Arginine deiminase with reduced cross-reactivity toward adi - peg 20 antibodies for cancer treatment

Abstract

The present invention relates generally to isolated to arginine deiminase (ADI) proteins that have reduced cross-reactivity with anti-ADI-PEG 20 antibodies as compared to ADI-PEG 20, but which can have functional characteristics comparable to or better than ADI-PEG 20, compositions comprising the ADI proteins, and related methods of treating arginine-dependent diseases or related diseases such as cancer.

1 .- 28 . (canceled) 
     
     
         29 . An isolated arginine deiminase, or a fragment thereof having ADI activity, wherein the isolated arginine deiminase has reduced cross-reactivity with patient anti-ADI-PEG 20 antibodies. 
     
     
         30 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase is not from  M. hominis.    
     
     
         31 . The isolated arginine deiminase of  claim 30  wherein the isolated arginine deiminase is from an organism listed in Table 1. 
     
     
         32 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase has one or more properties comparable to or better than those of ADI-PEG 20. 
     
     
         33 . The isolated arginine deiminase of  claim 32  wherein the one or more properties is Keat, Km, pH optimum, stability, in vivo proteolytic stability, or no requirement for ions or cofactors that are not already present in blood, or any combination thereof. 
     
     
         34 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase has at least 20 surface residue changes as compared to  M. hominis  arginine deiminase. 
     
     
         35 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase has between 20 and 135 surface residue changes as compared to  M. hominis  arginine deiminase. 
     
     
         36 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase has between 40 and 100 surface residue changes as compared to  M. hominis  arginine deiminase. 
     
     
         37 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase has between 30 and 60 surface residue changes as compared to  M. hominis  arginine deiminase. 
     
     
         38 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase has between 80 and 100 surface residues changes as compared to  M. hominis  arginine deiminase. 
     
     
         39 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase has between 100 and 120 surface residues changes as compared to  M. hominis  arginine deiminase. 
     
     
         40 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase is from  M. arginini, M. arthritidis, M. phocicerebrale, M. gateae, M. phocidae, M. columbinum, M. iowae, M. crocodyli, M. alligatoris, H. orenii , or  M. bovis.    
     
     
         41 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase comprises the amino acid sequence set forth in any one of SEQ ID NOs:2-32. 
     
     
         42 . The isolated arginine deiminase of  claim 29  wherein the isolated arginine deiminase has been modified to remove at least one pegylation site. 
     
     
         43 . The isolated arginine deiminase of  claim 29  wherein at least one lysine residue has been modified by an amino acid substitution. 
     
     
         44 . The isolated arginine deiminase of  claim 43  wherein at least 5 lysine residues have been modified by an amino acid substitution. 
     
     
         45 . The isolated arginine deiminase of  claim 43  wherein at least 10 lysine residues have been modified by an amino acid substitution. 
     
     
         46 . The isolated arginine deiminase of  claim 43  wherein at least 15 lysine residues have been modified by an amino acid substitution. 
     
     
         47 . The isolated arginine deiminase of  claim 43  wherein at least 20 lysine residues have been modified by an amino acid substitution. 
     
     
         48 . The isolated arginine deiminase of  claim 29  wherein the arginine deiminase is covalently bonded via a linker to a PEG molecule. 
     
     
         49 . The isolated arginine deiminase of  claim 48  wherein the arginine deiminase is covalently bonded to more than one PEG molecule. 
     
     
         50 . The isolated arginine deiminase of  claim 48  wherein the arginine deiminase is covalently bonded to about 1 to about 10 PEG molecules. 
     
     
         51 . The isolated arginine deiminase of  claim 48  wherein the arginine deiminase is covalently bonded to about 2 to about 8 PEG molecules. 
     
     
         52 . The isolated arginine deiminase of  claim 48  wherein the PEG molecules are straight chain or branch chain PEG molecules. 
     
     
         53 . The isolated arginine deiminase of  claim 48  wherein the PEG has a total weight average molecular weight of from about 1,000 to about 40,000. 
     
     
         54 . The isolated arginine deiminase of  claim 48  wherein the PEG has a total weight average molecular weight of from about 10,000 to about 30,000. 
     
     
         55 . The isolated arginine deiminase of  claim 48  wherein the linker is a succinyl group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, a histidine group, a methylene group, or any combinations thereof. 
     
     
         56 . The isolated arginine deiminase of  claim 55  wherein the source of the succinyl group is succinimidyl succinate. 
     
     
         57 . A polynucleotide encoding the isolated arginine deiminase of  claim 29 . 
     
     
         58 . A vector comprising the polynucleotide of  claim 57 . 
     
     
         59 . An isolated host cell comprising the vector of  claim 58 . 
     
     
         60 .- 68 . (canceled)