O-Mannosyltransferase Deficient Filamentous Fungal Cells and Methods of Use Thereof

Abstract

The present disclosure relates to compositions and methods useful for the production of heterologous proteins with reduced O-mannosylation in filamentous fungal cells, such as Trichoderma cells. More specifically, the invention provides a PMT-deficient filamentous fungal cell comprising a) at least a first mutation that reduces an endogenous protease activity compared to a parental filamentous fungal cell which does not have said first mutation, and, b) at least a second mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity compared to a parental filamentous fungal cell which does not have said second mutation, wherein said filamentous fungal cell is selected from the group consisting of Trichoderma, Neurospora, Myceliophthora or Chrysosporium cell.

1 - 16 . (canceled) 
     
     
         17 . A Protein O-mannosyltransferase (PMT)-deficient filamentous fungal cell comprising
 a) a first mutation in a gene encoding an endogenous protease that eliminates an endogenous protease activity as compared to a parental filamentous fungal cell which does not have said first mutation;   b) a second mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity compared to a parental filamentous fungal cell which does not have said second mutation; and   c) a polynucleotide encoding a light chain of an antibody and a polynucleotide encoding a heavy chain of an antibody;
 wherein: 
   the antibody is secreted by the PMT-deficient filamentous fungal cell upon expression of the polynucleotides encoding the light chain and the heavy chain of the antibody;   said antibody comprising reduced O-mannosylation on the light chain, said reduced O-mannosylation being less than about 10% that of a light chain secreted by said parental filamentous fungal cell which does not have said second mutation, O-mannosylation being defined as mole % of mannose residues per polypeptide chain; and
 said filamentous fungal cell is a  Trichoderma  or  Myceliophthora  cell. 
   
     
     
         18 . The PMT-deficient filamentous fungal cell of  claim 17 , wherein said second mutation that reduces the endogenous O-mannosyltransferase activity is a deletion or a disruption of a PMT gene encoding the endogenous protein O-mannosyltransferase activity. 
     
     
         19 . The PMT-deficient filamentous fungal cell of  claim 17 , wherein said second mutation in a PMT gene is a mutation in either:
 a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,   b) a functional homologous gene of PMT1 gene, which functional homologous gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a  T. reesei  strain having a disruption in said PMT1 gene, or   c) a polynucleotide encoding a polypeptide having at least 50% identity with SEQ ID NO:2, said polypeptide having O-mannosyltransferase activity.   
     
     
         20 . The PMT-deficient filamentous fungal cell of  claim 17 , wherein the reduced endogenous O-mannosyltransferase activity results from a deletion or a disruption of a PMT1 gene encoding a polypeptide of SEQ ID NO:2 or SEQ ID NO:12. 
     
     
         21 . The PMT-deficient filamentous fungal cell of  claim 17 , wherein said cell has a third mutation that reduces or eliminates the level of expression of a dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (ALG3) gene compared to the level of expression in a parental cell which does not have such third mutation. 
     
     
         22 . The PMT-deficient filamentous fungal cell of  claim 17 , further comprising one or more polynucleotides encoding a polypeptide selected from the group consisting of:
 a) α1,2 mannosidase;   b) N-acetylglucosaminyltransferase I catalytic domain;   c) α mannosidase II;   d) N-acetylglucosaminyltransferase II catalytic domain;   e) β1,4 galactosyltransferase; and,   f) fucosyltransferase.   
     
     
         23 . The PMT-deficient filamentous fungal cell of  claim 17 , wherein said cell is a  Trichoderma  cell comprising a mutation that reduces or eliminates the protein O-mannosyltransferase activity of  Trichoderma  pmt1. 
     
     
         24 . The PMT-deficient filamentous fungal cell of  claim 17 , wherein said cell is a  Trichoderma reesei  cell, and said cell comprises mutations that reduce or eliminate the activity of
 a) the three endogenous proteases pep1, tsp1 and slp1;   b) the three endogenous proteases gap1, slp1 and pep1;   c) the three endogenous proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp7, gap1 and gap2;   d) three to six proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, tsp1, slp1, slp2, slp3, gap1 and gap2; or   e) seven to ten proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep7, pep8, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, slp8, tpp1, gap1 and gap2.   
     
     
         25 . The PMT-deficient filamentous fungal cell of  claim 17 , wherein the O-mannosylation level on the expressed light chain of the antibody is reduced to 0%. 
     
     
         26 . The PMT-deficient filamentous fungal cell of  claim 17 , wherein said produced antibody is a mammalian antibody selected from the group consisting of:
 a) an immunoglobulin, such as IgG,   b) a light chain or heavy chain of an immunoglobulin,   c) a heavy chain or a light chain of an antibody,   d) a single chain antibody,   e) a camelid antibody,   f) a monomeric or multimeric single domain antibody,   g) a Fab-fragment, a Fab 2 -fragment, and,   h) their antigen-binding fragments.   
     
     
         27 . A method for producing an antibody having reduced O-mannosylation, comprising:
 a) providing the PMT-deficient filamentous fungal cell of  claim 17 , and   b) culturing the cell to produce said antibody, consisting of heavy and light chains, having reduced O-mannosylation.   
     
     
         28 . The method of  claim 27 , wherein said produced antibody is a mammalian antibody selected from the group consisting of:
 a) an immunoglobulin, optionally IgG;   b) a light chain or heavy chain of an immunoglobulin;   c) a heavy chain or a light chain of an antibody;   d) a single chain antibody;   e) a camelid antibody;   f) a monomeric or multimeric single domain antibody;   g) a Fab-fragment, a Fab 2 -fragment; and,   h) an antigen-binding fragment of one of a) through g).