Modified pseudomonas exotoxin a

Abstract

The invention provides a Pseudomonas exotoxin A (PE) comprising an amino acid sequence having a substitution of one or more B-cell and/or T-cell epitopes. The invention further provides related chimeric molecules, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. Methods of treating or preventing cancer in a mammal, methods of inhibiting the growth of a target cell, methods of producing the PE, and methods of producing the chimeric molecule are further provided by the invention.

1 . A  Pseudomonas  exotoxin A (PE) comprising a PE amino acid sequence wherein one or more of amino acid residues F443, R456, L477, R494, and L552 as defined by reference to SEQ ID NO: 1 are, independently, substituted, wherein the PE optionally has:
 (i) a further substitution of one or more amino acid residues within one or more B cell epitopes, and the further substitution for an amino acid within one or more B-cell epitopes is a substitution of, independently, one or more of amino acid residues D403, D406, R412, R427, E431, R432, D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592, and L597 as defined by reference to SEQ ID NO: 1,   (ii) a further substitution of one or more amino acid residues within one or more T-cell epitopes,   (iii) a deletion of one or more continuous amino acid residues of residues 1-273 and 285-394 as defined by SEQ ID NO: 1, or   (iv) a combination of any one, two, or three of (i)-(iii).   
     
     
         2 . An isolated, mutated  Pseudomonas  exotoxin A (PE), comprising a sequence of the following formula:
   R 1   n -FCS-R 2   n —R 3   n -PE functional domain II
   wherein:   n=0 or 1 independently for each of R 1 , R 2  and R 3 ,   R 1 =1 to 10 amino acid residues   FCS=a furin cleavage sequence of amino acid residues, which sequence is cleavable by furin and has an amino end and a carboxyl end,   R 2 =1 to 10 amino acid residues;   R 3 =1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1; and,   PE functional domain III=residues 395-613 of SEQ ID NO:1, wherein one or more of amino acid residues F443, R456, L477, R494, and L552 as defined by reference to SEQ ID NO: 1 are, independently, substituted, wherein the PE optionally has:   (i) a further substitution of one or more amino acid residues within one or more B cell epitopes, and the further substitution for an amino acid within one or more B-cell epitopes is a substitution of, independently, one or more of amino acid residues D403, D406, R412, R427, E431, R432, D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592, and L597 as defined by reference to SEQ ID NO: 1,   (ii) a further substitution of one or more amino acid residues within one or more T-cell epitopes, or   (iii) both (i) and (ii).   
     
     
         3 . The mutated PE of  claim 2 , further wherein the FCS is represented by the formula P4-P3-P2-P1, wherein P4 is an amino acid residue at the amino end, P1 is an amino acid residue at the carboxyl end, P1 is an arginine or a lysine residue, and the sequence is cleavable at the carboxyl end of P1 by furin. 
     
     
         4 . The mutated PE of  claim 3 , further wherein the FCS (i) further comprises amino acid residues represented by P6-P5 at the amino end, (ii) further comprises amino acid residues represented by P1′-P2′ at the carboxyl end, (iii) wherein if P1 is an arginine or a lysine residue, P2′ is tryptophan, and P4 is arginine, valine or lysine, provided that if P4 is not arginine, then P6 and P2 are basic residues, and (iv) the sequence is cleavable at the carboxyl end of P1 by furin. 
     
     
         5 . The mutated PE of any one of  claims 2 - 4 , further wherein the PE functional domain III consists of the sequence of residues 395 to 613 of SEQ ID NO: 1. 
     
     
         6 . The mutated PE of any one of  claims 2 - 5 , wherein the mutated PE comprises one or more contiguous residues of residues 365-394 of SEQ ID NO: 1 between the FCS and the PE domain III. 
     
     
         7 . The mutated PE of any one of  claims 2 - 6 , wherein n is 1 for R 1  and R 2 . 
     
     
         8 . The mutated PE of any one of  claims 2 - 7 , wherein the FCS is SEQ ID NO: 8. 
     
     
         9 . The mutated PE of any one of  claims 2 - 8 , wherein R 1 =a linker of the amino acid sequence of SEQ ID NO: 282, R 2 =a linker of the amino acid sequence SEQ ID NO: 284, and the FCS=SEQ ID NO: 8. 
     
     
         10 . The mutated PE of any one of  claims 2 - 9 , wherein n is 0 for R 3 . 
     
     
         11 . The mutated PE of any one of  claims 2 - 10 , wherein PE functional domain III comprises the amino acid sequence of SEQ ID NO: 37. 
     
     
         12 . The mutated PE of any one of  claims 2 - 11 , wherein R 1   n -FCS-R 2   n =SEQ ID NO: 36. 
     
     
         13 . The PE of any one of  claims 1 - 12 , wherein the substitution of one or more of amino acid residues F443, R456, L477, R494, and L552 is a substitution of, independently, alanine, glutamic acid, histidine, or asparagine in place of one or more of amino acid residues F443, R456, L477, R494, and L552. 
     
     
         14 . The PE of any one of  claims 1 - 13 , wherein the substitution of L552 is a substitution of glutamic acid or asparagine in place of L552 and the substitution of L4A77 is a substitution of histidine in place of L477. 
     
     
         15 . The PE of any one of  claims 1 - 14 , wherein the further substitution of an amino acid within one or more B-cell epitopes is a substitution of, independently, alanine, glycine, serine, or glutamine in place of one or more of amino acid residues E282, E285, P290, R313, N314, P319, D324, E327, E331, Q332, D403, D406, R412, R427, E431, R432, D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, K590, Q592, and L597, as defined by reference to SEQ ID NO: 1. 
     
     
         16 . The PE of any one of  claims 1 - 15 , wherein the PE has the further substitution of an amino acid within one or more T-cell epitopes, and the further substitution of an amino acid within one or more T-cell epitopes is a substitution of, independently, alanine, glycine, serine, or glutamine in place of one or more of amino acid residues R421, L422, L423, A425, R427, L429, Y439, H440, F443, L444, A446, A447, 1450, 463-519, R551, L552, T554, 1555, L556, and W558 as defined by reference to SEQ ID NO: 1. 
     
     
         17 . The PE of any one of  claims 1 - 16 , wherein the substitution of one or more of amino acid residues F443, R456, L477, R494, and L552 is a substitution of alanine in place of amino acid residue F443; a substitution of alanine in place of amino acid residue R456; a substitution of histidine in place of amino acid residue L477; a substitution of alanine in place of amino acid residue R494; and a substitution of glutamic acid in place of amino acid residue L552, the PE has an arginine residue at position 458, and the further substitution of an amino acid within one or more B-cell epitopes is:
 (a) a substitution of alanine for amino acid residue R427;   (b) a substitution of alanine for amino acid residue R463;   (c) a substitution of alanine for amino acid residue R467;   (d) a substitution of alanine for amino acid residue R490;   (e) a substitution of alanine for amino acid residue R505; and   (f) a substitution of alanine for amino acid residue R538;   as defined by reference to SEQ ID NO: 1.   
     
     
         18 . The PE of any one of  claims 1 - 16 , wherein the substitution of one or more of amino acid residues F443, R456, L477, R494, and L552 is a substitution of alanine in place of amino acid residue R456, the PE has an arginine residue at position 458, and the further substitution of an amino acid within one or more B-cell epitopes is:
 (a) a substitution of alanine for amino acid residue R427;   (b) a substitution of alanine for amino acid residue R463;   (c) a substitution of alanine for amino acid residue R467;   (d) a substitution of alanine for amino acid residue R490;   (e) a substitution of alanine for amino acid residue R505; and   (f) a substitution of alanine for amino acid residue R538;   as defined by reference to SEQ ID NO: 1.   
     
     
         19 . The PE of any one of  claims 1 - 16 , wherein the substitution of one or more of amino acid residues F443, R456, L477, R494, and L552 is a substitution of alanine in place of amino acid residue F443; a substitution of alanine in place of amino acid residue R456; a substitution of histidine in place of amino acid residue L477; a substitution of alanine in place of amino acid residue R494; and a substitution of asparagine in place of amino acid residue L552, the PE has an arginine residue at position 458, and the further substitution of an amino acid within one or more B-cell epitopes is:
 (a) a substitution of alanine for amino acid residue R427;   (b) a substitution of alanine for amino acid residue R463;   (c) a substitution of alanine for amino acid residue R467;   (d) a substitution of alanine for amino acid residue R490;   (e) a substitution of alanine for amino acid residue R505; and   (f) a substitution of alanine for amino acid residue R538;   
       as defined by reference to SEQ ID NO: 1. 
     
     
         20 . The mutated PE of  claim 2 , wherein one or more of amino acid residues F443, L477, R494, and L552 as defined by reference to SEQ ID NO: 1 are, independently, substituted. 
     
     
         21 . The mutated PE of  claim 20 , wherein the substitution of one or more of amino acid residues F443, L477, R494, and L552 is a substitution of alanine in place of amino acid residue F443, a substitution of histidine in place of amino acid residue L477, a substitution of alanine in place of amino acid residue R494, and a substitution of glutamic acid or asparagine in place of amino acid residue L552. 
     
     
         22 . An isolated, mutated  Pseudomonas  exotoxin A (PE), comprising a sequence of the following formula:
   R 1   n -FCS-R 2   n —R 3   n -PE functional domain III
   wherein:   n=0 or 1 independently for each of R 1 , R 2  and R 3 ,   R 1 =1 to 10 amino acid residues   FCS=a furin cleavage sequence of amino acid residues, which sequence is cleavable by furin and has an amino end and a carboxyl end,   R 2 =1 to 10 amino acid residues;   R 3 =1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1; and,   PE functional domain III=residues 395-613 of SEQ ID NO:1, wherein the PE includes an arginine at position 458, as defined by reference to SEQ ID NO: 1, and wherein the PE has:   (a) a substitution of alanine for amino acid residue R427;   (b) a substitution of alanine for amino acid residue R463;   (c) a substitution of alanine for amino acid residue R467;   (d) a substitution of alanine for amino acid residue R490;   (e) a substitution of alanine for amino acid residue R505; and   (f) a substitution of alanine for amino acid residue R538.   
     
     
         23 . A chimeric molecule comprising (a) a targeting moiety conjugated or fused to (b) the PE of any one of  claims 1 - 22 . 
     
     
         24 . The chimeric molecule of  claim 23 , wherein the targeting moiety is a monoclonal antibody or an antigen binding portion of the monoclonal antibody. 
     
     
         25 . The chimeric molecule of  claim 24 , wherein the monoclonal antibody or antigen binding portion of the monoclonal antibody specifically binds to a cell surface marker selected from the group consisting of CD19, CD21, CD22, CD25, CD30, CD79b, transferrin receptor, epidermal growth factor (EGF) receptor, mesothelin, cadherin, Lewis Y, glypican-3, FAP (fibroblast activation protein alpha), PSMA (prostate specific membrane antigen), CA9=CAIX (carbonic anhydrase IX), LICAM (neural cell adhesion molecule L1), endosialin, HER3 (activated conformation of epidermal growth factor receptor family member 3), Alkl/BMP9 complex (anaplastic lymphoma kinase 1/bone morphogenetic protein 9), TPBG=5T4 (trophoblast glycoprotein), CD33 (sialic acid binding Ig-like lectin 3, myeloid cell surface antigen), CD123 (interleukin 3 receptor alpha), MUC1 (tumor-associated epithelial mucin), ROR1 (receptor tyrosine kinase-like surface antigen), HER1 (activated conformation of epidermal growth factor receptor), and CLL1 (C-type lectin domain family 12, member A). 
     
     
         26 . The chimeric molecule of  claim 23 , wherein the targeting moiety is selected from the group consisting of B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21, MORAb-009, antigen binding portions thereof, and the antigen binding portion of HA22. 
     
     
         27 . The chimeric molecule of  claim 23 , wherein the targeting moiety is a humanized SS1 or an antigen binding portion of the humanized SS1. 
     
     
         28 . The chimeric molecule of  claim 23 , wherein the targeting moiety comprises:
 (a) SEQ ID NOs: 31 and 34;   (b) SEQ ID NOs: 45 and 46;   (c) SEQ ID NOs: 61 and 62;   (d) SEQ ID NOs: 77 and 78;   (e) SEQ ID NOs: 93 and 94;   (f) SEQ ID NOs: 109 and 110;   (g) SEQ ID NOs: 125 and 126;   (h) SEQ ID NOs: 141 and 142;   (i) SEQ ID NOs: 157 and 158;   (j) SEQ ID NOs: 173 and 174;   (k) SEQ ID NOs: 49, 50, 53, 54, 57, and 58;   (l) SEQ ID NOs: 65, 66, 69, 70, 73, and 74;   (m) SEQ ID NOs: 81, 82, 85, 86, 89, and 90;   (n) SEQ ID NOs: 97, 98, 101, 102, 105, and 106;   (o) SEQ ID NOs: 113, 114, 117, 118, 121, and 122;   (p) SEQ ID NOs: 129, 130, 133, 134, 137, and 138;   (q) SEQ ID NOs: 145, 146, 149, 150, 153, and 154;   (r) SEQ ID NOs: 161, 162, 165, 166, 169, and 170;   (s) SEQ ID NOs: 177, 178, 181, 182, 185, and 186;   (t) SEQ ID NOs: 31-32 and 34-36;   (u) SEQ ID NOs: 33 and 38; or   (v) SEQ ID NOs: 93 and 290.   
     
     
         29 . The chimeric molecule of any one of  claims 23 - 28 , wherein the chimeric molecule comprises a linker comprising SEQ ID NO: 36. 
     
     
         30 . The chimeric molecule of any one of  claims 23 - 28 , comprising
 (a) SEQ ID NOs: 39 and 40;   (b) SEQ ID NOs: 41 and 42;   (c) SEQ ID NOs: 43 and 44;   (d) SEQ ID NOs: 291 and 293;   (e) SEQ ID NOs: 291 and 294;   (f) SEQ ID NOs: 292 and 294;   (g) SEQ ID NOs: 295 and 297; or   (h) SEQ ID NOs:296 and 297.   
     
     
         31 . A nucleic acid comprising a nucleotide sequence encoding the PE of any one of  claims 1 - 22  or the chimeric molecule of any one of  claims 23 - 30 . 
     
     
         32 . A recombinant expression vector comprising the nucleic acid of  claim 31 . 
     
     
         33 . A host cell comprising the recombinant expression vector of  claim 32 . 
     
     
         34 . A population of cells comprising at least one host cell of  claim 33 . 
     
     
         35 . A pharmaceutical composition comprising (a) the PE of any one of  claims 1 - 22 , the chimeric molecule of any one of  claims 23 - 30 , the nucleic acid of  claim 31 , the recombinant expression vector of  claim 32 , the host cell of  claim 33 , or the population of cells of  claim 34 , and (b) a pharmaceutically acceptable carrier. 
     
     
         36 . The PE of any one of  claims 1 - 22 , the chimeric molecule of any one of  claims 23 - 30 , the nucleic acid of  claim 31 , the recombinant expression vector of  claim 32 , the host cell of  claim 33 , the population of cells of  claim 34 , or the pharmaceutical composition of  claim 35 , for use in treating or preventing cancer in the mammal. 
     
     
         37 . The PE of any one of  claims 1 - 22 , the chimeric molecule of any one of  claims 23 - 30 , the nucleic acid of  claim 31 , the recombinant expression vector of  claim 32 , the host cell of  claim 33 , the population of cells of  claim 34 , or the pharmaceutical composition of  claim 35 , for use in inhibiting growth of a target cell. 
     
     
         38 . The PE, chimeric molecule, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for the use of  claim 37 , wherein the target cell is a cancer cell. 
     
     
         39 . The PE, chimeric molecule, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for the use of  claim 37 , wherein the target cell expresses a cell surface marker selected from the group consisting of CD19, CD21, CD22, CD25, CD30, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin, Lewis Y, glypican-3, FAP (fibroblast activation protein alpha), PSMA (prostate specific membrane antigen), CA9=CAIX (carbonic anhydrase IX), LICAM (neural cell adhesion molecule L1), Endosialin, HER3 (activated conformation of epidermal growth factor receptor family member 3), Alkl/BMP9 complex (anaplastic lymphoma kinase 1/bone morphogenetic protein 9), TPBG=5T4 (trophoblast glycoprotein), CD33 (sialic acid binding Ig-like lectin 3, myeloid cell surface antigen), CD123 (interleukin 3 receptor alpha), MUC1 (tumor-associated epithelial mucin), ROR1 (receptor tyrosine kinase-like surface antigen), HER1 (activated conformation of epidermal growth factor receptor), and CLL1 (C-type lectin domain family 12, member A). 
     
     
         40 . A method of producing the PE of any one of  claims 1 - 22  comprising (a) recombinantly expressing the PE and (b) purifying the PE. 
     
     
         41 . A method of producing the chimeric molecule of any one of  claims 23 - 30  comprising (a) recombinantly expressing the chimeric molecule and (b) purifying the chimeric molecule. 
     
     
         42 . A method of producing the chimeric molecule of any one of  claims 23 - 30  comprising (a) recombinantly expressing the PE of any one of  claims 1 - 22 , (b) purifying the PE, and (c) covalently linking a targeting moiety to the purified PE.