Non-cytotoxic PAP mutants

Abstract

Disclosed are PAP mutants that are less toxic than wild type PAP and that exhibit depurination activity. Also disclosed are transgenic plants that produce the PAP mutants, and methods for preparing the plants. Further disclosed are bioconjugates containing the PAP mutants, pharmaceutical compositions containing the bioconjugates, and methods of administering the compositions to treat disease.

1 . A Pokeweed Antiviral Protein (PAP) mutant that is less toxic than wild-type PAP and which exhibits ribosome depurination activity. 
     
     
         2 . The PAP mutant of  claim 1  which comprises PAP (1-262, Y123A). 
     
     
         3 . The PAP mutant of  claim 1  which comprises PAP (1-262, S14M, Y16A). 
     
     
         4 . The PAP mutant of  claim 1  which comprises PAP (1-262, L71R). 
     
     
         5 . The PAP mutant of  claim 1  which comprises PAP (1-262, V73E). 
     
     
         6 . The PAP mutant of  claim 1  which comprises PAP (1-262, M74R). 
     
     
         7 . The PAP mutant of  claim 1  which comprises PAP (1-262, Y76A). 
     
     
         8 . The PAP mutant of  claim 1  which comprises PAP (1-262, Y1231). 
     
     
         9 . The PAP mutant of  claim 1  which differs from wild-type PAP substantially in that it is truncated at its C-terminus from 10 to 20 mature PAP amino acids. 
     
     
         10 . The PAP mutant of  claim 9  which is selected from the group of PAP mutants consisting of PAP (1-242), PAP (1-243), PAP (1-244), PAP (1-245), PAP (1-246), PAP (1-247), PAP (1-248), PAP (1-249), PAP (1-250) and PAP (1-251). 
     
     
         11 . The PAP mutant of  claim 1  further comprising N-terminal signal sequence of wild-type PAP. 
     
     
         12 . The PAP mutant of  claim 1  further comprising C-terminal extension of wild-type PAP. 
     
     
         13 . A PAP mutant that is less toxic than wild-type PAP and which exhibits ribosome depurination activity, wherein said mutant is an N-terminal domain mutant. 
     
     
         14 . A PAP mutant that is less toxic than wild-type PAP and which exhibits ribosome depurination activity, wherein said mutant is a central domain mutant. 
     
     
         15 . A fusion protein comprising the PAP mutant of  claim 1  and a targeting moiety that binds a cell surface receptor. 
     
     
         16 . An immunoconjugate comprising the PAP mutant of  claim 1  and a targeting moiety that binds a cell surface receptor. 
     
     
         17 . A DNA molecule encoding the fusion protein of  claim 15 . 
     
     
         18 . A DNA molecule encoding the PAP mutant of  claim 1 . 
     
     
         19 . A chimeric DNA molecule comprising the DNA molecule of  claim 17  or  claim 18  operably linked to a promoter. 
     
     
         20 . The chimeric DNA molecule of  claim 19  wherein said promoter is functional in a yeast cell. 
     
     
         21 . The chimeric DNA molecule of  claim 19  wherein said promoter is functional in a plant cell. 
     
     
         22 . The chimeric DNA molecule of  claim 19  wherein the promoter is functional in an animal cell. 
     
     
         23 . The chimeric DNA molecule of  claim 19  wherein said promoter is an inducible promoter. 
     
     
         24 . The chimeric DNA molecule of  claim 19  wherein said promoter is a constitutive promoter. 
     
     
         25 . The DNA molecule of  claim 19  wherein said PAP mutant comprises the N-terminal signal sequence of wild-type PAP. 
     
     
         26 . The DNA molecule of  claim 19  wherein said PAP mutant comprises the C-terminal extension of wild-type PAP. 
     
     
         27 . A recombinant vector comprising the DNA molecule of  claim 17  or  claim 18 . 
     
     
         28 . A protoplast stably transformed with the DNA molecule of  claim 17  or  claim 18 . 
     
     
         29 . A non-human host transformed with the chimeric DNA molecule of  claim 17  or  claim 18 . 
     
     
         30 . The host of  claim 29  which is an  E. coli  cell. 
     
     
         31 . The host of  claim 29  which is a yeast cell. 
     
     
         32 . The yeast cell of  claim 29  which is a  Saccharomyces cerevisiae  cell. 
     
     
         33 . The host of  claim 29  which is a plant cell. 
     
     
         34 . The host of  claim 29  wherein said DNA molecule is operably linked to an inducible promoter functional in said host. 
     
     
         35 . A transgenic plant regenerated from the protoplast of  claim 28 . 
     
     
         36 . A transgenic plant comprising the chimeric DNA molecule of  claim 17  or  claim 18 , wherein the DNA molecule is expressed in said plant. 
     
     
         37 . The transgenic plant of  claim 35  or  claim 36  which is a monocot plant. 
     
     
         38 . The transgenic plant of  claim 37  wherein said monocot is a cereal crop plant. 
     
     
         39 . The transgenic plant of  claim 35  or  claim 36  which is a dicot plant. 
     
     
         40 . Seed derived from the transgenic plant of  claim 35  or  36 . 
     
     
         41 . A method of making a plant that has increased resistance to viruses and/or fungi, comprising preparing a plant having a genome that contains the DNA molecule of  claim 18  wherein said sequence is expressed. 
     
     
         42 . The method of  claim 41  comprising transforming a protoplast with the DNA molecule, and generating the transgenic plant from the transformed protoplast. 
     
     
         43 . The method of  claim 41  comprising introducing the DNA molecule into plant tissue, and regenerating the plant from the plant tissue containing the DNA molecule. 
     
     
         44 . A pharmaceutical composition comprising the fusion protein of  claim 15  or the immunoconjugate of  claim 16  and a pharmaceutically acceptable carrier. 
     
     
         45 . A method of treating a mammal suffering from cancer comprising administering the composition of  claim 44  to the mammal. 
     
     
         46 . A method of treating a mammal suffering from AIDS comprising administering the composition of  claim 44  to the mammal.