Enzyme-mediated depletion of adenosine and/or methylthioadenosine

Abstract

Methods and compositions related to the engineering of a protein with MTA/ADO-degrading enzyme activity are described. For example, in certain aspects there may be disclosed an MTase capable of degrading MTA/ADO. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer or SCID with an MTase using the disclosed proteins or nucleic acids.

What is claimed is: 
     
         1 . An isolated, modified MTase enzyme, said modified enzyme being conjugated to an antibody. 
     
     
         2 . The enzyme of  claim 1 , wherein the MTase is a prokaryotic MTAN. 
     
     
         3 . The enzyme of  claim 2 , wherein the prokaryotic MTAN comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3. 
     
     
         4 . The enzyme of  claim 3 , wherein the prokaryotic MTAN comprises an amino acid sequence at least 95% identical to SEQ ID NO: 3. 
     
     
         5 . The enzyme of  claim 4 , wherein the prokaryotic MTAN comprises an amino acid sequence according to SEQ ID NO: 3. 
     
     
         6 . The enzyme of  claim 1 , wherein the MTase is a mammalian MTAP. 
     
     
         7 . The enzyme of  claim 6 , wherein the mammalian MTAP comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. 
     
     
         8 . The enzyme of  claim 7 , wherein the mammalian MTAP comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1. 
     
     
         9 . The enzyme of  claim 8 , wherein the mammalian MTAP comprises an amino acid sequence according to SEQ ID NO: 1. 
     
     
         10 . The enzyme of any one of  claims 1 - 9 , wherein the antibody is a scFv antibody. 
     
     
         11 . The enzyme of  claim 1  or  10 , wherein the antibody or scFv antibody is an anti-MUC1 antibody, an anti-HER2 antibody, an anti-CTLA4 antibody, an anti-PD1 antibody, or an anti-PDL1 antibody. 
     
     
         12 . The enzyme of any one of  claims 1 - 11 , further comprising a heterologous peptide segment. 
     
     
         13 . The enzyme of  claim 12 , wherein the heterologous peptide segment is an XTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide. 
     
     
         14 . The enzyme of any one of  claims 1 - 13 , wherein the enzyme is coupled to polyethylene glycol (PEG). 
     
     
         15 . The enzyme of  claim 14 , wherein the enzyme is coupled to PEG via one or more Lys or Cys residues. 
     
     
         16 . A nucleic acid comprising a nucleotide sequence encoding the enzyme of any one of  claims 1 - 15 . 
     
     
         17 . The nucleic acid of  claim 16 , wherein the nucleic acid is codon optimized for expression in bacteria, fungus, insects, or mammals. 
     
     
         18 . An expression vector comprising the nucleic acid of  claim 16  or  17 . 
     
     
         19 . A host cell comprising the nucleic acid of  claim 16  or  17 . 
     
     
         20 . The host cell of  claim 19 , wherein the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell. 
     
     
         21 . A pharmaceutical formulation comprising an MTase-antibody conjugate of any one of  claims 1 - 15 . 
     
     
         22 . A method of treating a patient having a tumor comprising administering to the patient an effective amount of a pharmaceutical formulation comprising an MTase polypeptide in a pharmaceutically acceptable carrier, wherein the MTase polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 1 or 3, wherein the MTase polypeptide has MTase activity. 
     
     
         23 . The method of  claim 22 , wherein the MTase polypeptide has at least 95% identity to SEQ ID NO: 1 or 3, wherein the polypeptide has MTase activity. 
     
     
         24 . The method of  claim 23 , wherein the MTase polypeptide has the sequence of SEQ ID NO: 1 or 3. 
     
     
         25 . The method of  claim 23 , wherein the MTase is a prokaryotic MTAN, wherein the prokaryotic MTAN comprises an amino acid sequence at least 95% identical to SEQ ID NO: 3 and has MTase activity. 
     
     
         26 . The method of  claim 23 , wherein the MTase is a human MTAP, wherein the human MTAP comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1 and has MTase activity. 
     
     
         27 . The method of  claim 24 , wherein the MTase polypeptide is a prokaryotic MTAN having a sequence according to SEQ ID NO: 3. 
     
     
         28 . The method of  claim 24 , wherein the MTase polypeptide is a prokaryotic MTAP having a sequence according to SEQ ID NO: 1. 
     
     
         29 . The method of any one of  claims 22 - 28 , wherein the MTase is coupled to polyethylene glycol (PEG). 
     
     
         30 . The method of  claim 29 , wherein the MTase is coupled to PEG via one or more Lys or Cys residues. 
     
     
         31 . The method of any one of  claims 22 - 30 , wherein the MTase further comprises a heterologous peptide segment. 
     
     
         32 . The method of  claim 31 , wherein the heterologous peptide segment is an XTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide. 
     
     
         33 . The method of any one of  claims 22 - 32 , wherein the MTase is conjugated to an antibody. 
     
     
         34 . The enzyme of  claim 33 , wherein the antibody is a scFv antibody. 
     
     
         35 . The enzyme of  claim 33  or  34 , wherein the antibody or scFv antibody is an anti-MUC1 antibody, an anti-HER2 antibody, an anti-CTLA4 antibody, an anti-PD1 antibody, or an anti-PDL1 antibody. 
     
     
         36 . The method of any one of  claims 22 - 35 , wherein the tumor is a solid tumor. 
     
     
         37 . The method of any one of  claims 22 - 35 , wherein the tumor is a hematological tumor. 
     
     
         38 . The method of any one of  claims 22 - 35 , wherein the tumor is an osteosarcoma, a pancreatic cancer, a chordoma, a mesothelioma, a T-cell ALL, a glioma, a renal cell carcinoma, a melanoma, a squamous cell carcinoma, a gallbladder cancer, a gastric cancer, or a hepatocellular carcinoma. 
     
     
         39 . The method of any one of  claims 22 - 38 , wherein the tumor has a decreased level of MTAP relative to a reference level. 
     
     
         40 . The method of  claim 39 , wherein the tumor has an MTAP deletion. 
     
     
         41 . The method of any one of  claims 22 - 38 , wherein the tumor has an increased level of CD73 relative to a reference sample. 
     
     
         42 . The method of  claim 41 , wherein the tumor has a decreased level of MTAP relative to a reference level. 
     
     
         43 . The method of any one of  claims 22 - 38 , wherein the tumor has an increased level of CD39 relative to a reference sample. 
     
     
         44 . The method of any one of  claims 22 - 38 , wherein the tumor has an increased level of MTA relative to a reference level. 
     
     
         45 . The method of any one of  claims 22 - 38 , wherein the tumor has an increased level of ADO relative to a reference level. 
     
     
         46 . The method of any one of  claims 39  and  41 - 45 , wherein the reference level is a level in a healthy tissue in the patient. 
     
     
         47 . The method of any one of  claims 39  and  41 - 45 , wherein the reference level is a level in a healthy subject. 
     
     
         48 . The method of any one of  claims 22 - 47 , wherein the patient is a human patient. 
     
     
         49 . The method of any one of  claims 22 - 48 , wherein the formulation is administered intratumorally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage. 
     
     
         50 . The method of any one of  claims 22 - 49 , further defined as a method for increasing sensitivity to immunotherapy. 
     
     
         51 . The method of  claim 49 , wherein the patient has previously failed to respond to the administration of an immune checkpoint inhibitor. 
     
     
         52 . The method of any one of  claims 22 - 51 , further comprising administering at least a second anticancer therapy to the subject. 
     
     
         53 . The method of  claim 52 , wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy. 
     
     
         54 . The method of  claim 52 , wherein the second anticancer therapy comprises an adoptive T cell therapy, an anti-PD1 antibody, an anti-CTLA-4 antibody, and/or an anti-PD-L1 antibody. 
     
     
         55 . The method of  claim 54 , wherein the anti-PD-L1 antibody comprises atezolizumab, avelumab, durvalumab, BMS-036559, or CK-301. 
     
     
         56 . The method of  claim 54 , wherein the anti-PD1 antibody comprises nivolumab, pembrolizumab, pidilizumab, AMP-223, AMP-514, cemiplimab, or PDR-001. 
     
     
         57 . The method of  claim 54 , wherein the anti-CTLA-4 therapy comprises ipilimumab or tremelimumab. 
     
     
         58 . The method of any one of  claims 22 - 57 , further defined as a method of preventing metastasis. 
     
     
         59 . A method of selecting a patient having a tumor for treatment with a combined effective amount of an MTase polypeptide and an immune checkpoint inhibitor, the method comprising (a) determining whether the patient's tumor has a decreased level of MTAP, an increased level of CD73, an increased level of CD39, an increased level of MTA, or an increased level of ADO relative to a reference level, and (b) selecting the patient for treatment if the patient's tumor has a decreased level of MTAP, an increased level of CD73, an increased level of CD39, an increased level of MTA, or an increased level of ADO relative to a reference level, wherein the MTase polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 1 or 3, wherein the MTase polypeptide has MTase activity. 
     
     
         60 . The method of  claim 59 , further comprising administering a combined effective amount of an MTase polypeptide and an immune checkpoint inhibitor to the selected patient. 
     
     
         61 . The method of  claim 59 , further comprising selecting the patient for treatment if the patient has previously failed to respond to the administration of an immune checkpoint inhibitor. 
     
     
         62 . The method of  claim 59 , wherein the patient has previously undergone at least one round of anti-cancer therapy. 
     
     
         63 . The method of any one of  claims 59 - 62 , wherein the MTase polypeptide has at least 95% identity to SEQ ID NO: 1 or 3, wherein said polypeptide has MTase activity. 
     
     
         64 . The method of  claim 63 , wherein the MTase polypeptide has the sequence of SEQ ID NO: 1 or 3. 
     
     
         65 . The method of  claim 63 , wherein the MTase is a prokaryotic MTAN, wherein the prokaryotic MTAN comprises an amino acid sequence at least 95% identical to SEQ ID NO:
 3 and has MTase activity.   
     
     
         66 . The method of  claim 63 , wherein the MTase is a human MTAP, wherein the human MTAP comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1 and has MTase activity. 
     
     
         67 . The method of  claim 64 , wherein the MTase polypeptide is a prokaryotic MTAN having a sequence according to SEQ ID NO: 3. 
     
     
         68 . The method of  claim 64 , wherein the MTase polypeptide is a prokaryotic MTAP having a sequence according to SEQ ID NO: 1. 
     
     
         69 . The method of any one of  claims 59 - 68 , wherein the MTase is coupled to polyethylene glycol (PEG). 
     
     
         70 . The method of  claim 69 , wherein the MTase is coupled to PEG via one or more Lys or Cys residues. 
     
     
         71 . The method of any one of  claims 59 - 70 , wherein the MTase further comprises a heterologous peptide segment. 
     
     
         72 . The method of  claim 71 , wherein the heterologous peptide segment is an XTEN peptide, an IgG Fe, an albumin, or an albumin binding peptide. 
     
     
         73 . The method of any one of  claims 59 - 72 , wherein the MTase is conjugated to an antibody. 
     
     
         74 . The enzyme of  claim 73 , wherein the antibody is a scFv antibody. 
     
     
         75 . The enzyme of  claim 73  or  74 , wherein the antibody or scFv antibody is an anti-MUC1 antibody, an anti-HER2 antibody, an anti-CTLA4 antibody, an anti-PD1 antibody, or an anti-PDL1 antibody. 
     
     
         76 . The method of any one of  claims 59 - 75 , wherein the tumor is a solid tumor. 
     
     
         77 . The method of any one of  claims 59 - 75 , wherein the tumor is a hematological tumor. 
     
     
         78 . The method of any one of  claims 59 - 75 , wherein the tumor is an osteosarcoma, a pancreatic cancer, a chordoma, a mesothelioma, a T-cell ALL, a glioma, a renal cell carcinoma, a melanoma, a squamous cell carcinoma, a gallbladder cancer, a gastric cancer, or a hepatocellular carcinoma. 
     
     
         79 . The method of any one of  claims 59 - 78 , wherein the tumor has a decreased level of MTAP relative to a reference level. 
     
     
         80 . The method of  claim 79 , wherein the tumor has an MTAP deletion. 
     
     
         81 . The method of any one of  claims 59 - 78 , wherein the tumor has an increased level of CD73 relative to a reference sample. 
     
     
         82 . The method of  claim 81 , wherein the tumor has a decreased level of MTAP relative to a reference level. 
     
     
         83 . The method of any one of  claims 59 - 78 , wherein the tumor has an increased level of CD39 relative to a reference sample. 
     
     
         84 . The method of any one of  claims 59 - 78 , wherein the tumor has an increased level of MTA relative to a reference level. 
     
     
         85 . The method of any one of  claims 59 - 78 , wherein the tumor has an increased level of ADO relative to a reference level. 
     
     
         86 . The method of any one of  claims 79  and  81 - 85 , wherein the reference level is a level in a healthy tissue in the patient. 
     
     
         87 . The method of any one of  claims 79  and  81 - 85 , wherein the reference level is a level in a healthy subject. 
     
     
         88 . The method of any one of  claims 59 - 87 , wherein the patient is a human patient. 
     
     
         89 . The method of any one of  claims 60 - 88 , wherein the MTase polypeptide is administered intratumorally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage. 
     
     
         90 . The method of any one of  claims 60 - 88 , further defined as a method for increasing sensitivity to immunotherapy. 
     
     
         91 . The method of  claim 90 , wherein the patient has previously failed to respond to the administration of an immune checkpoint inhibitor. 
     
     
         92 . The method of any one of  claims 59 - 91 , further comprising administering at least a second anticancer therapy to the subject. 
     
     
         93 . The method of  claim 92 , wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy. 
     
     
         94 . The method of  claim 92 , wherein the second anticancer therapy comprises an adoptive T cell therapy, an anti-PD1 antibody, an anti-CTLA-4 antibody, and/or an anti-PD-L1 antibody. 
     
     
         95 . The method of  claim 94 , wherein the anti-PD-L1 antibody comprises atezolizumab, avelumab, durvalumab, BMS-036559, or CK-301. 
     
     
         96 . The method of  claim 94 , wherein the anti-PD1 antibody comprises nivolumab, pembrolizumab, pidilizumab, AMP-223, AMP-514, cemiplimab, or PDR-001. 
     
     
         97 . The method of  claim 94 , wherein the anti-CTLA-4 therapy comprises ipilimumab or tremelimumab. 
     
     
         98 . The method of any one of  claims 59 - 97 , further defined as a method of preventing metastasis. 
     
     
         99 . A method of treating a patient having severe combined immunodeficiency (SCID), the method comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an MTase polypeptide in a pharmaceutically acceptable carrier, wherein the MTase polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 1 or 3, wherein the MTase polypeptide has MTase activity. 
     
     
         100 . The method of  claim 99 , wherein the patient has at least one mutation in an adenosine deaminase gene. 
     
     
         101 . The method of  claim 99  or  100 , wherein the MTase polypeptide has at least 95% identity to SEQ ID NO: 1 or 3, wherein the polypeptide has MTase activity. 
     
     
         102 . The method of  claim 101 , wherein the MTase polypeptide has the sequence of SEQ ID NO: 1 or 3. 
     
     
         103 . The method of  claim 101 , wherein the MTase is a prokaryotic MTAN, wherein the prokaryotic MTAN comprises an amino acid sequence at least 95% identical to SEQ ID NO: 3 and has MTase activity. 
     
     
         104 . The method of  claim 101 , wherein the MTase is a human MTAP, wherein the human MTAP comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1 and has MTase activity. 
     
     
         105 . The method of  claim 102 , wherein the MTase polypeptide is a prokaryotic MTAN having a sequence according to SEQ ID NO: 3. 
     
     
         106 . The method of  claim 102 , wherein the MTase polypeptide is a prokaryotic MTAP having a sequence according to SEQ ID NO: 1. 
     
     
         107 . The method of any one of  claims 99 - 106 , wherein the MTase is coupled to polyethylene glycol (PEG). 
     
     
         108 . The method of  claim 107 , wherein the MTase is coupled to PEG via one or more Lys or Cys residues. 
     
     
         109 . The method of any one of  claims 99 - 108 , wherein the MTase further comprises a heterologous peptide segment. 
     
     
         110 . The method of  claim 109 , wherein the heterologous peptide segment is an XTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide. 
     
     
         111 . The method of any one of  claims 99 - 110 , wherein the MTase is conjugated to an antibody. 
     
     
         112 . The method of any one of  claims 99 - 111 , wherein the patient is a human patient. 
     
     
         113 . The method of any one of  claims 99 - 112 , wherein the formulation is administered intratumorally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage. 
     
     
         114 . A method of selecting a patient having SCID for treatment with an effective amount of a pharmaceutical formulation comprising an MTase polypeptide in a pharmaceutically acceptable carrier, the method comprising (a) determining whether the patient has at least one mutation in an adenosine deaminase gene, and (b) selecting the patient for treatment if the patient has at least one mutation in an adenosine deaminase gene, wherein the MTase polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 1 or 3, wherein the MTase polypeptide has MTase activity. 
     
     
         115 . The method of  claim 114 , further comprising administering an effective amount of an MTase polypeptide in a pharmaceutically acceptable carrier to the selected patient. 
     
     
         116 . The method of  claim 114  or  115 , wherein the MTase polypeptide has at least 95% identity to SEQ ID NO: 1 or 3, wherein said polypeptide has MTase activity. 
     
     
         117 . The method of  claim 116 , wherein the MTase polypeptide has the sequence of SEQ ID NO: 1 or 3. 
     
     
         118 . The method of  claim 116 , wherein the MTase is a prokaryotic MTAN, wherein the prokaryotic MTAN comprises an amino acid sequence at least 95% identical to SEQ ID NO: 3 and has MTase activity. 
     
     
         119 . The method of  claim 116 , wherein the MTase is a human MTAP, wherein the human MTAP comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1 and has MTase activity. 
     
     
         120 . The method of  claim 117 , wherein the MTase polypeptide is a prokaryotic MTAN having a sequence according to SEQ ID NO: 3. 
     
     
         121 . The method of  claim 117 , wherein the MTase polypeptide is a prokaryotic MTAP having a sequence according to SEQ ID NO: 1. 
     
     
         122 . The method of any one of  claims 114 - 121 , wherein the MTase is coupled to polyethylene glycol (PEG). 
     
     
         123 . The method of  claim 122 , wherein the MTase is coupled to PEG via one or more Lys or Cys residues. 
     
     
         124 . The method of any one of  claims 99 - 123 , wherein the MTase further comprises a heterologous peptide segment. 
     
     
         125 . The method of  claim 124 , wherein the heterologous peptide segment is an XTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide. 
     
     
         126 . The method of any one of  claims 99 - 125 , wherein the MTase is conjugated to an antibody. 
     
     
         127 . The method of any one of  claims 99 - 126 , wherein the patient is a human patient. 
     
     
         128 . The method of any one of  claims 99 - 127 , wherein the formulation is administered intratumorally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage.