CELL-TARGETED MOLECULES COMPRISING AMINO-TERMINUS PROXIMAL OR AMINO-TERMINAL SHIGA TOXIN A SUBUNIT EFFECTOR REGIONS
The present invention provides cell-targeted molecules comprising binding regions for cell-type specific targeting and Shiga toxin A Subunit effector regions for Shiga toxin effector functions, wherein the Shiga toxin effector regions are at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeted molecule, and optionally comprising a disrupted, furin-cleavage motif between the Shiga toxin effector region and the binding region. The cell-targeted molecules of the invention exhibit a more optimized cytotoxicity and/or improved, in vivo tolerability as compared to related molecules comprising less amino-terminus proximal, Shiga toxin effector regions and/or furin-cleavage sensitive, wild-type, Shiga toxin effector regions. The cell-targeted molecules of the invention have uses, such as, e.g., in methods involving targeted killing of cells, delivering exogenous materials into cells, labeling subcellular compartments of cells, and diagnosing and/or treating a variety of conditions, including cancers, tumors, other growth abnormalities, immune disorders, and microbial infections.
1 . A cell-targeted molecule having an amino-terminus and comprising
a) a binding region comprising a peptide or polypeptide and capable of specifically binding at least one extracellular target biomolecule and
b) a Shiga toxin effector polypeptide region;
wherein the Shiga toxin effector polypeptide region is positioned at or proximal to the amino-terminus of the cell-targeted molecule.
2 . The cell-targeted molecule of claim 1 , wherein the cell-targeted molecule is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a second cell-targeted molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the second cell-targeted molecule.
3 . The cell-targeted molecule of claim 2 , wherein the binding region is linked to the cell-targeted molecule at a position carboxy-terminal to the Shiga toxin effector polypeptide region.
4 . The cell-targeted molecule of claim 3 , wherein the binding region comprises an immunoglobulin-type binding region comprising a polypeptide selected from the group consisting of:
single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronectin-derived 10 th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystalline-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality.
5 . The cell-targeted molecule of claim 4 , whereby administration of the cell-targeted molecule to a first population of cells whose members are physically coupled to extracellular target biomolecules of the immunoglobulin-type binding region of the cell-targeted molecule, and a second population of cells whose members are not physically coupled to any extracellular target biomolecule of the immunoglobulin-type binding region, the cytotoxic effect of the cell-targeted molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
6 . The cell-targeted molecule of claim 5 , wherein the Shiga toxin effector polypeptide region comprises or consists essentially of a polypeptide represented by the amino acid sequence selected from the group consisting of:
i) amino acids 75 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;
ii) amino acids 1 to 241 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; and
iii) amino acids 1 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
7 . The cell-targeted molecule of claim 5 , wherein the immunoglobulin-type binding region is capable of binding to an extracellular target biomolecule selected from the group consisting of:
CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72, tyrosine-protein kinase transmembrane receptor, carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha Vbeta3, Alpha5beta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen, prostate stem cell antigen, human aspartyl (asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gp100, tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, CD38, CD15, CD23, CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, galectin-9, mrp-14, PD-L1, Siglec-8, Siglec-10, CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86, CD105, CD115, F4/80, ILT-3, galectin-3, CD11a-c, GITRL, MHC class I molecule, MHC class II molecule, CD284, CD107-Mac3, CD195, HLA-DR, CD16/32, CD282, and any immunogenic fragment of any of the foregoing.
8 . The cell-targeted molecule of claim 7 , wherein the immunoglobulin-type binding region comprises or consists essentially of the polypeptide sequence of amino acids 269-508 of any one of SEQ ID NOs: 4, 8, 12, or 16.
9 . The cell-targeted molecule of claim 8 , which comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 4-31.
10 . The cell-targeted molecule of any one of claims 3 - 8 , wherein the Shiga toxin effector polypeptide region comprises
a) a Shiga toxin A1 fragment region having a carboxy-terminus and
b) a disrupted furin-cleavage motif at the carboxy-terminus of the Shiga toxin A1 fragment region.
11 . The cell-targeted molecule of claim 10 , wherein the cell-targeted molecule is capable of exhibiting improved, in vivo tolerability compared to in vivo tolerability of a second cell-targeted molecule comprising the binding region and a second, Shiga toxin effector polypeptide region consisting of a wild-type, Shiga toxin A1 fragment, wherein the binding region is linked to the second cell targeted molecule carboxy-terminal to the wild-type, Shiga toxin A1 fragment.
12 . The cell-targeted molecule of claim 10 or claim 11 , wherein the cell-targeted molecule is capable of exhibiting cytotoxicity equivalent to a second cell-targeted molecule comprising the binding region and a second, Shiga toxin effector polypeptide region consisting of a wild-type, Shiga toxin A1 fragment, wherein the binding region is linked to the second cell-targeted molecule carboxy-terminal to the wild-type, Shiga toxin A1 fragment.
13 . A cell-targeted molecule comprising
a) a binding region comprising one or more polypeptides and capable of specifically binding at least one extracellular target biomolecule,
b) a Shiga toxin effector polypeptide region having an amino-terminus and comprising a Shiga toxin A1 fragment region having a carboxy-terminus, and
c) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region;
wherein the binding region is positioned within the cell-targeted molecule carboxy-terminal to the Shiga toxin A1 fragment region; and
wherein the cell-targeted molecule is capable when introduced to cells of exhibiting cytotoxicity that is in a range of from 0.1-fold to 1.5-fold of cytotoxicity exhibited by a second cell-targeted molecule comprising the binding region and a second, Shiga toxin effector polypeptide region consisting of a wild-type, Shiga toxin A1 fragment, and wherein the binding region is positioned within the second cell targeted molecule carboxy-terminal to the wild-type, Shiga toxin A1 fragment.
14 . The cell-targeted molecule of claim 13 , wherein the binding region comprises an immunoglobulin-type binding region comprising a polypeptide selected from the group consisting of:
single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronectin-derived 10 th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystalline-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality.
15 . The cell-targeted molecule of claim 14 , whereby administration of the cell-targeted molecule to a first population of cells whose members are physically coupled to extracellular target biomolecules of the immunoglobulin-type binding region of the cell-targeted molecule, and a second population of cells whose members are not physically coupled to any extracellular target biomolecule of the immunoglobulin-type binding region, the cytotoxic effect of the cell-targeted molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
16 . The cell-targeted molecule of claim 14 , wherein the Shiga toxin effector polypeptide region comprises or consists essentially of a polypeptide represented by the amino acid sequence selected from the group consisting of:
1) amino acids 75 to 246 of SEQ ID NO:3;
2) amino acids 75 to 247 of SEQ ID NO:1 or SEQ ID NO:2;
3) amino acids 1 to 241 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;
4) amino acids 1 to 246 of SEQ ID NO:3; and
5) amino acids 1 to 247 of SEQ ID NO:1 or SEQ ID NO:2.
17 . The cell-targeted molecule of claim 16 , wherein the immunoglobulin-type binding region is capable of binding to an extracellular target biomolecule selected from the group consisting of:
CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72, tyrosine-protein kinase transmembrane receptor, carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha Vbeta3, Alpha5beta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen, prostate stem cell antigen, human aspartyl (asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gp100, tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, CD38, CD15, CD23, CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, galectin-9, mrp-14, PD-L1, Siglec-8, Siglec-10, CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86, CD105, CD115, F4/80, ILT-3, galectin-3, CD11a-c, GITRL, MHC class I molecule, MHC class II molecule, CD284, CD107-Mac3, CD195, HLA-DR, CD16/32, CD282, and any immunogenic fragment of any of the foregoing.
18 . The cell-targeted molecule of any one of claims 13 - 17 , wherein the cell-targeted molecule is capable when introduced to cells of exhibiting a cytotoxicity that is greater than that of a third cell-targeted molecule having an amino-terminus, and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the third cell-targeted molecule.
19 . The cell-targeted molecule of claim 17 , wherein the immunoglobulin-type binding region comprises or consists essentially of the polypeptide sequence represented by amino acids 269-508 of any one of SEQ ID NOs: 4, 8, 12, or 16.
20 . The cell-targeted molecule of claim 17 , which comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 32-35.
21 . The cell-targeted molecule of any one of claims 13 - 17 , wherein the cell-targeted molecule is capable of exhibiting improved, in vivo tolerability compared to in vivo tolerability of the second cell-targeted molecule.
22 . A pharmaceutical composition comprising the cell-targeted molecule of any one of claims 1 - 21 and at least one pharmaceutically acceptable excipient or carrier.
23 . A diagnostic composition comprising
the cell-targeted molecule of any one of claims 1 - 21 and
a detection promoting agent.
24 . A polynucleotide capable of encoding the cell-targeted molecule of any one of claims 1 - 21 or a complement thereof, or a fragment of any of the foregoing.
25 . An expression vector comprising the polynucleotide of claim 24 .
26 . A host cell comprising any one of the polynucleotides or expression vectors of claims 24 - 25 .
27 . A method of conferring greater cytotoxicity to a protein which comprises
a) an immunoglobulin-type binding region comprising one or more polypeptides and capable of specifically binding at least one extracellular target biomolecule, and
b) a Shiga toxin effector polypeptide with an amino-terminus;
the method comprising the step of arranging the amino-terminus of the Shiga toxin effector polypeptide more proximally to an amino-terminus of a polypeptide component of the protein.
28 . A method of killing a cell, the method comprising the step of contacting the cell with the cell-targeted molecule of any one of claims 1 - 21 or the pharmaceutical composition of claim 22 .
29 . The method of claim 28 , wherein the contacting occurs in vitro.
30 . The method of claim 28 , wherein the contacting occurs in vivo.
31 . A method of treating a disease, disorder, or condition in a patient, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of the cell-targeted molecule of any one of claims 1 - 21 or the pharmaceutical composition of claim 22 .
32 . The method of claim 31 , wherein the disease, disorder, or condition is selected from the group consisting of: cancer, tumor, immune disorder, and microbial infection.
33 . The method of claim 32 , wherein the cancer selected from the group consisting of:
bone cancer, breast cancer, central/peripheral nervous system cancer, gastrointestinal cancer, germ cell cancer, glandular cancer, head-neck cancer, hematological cancer, kidney-urinary tract cancer, liver cancer, lung/pleura cancer, prostate cancer, sarcoma, skin cancer, and uterine cancer; or
wherein the immune disorder is associated with a disease selected from the group consisting of:
amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes, graft rejection, graft-versus-host disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis, polyarteritis nodosa , polyarthritis, psoriasis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjorgren's syndrome, ulcerative colitis, and vasculitis.
34 . A method of detecting a cell, the method comprising the steps of:
contacting the cell with the diagnostic composition of claim 23 and
detecting the presence of the diagnostic composition.
35 . A kit comprising the composition of matter of any one of claims 1 - 26 and an additional reagent and/or pharmaceutical delivery device.