Production of functional antibodies in filamentous fungi

Abstract

Described herein are methods for the production of monoclonal antibodies in filamentous fungi host cells. The monoclonal antibodies are expressed as full-length fusion proteins that retain functional antigen binding and antibody-dependent cellular cytotoxicity capabilities. Improvements in the cleavage of the glucoamylase-light chain fusion protein to yield a mature antibody are also provided. The antibodies produced in filamentous fungi show equivalent pharmacokinetic disposition to antibodies produced in mammalian cells.

What is claimed is:  
     
         1 . A monoclonal antibody comprising two heavy chains and two light chains wherein each of the chains comprising the monoclonal antibody has been expressed in a filamentous fungal host cell as a fusion protein, wherein the fusion protein comprises a secreted polypeptide or portion thereof normally secreted from a filamentous fungus and an immunoglobulin chain.  
     
     
         2 . The monoclonal antibody of  claim 1  wherein the secreted polypeptide, or functional part thereof, is selected from the group consisting of an Aspergillus glucoamylase, Aspergillus alpha-amylase, Aspergillus aspartyl protease, Trichoderma cellobiohydrolase and Trichoderma endoglucanase.  
     
     
         3 . The monoclonal antibody of  claim 1  wherein the fusion protein comprises an antibody chain and a glucoamylase.  
     
     
         4 . The monoclonal antibody of  claim 1  wherein the filamentous fungus host is an Aspergillus, Neurospora, Fusarium, Trichoderma, Cephalosporium Penicillium or Chrysosporium.  
     
     
         5 . The monoclonal antibody of  claim 1  wherein the filamentous fungus is an Aspergillus.  
     
     
         6 . The monoclonal antibody of claim wherein the filamentous fungus is an  Aspergillus niger.    
     
     
         7 . The monoclonal antibody of  claim 1  which is conjugated to a label.  
     
     
         8 . The monoclonal antibody of  claim 7  wherein the label is a cytotoxic agent, an antibiotic, or a detectable marker.  
     
     
         9 . The monoclonal antibody of  claim 8  wherein the detectable marker is a radioisotope or an enzyme.  
     
     
         10 . A monoclonal antibody comprising two heavy chains and two light chains wherein at least one of the heavy chains and at least one of the light chains comprising the monoclonal antibody has been expressed in a filamentous fungal host cell as a fusion protein, wherein the fusion protein comprises a secreted polypeptide or portion thereof normally secreted from a filamentous fungus and an immunoglobulin chain.  
     
     
         11 . The monoclonal antibody of  claim 10  wherein the secreted polypeptide, or functional part thereof, is selected from the group consisting of an Aspergillus glucoamylase, Aspergillus alpha-amylase, Aspergillus aspartyl protease, Trichoderma cellobiohydrolase and Trichoderma endoglucanase.  
     
     
         12 . The monoclonal antibody of  claim 10  wherein the fusion protein comprises an immunoglobulin chain and a glucoamylase.  
     
     
         13 . A monoclonal antibody fragment selected from the group consisting of F(ab′) 2 , Fab′ and Fab, said fragment comprising at least one heavy chain and at least one light chain wherein each of the chains comprising the monoclonal antibody fragment has been expressed in a filamentous fungal host cell as a fusion protein, wherein the fusion protein comprises a secreted polypeptide or portion thereof normally secreted from a filamentous fungus and an immunoglobulin chain.  
     
     
         14 . The monoclonal antibody of  claim 13  wherein the secreted polypeptide, or functional part thereof, is selected from the group consisting of an Aspergillus glucoamylase, Aspergillus alpha-amylase, Aspergillus aspartyl protease, Trichoderma cellobiohydrolase and Trichoderma endoglucanase.  
     
     
         15 . The monoclonal antibody fragment of  claim 13  wherein the fusion protein comprises an immunoglobulin chain and a glucoamylase.  
     
     
         16 . The monoclonal antibody of  claim 13  which is conjugated to a label.  
     
     
         17 . The monoclonal antibody of  claim 16  wherein the label is a cytotoxic agent, an antibiotic, or a detectable marker.  
     
     
         18 . The monoclonal antibody of  claim 17  wherein the detectable marker is a radioisotope or an enzyme.  
     
     
         19 . A monoclonal antibody according to  claim 1  that is non-glycosylated.  
     
     
         20 . The monoclonal antibody of  claim 19 , wherein the antibody has been treated with an enzyme.  
     
     
         21 . The monoclonal antibody according to  claim 20 , wherein the enzyme is selected from the group consisting of endoglycosidase H, endoglycosidase F1, endoglycosidase F2, endoglycosidase A, PNGase F, PNGase A, and PNGase At.  
     
     
         22 . The monoclonal antibody of  claim 19 , wherein the antibody is aglycosylated.  
     
     
         23 . The monoclonal antibody of  claim 22 , wherein aglycosylation is due to an amino acid substitution.  
     
     
         24 . The monoclonal antibody of  claim 23 , wherein aglycosylation is due to the amino acid substitution N297Q.  
     
     
         25 . The monoclonal antibody of  claim 1  having a fungal glycosylation pattern.  
     
     
         26 . The monoclonal antibody of  claim 25 , having a high mannose glycosylation pattern.  
     
     
         27 . A fusion nucleic acid encoding a fusion polypeptide comprising, from a 5′ end of said fusion nucleic acid, first, second, third and fourth nucleic acids, wherein said first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus, said second nucleic acid encodes a secreted polypeptide or functional portion thereof normally secreted from said first or a second filamentous fungus, said third nucleic acid encodes a cleavable linker and said fourth nucleic acid encodes an immunoglobulin light chain or fragment thereof.  
     
     
         28 . The fusion nucleic acid according to  claim 27  wherein the fourth nucleic acid encodes an immunoglobin light chain.  
     
     
         29 . The fusion nucleic acid according to  claim 27  wherein the fourth nucleic acid encodes a fragment of an immunoglobin light chain.  
     
     
         30 . A composition comprising the nucleic acid of  claim 27 .  
     
     
         31 . A composition according to  claim 30  further comprising a fusion nucleic acid encoding a fusion polypeptide comprising, from a 5′ end of said fusion nucleic acid, first, second, third and fourth nucleic acids, wherein said first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus, said second nucleic acid encodes a secreted polypeptide or functional portion thereof normally secreted from said first or a second filamentous fungus, said third nucleic acid encodes a cleavable linker and said fourth nucleic acid encodes an immunoglobulin heavy chain or fragment thereof.  
     
     
         32 . An expression vector comprising the nucleic acid of any one of claims  27 - 29 .  
     
     
         33 . The expression vector according to  claim 32  further comprising a fusion nucleic acid encoding a fusion polypeptide comprising, from a 5′ end of said fusion nucleic acid, first, second, third and fourth nucleic acids, wherein said first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus, said second nucleic acid encodes a secreted polypeptide or functional portion thereof normally secreted from said first or a second filamentous fungus, said third nucleic acid encodes a cleavable linker and said fourth nucleic acid encodes an immunoglobulin heavy chain or fragment thereof.  
     
     
         34 . A process for producing an immunoglobulin molecule or an immunologically functional immunoglobulin fragment comprising at least the variable domains of the immunoglobulin heavy and light chains, in a host filamentous fungus, comprising the steps of: 
 a. transforming said host with a first expression vector containing the fusion DNA sequence of  claim 27;     b. transforming said host with a second expression vector containing the a fusion nucleic acid encoding a fusion polypeptide comprising, from a 5′ end of said fusion nucleic acid, first, second, third and fourth nucleic acids, wherein said first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus, said second nucleic acid encodes a secreted polypeptide or functional portion thereof normally secreted from said first or a second filamentous fungus, said third nucleic acid encodes a cleavable linker and said fourth nucleic acid encodes an immunoglobulin heavy chain or fragment thereof;    c. growing said host under conditions which permit expression of said fusion DNA sequences to cause the expression of the desired polypeptides encoded by said fusion DNA sequences; and    d. isolating said immunoglobulin or molecule or immunologically functional immunoglobulin fragment therefrom.    
     
     
         35 . The process of  claim 34 , wherein the immunoglobulin or fragment thereof is secreted.  
     
     
         36 . A process for producing an immunoglobulin molecule or an immunologically functional immunoglobulin fragment comprising at least the variable domains of the immunoglobulin heavy and light chains, in a host filamentous fungus, comprising the steps of: 
 a. transforming said host with a composition according to  claim 31;     b. growing said host under conditions which permit expression of said fusion DNA sequences to cause the expression of the desired polypeptides encoded by said fusion DNA sequences; and    c. isolating said immunoglobulin or molecule or immunologically functional immunoglobulin fragment therefrom.    
     
     
         37 . The process of  claim 36 , wherein the immunoglobulin or fragment thereof is secreted.  
     
     
         38 . A filamentous fungal host cell capable of secreting a monoclonal antibody wherein the monoclonal antibody is expressed by the process of  claim 34  or  claim 36 .  
     
     
         39 . A filamentous fungal host cell that has been transformed with a first and second fusion nucleic acid wherein 
 a. the first fusion nucleic acid encodes at least the variable domain of either the immunoglobulin heavy or light chain one chain; and    b. the second fusion nucleic acid encodes the other chain.    
     
     
         40 . A filamentous fungal host cell that has been transformed with a composition according to  claim 31 .  
     
     
         41 . A therapeutic composition comprising a monoclonal antibody or fragment thereof, wherein the antibody is produced by the method of  claim 34  or  claim 36 .  
     
     
         42 . A therapeutic composition comprising a monoclonal antibody or fragment thereof according to  claim 1 .  
     
     
         43 . A therapeutic composition comprising a monoclonal antibody conjugated to a label according to  claim 7 .  
     
     
         44 . A diagnostic or assay kit comprising an antibody or fragment thereof, wherein the antibody is produced by the method of  claim 34  or  claim 36 .  
     
     
         45 . The kit of  claim 44  wherein the monoclonal antibody is conjugated to a label.  
     
     
         46 . The kit of  claim 45  wherein the label is a cytotoxic agent, an antibiotic, or a detectable marker.  
     
     
         47 . The kit of  claim 46  wherein the detectable marker is a radioisotope or an enzyme.