1, CEA, FGFR3, IGFBP2 or IGF-1R. 
     
     
         29 . The polypeptide of  claim 20 , wherein the polypeptide or cell binding moiety is further conjugated to an imaging agent. 
     
     
         30 . A recombinant polypeptide comprising:
 (a) a recombinant Granzyme B (GrB) polypeptide at least 80% identical to SEQ ID NO: 1;   (b) a cell penetrating peptide (CPP); and   (c) a cell-targeting polypeptide,   wherein the CPP is positioned between the GrB polypeptide and the cell-targeting polypeptide or wherein the CPP is positioned C-terminal relative to the cell-targeting polypeptide.   
     
     
         31 . The recombinant polypeptide of  claim 30 , wherein the CPP is T1, T2, INF7 or 26. 
     
     
         32 . The recombinant polypeptide of  claim 30 , wherein the cell-targeting polypeptide is ZME or 4D5. 
     
     
         33 . The recombinant polypeptide of  claim 30 , wherein the polypeptide comprises from N-terminus to C-terminus the GrB polypeptide; a first linker; a T1 or INF7 CPP; a second linker and a ZME antibody. 
     
     
         34 . The recombinant polypeptide of  claim 30 , wherein the polypeptide comprises from N-terminus to C-terminus the GrB polypeptide; a first linker; a 4D5 antibody; a second linker and a 26 CPP. 
     
     
         35 . A composition comprising a polypeptide of  claim 1  in a pharmaceutically acceptable carrier. 
     
     
         36 . A polynucleotide molecules comprising a nucleic acid sequence encoding the polypeptide of  claim 1 . 
     
     
         37 . A host cell comprising the polynucleotide sequence of  claim 36 . 
     
     
         38 . The host cell of  claim 37 , wherein the host cell expresses a polypeptide according to  claim 1 . 
     
     
         39 . The host cell of  claim 37 , wherein the host cell is a mammalian cell, a yeast cell, a bacterial cell, a ciliate cell or an insect cell. 
     
     
         40 . A method of manufacturing a polypeptide comprising:
 (a) expressing a polynucleotide molecule of  claim 36  in a cell under conditions to produce the encoded polypeptide; and   (b) purifying the polypeptide from the cell.   
     
     
         41 . A method of treating a subject with a cell proliferative disease comprising administering to the subject an effective amount of a polypeptide of  claim 1 , wherein the polypeptide is conjugated to a cell-targeting moiety. 
     
     
         42 . The method of  claim 41 , wherein the cell proliferative disease is an autoimmune disease. 
     
     
         43 . The method of  claim 41 , wherein the cell proliferative disease is a cancer or precancerous condition. 
     
     
         44 . The method of  claim 43 , wherein the cancer is lung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck, esophageal, liver, skin, kidney, leukemia, bone, testicular, colon, or bladder cancer. 
     
     
         45 . The method of  claim 43 , further comprising administering at least a second anticancer therapy to the subject. 
     
     
         46 . The method of  claim 45 , wherein the second anticancer therapy is surgical therapy, chemotherapy, radiation therapy, gene therapy or immunotherapy. 
     
     
         47 . A method of treating a bacterial or viral infection comprising administering to the subject an effective amount of a polypeptide of  claim 1 , wherein the polypeptide is conjugated to a cell-targeting moiety. 
     
     
         48 . A composition for using in treatment of a subject comprising a polypeptide according to  claim 1 . 
     
     
         49 . The composition of  claim 48 , wherein the polypeptide is conjugated to a cell binding moiety. 
     
     
         50 . A targeting agent comprising:
 (a) a recombinant Granzyme B (GrB) coding sequence;   (b) a targeting polypeptide; and   (c) a cell penetrating peptide (CPP), having the sequence of SEQ ID NO: 22.   
     
     
         51 . The targeting agent of  claim 50 , wherein the targeting polypeptide binds to Her2/neu. 
     
     
         52 . The targeting agent of  claim 51 , wherein the targeting polypeptide is scFv 4D5. 
     
     
         53 . The targeting agent of  claim 50 , comprising from N-terminus to C-terminus (a) a recombinant GrB coding sequence; (i) a first linker peptide; (b) a targeting polypeptide; (ii) a second linker peptide; and (c) a cell penetrating peptide (CPP), having the sequence of SEQ ID NO: 22. 
     
     
         54 . The targeting agent of  claim 53 , wherein the first or second linker peptides comprises the sequence of SEQ ID NO: 13. 
     
     
         55 . The targeting agent of  claim 50 , wherein the recombinant GrB comprises a sequence in accordance with  claim 1 . 
     
     
         56 . The targeting agent of  claim 50 , comprising an amino acid sequence at least 90% identical to SEQ ID NO: 24. 
     
     
         57 . The targeting agent of  claim 56 , comprising the amino acid sequence of SEQ ID NO: 24. 
     
     
         58 . A method of treating a lapatinib or trastuzumab-resistant cancer in a subject comprising:
 (a) identifying a subject having a lapatinib or trastuzumab-resistant cancer; and   (b) administering a Her2/neu-targeted Granzyme therapeutic to the subject.   
     
     
         59 . The method of  claim 58 , wherein the subject has a breast cancer. 
     
     
         60 . The method of  claim 58 , wherein the subject has been previously treated with lapatinib or trastuzumab. 
     
     
         61 . The method of  claim 58 , wherein the subject has a lapatinib-resistance cancer. 
     
     
         62 . The method of  claim 58 , wherein the subject has a trastuzumab-resistance cancer. 
     
     
         63 . The method of  claim 58 , wherein the Her2/neu-targeted Granzyme therapeutic comprises a cell penetrating peptide (CPP). 
     
     
         64 . The method of  claim 58 , wherein the Her2/neu-targeted Granzyme therapeutic is a targeting agent in accordance with any one of  claims 50 - 57 . 
     
     
         65 . A recombinant polypeptide comprising a cleavage site that is susceptible to cleavage by a selected protease fused to a truncated serine protease having an IIGG, IVGG or ILGG at its N-terminus, such that, upon cleavage of the polypeptide by selected protease, the truncated serine protease having an N-terminal isoleucine will be released from the polypeptide. 
     
     
         66 . The polypeptide of  claim 65 , wherein the protease cleavage site is a caspase, furin, granzyme B or factor Xa cleavage sequence. 
     
     
         67 . The polypeptide of  claim 66 , wherein the protease cleavage sequence is a caspase-3 cleavage sequence. 
     
     
         68 . The polypeptide of  claim 65 , further comprising a cell-binding moiety, positioned N-terminally relative to the cleavage site. 
     
     
         69 . The polypeptide of  claim 68 , wherein the cell-binding moiety binds to GP240, 5T4, HER1, HER2, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, Fn14 or IGF-1R. 
     
     
         70 . The polypeptide of  claim 68 , wherein the cell-binding moiety is VEGF, BLyS, an antibody or a cell-binding portion of any of the foregoing. 
     
     
         71 . The polypeptide of  claim 70 , wherein the cell-binding moiety is an antibody heavy chain or an antibody light chain. 
     
     
         72 . The polypeptide of  claim 71 , wherein the antibody heavy chain or antibody light chain is a human IgG antibody heavy chain or antibody light chain. 
     
     
         73 . The polypeptide of  claim 65 , wherein the serine protease is a granzyme. 
     
     
         74 . The polypeptide of  claim 73 , wherein the granzyme is granzyme B (GrB), having at least 80% identical to SEQ ID NO:1. 
     
     
         75 . The polypeptide of  claim 74 , wherein upon cleavage by the selected protease the GrB polypeptide produced comprises the sequence IIGGHEAK (SEQ ID NO: 27) at its amino terminus. 
     
     
         76 . The polypeptide of  claim 75 , wherein the polypeptide comprises the sequence YVDEVDIIGGHEAK (SEQ ID NO: 26); RVRRIIGGHEAK (SEQ ID NO: 29); RVRRIIGGHEAK (SEQ ID NO: 30); (I/A)(E/D)GRIIGGHEAK (SEQ ID NO: 31); YEVDIIGGHEAK (SEQ ID NO: 32); WEHDIIGGHEAK (SEQ ID NO: 33); DVADIIGGHEAK (SEQ ID NO: 34); DEHDIIGGHEAK (SEQ ID NO: 35); DEVDIIGGHEAK (SEQ ID NO: 36); DMQDIIGGHEAK (SEQ ID NO: 37); LEVDIIGGHEAK (SEQ ID NO: 38); LEHDIIGGHEAK (SEQ ID NO: 39); VEIDIIGGHEAK (SEQ ID NO: 40); VEHDIIGGHEAK (SEQ ID NO: 41); IETDIIGGHEAK (SEQ ID NO: 42); LETDIIGGHEAK (SEQ ID NO: 43) or IEADIIGGHEAK (SEQ ID NO: 44). 
     
     
         77 . The polypeptide of  claim 76 , wherein the polypeptide comprises the sequence YVDEVDIIGGHEAK (SEQ ID NO: 26). 
     
     
         78 . The polypeptide of  claim 65 , further comprising a cell penetrating peptide (CPP). 
     
     
         79 . The polypeptide of  claim 74 , wherein the recombinant GrB comprises a sequence in accordance with  claim 1 . 
     
     
         80 . An antibody targeting moiety comprising a human antibody heavy chain and light chain, wherein the antibody light chain, heavy chain or both comprise a truncated serine protease positioned C-terminally relative to the antibody light chain and/or heavy chain. 
     
     
         81 . The antibody targeting agent of  claim 80 , wherein the truncated serine protease is a GrB polypeptide in accordance with  claim 74 . 
     
     
         82 . The antibody targeting agent of  claim 80 , wherein the antibody is a human IgG. 
     
     
         83 . The antibody targeting agent of  claim 82 , wherein the antibody is a human IgG1. 
     
     
         84 . A method of providing a serum-stable GrB polypeptide comprising obtaining a recombinant polypeptide of  claim 74 . 
     
     
         85 . A cell-targeting construct comprising:
 (a) a cell-targeting scFv antibody domain;   (b) an antibody heavy chain constant (Fc) domain; and   (c) a truncated serine protease.   
     
     
         86 . The cell-targeting construct of  claim 85 , wherein the polypeptide comprises from N- to C-terminus (c) a truncated serine protease; (b) a Fc domain; and (a) a scFv domain. 
     
     
         87 . The cell-targeting construct of  claim 85 , wherein the polypeptide comprises from N- to C-terminus (a) a scFv domain; (b) a Fc domain; (d) a peptide comprising a protease cleavage site and (c) a truncated serine protease. 
     
     
         88 . The cell-targeting construct of  claim 85 , wherein the truncated serine protease is a Granzyme B (GrB) polypeptide at least 80% identical to SEQ ID NO: 1. 
     
     
         89 . The cell-targeting construct of  claim 88 , wherein upon cleavage by the protease the GrB polypeptide produced comprises the sequence IIGGHEAK (SEQ ID NO: 27) at its amino terminus. 
     
     
         90 . The cell-targeting construct of  claim 87 , wherein the protease cleavage site is a caspase, furin, granzyme B or factor Xa cleavage sequence. 
     
     
         91 . The cell-targeting construct of  claim 88 , wherein the polypeptide comprises the sequence YVDEVDIIGGHEAK (SEQ ID NO: 26); RVRRIIGGHEAK (SEQ ID NO: 29); RVRRIIGGHEAK (SEQ ID NO: 30); (I/A)(E/D)GRIIGGHEAK (SEQ ID NO: 31); YEVDIIGGHEAK (SEQ ID NO: 32); WEHDIIGGHEAK (SEQ ID NO: 33); DVADIIGGHEAK (SEQ ID NO: 34); DEHDIIGGHEAK (SEQ ID NO: 35); DEVDIIGGHEAK (SEQ ID NO: 36); DMQDIIGGHEAK (SEQ ID NO: 37); LEVDIIGGHEAK (SEQ ID NO: 38); LEHDIIGGHEAK (SEQ ID NO: 39); VEIDIIGGHEAK (SEQ ID NO: 40); VEHDIIGGHEAK (SEQ ID NO: 41); IETDIIGGHEAK (SEQ ID NO: 42); LETDIIGGHEAK (SEQ ID NO: 43) or IEADIIGGHEAK (SEQ ID NO: 44). 
     
     
         92 . The cell-targeting construct of  claim 87 , wherein the protease cleavage site is a caspase-3 cleavage sequence. 
     
     
         93 . The cell-targeting construct of  claim 88 , wherein the polypeptide comprises the sequence YVDEVDIIGGHEAK (SEQ ID NO: 26). 
     
     
         94 . The cell-targeting construct of  claim 85 , wherein the Fc domain is a human IgG Fc domain. 
     
     
         95 . The cell-targeting construct of  claim 94 , wherein the Fc domain is a human IgG1 Fc domain. 
     
     
         96 . The cell-targeting construct of  claim 95 , wherein the scFv domain binds to GP240, 5T4, HER1, HER2, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, Fn14 or IGF-1R. 
     
     
         97 . The cell-targeting construct of  claim 96 , wherein the scFv domain binds to Fn14. 
     
     
         98 . The cell-targeting construct of  claim 97 , wherein the polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 45. 
     
     
         99 . The cell-targeting construct of  claim 88 , wherein the recombinant GrB comprises a sequence in accordance with  claim 1 . 
     
     
         100 . The cell-targeting construct of  claim 88 , wherein the GrB coding sequence comprises an amino acid substitution at the positions corresponding to Lys 27 and Arg 28. 
     
     
         101 . The cell-targeting construct of  claim 100 , wherein the amino acid substitutions at the positions corresponding to Lys 27 and Arg 28 are K27E or K27L and R28A. 
     
     
         102 . The cell-targeting construct of  claim 85 , wherein the GrB coding sequence comprises the sequence PVPN substituted at the positions corresponding to  82 PKN 84 .