Recombinant factor h and variants and conjugates thereof

Abstract

The present invention relates to recombinant factor H and variants and conjugates thereof and methods of their production, as well as uses and methods of treatment involving the materials.

1 .- 20 . (canceled) 
     
     
         21 . A method for making a recombinant mammalian factor H (FH), the method comprising the step of:
 expressing a codon-optimized nucleic acid sequence encoding the mammalian FH in cells in culture,   wherein the codon-optimized nucleic acid sequence comprises a sequence at least 80% identical to any one of SEQ ID NO: 1-13 or at least 80% identical to a deletion variant of any one of SEQ ID NO: 1-13, wherein the deletion variant comprises a deletion of one or more domains, and   wherein the nucleic acid sequence has been codon optimized to enhance expression in the cells and inserted into an expression vector.   
     
     
         22 . The method according to  claim 21 , wherein at least 1 mg of the mammalian FH or variants thereof is produced per liter of culture medium. 
     
     
         23 . The method according to  claim 21  further comprising the step of purifying the mammalian FH or variant thereof from the cell and/or culture medium in which the cells are grown. 
     
     
         24 . The method according to  claim 21 , wherein the cell is a yeast cell, and wherein the yeast cell is  Pichia pastoris.    
     
     
         25 . The method according to  claim 21 , wherein the mammalian FH is selected from the group consisting of human FH, mouse FH, rat FH, hamster FH, rabbit FH, dog FH, horse FH, cow FH, pig FH, sheep FH, camel FH, cat FH, and guinea pig FH. 
     
     
         26 . The method according to  claim 21 , wherein the codon-optimized nucleic acid sequence includes one or more single-nucleotide polymorphisms. 
     
     
         27 . The method according to  claim 27 , wherein the one or more single-nucleotide polymorphisms are selected from the group consisting of Ile62Val, Tyr402H is, and Arg1210Cys. 
     
     
         28 . The method according to  claim 21 , wherein the protein encoded by the codon-optimized nucleic acid sequence includes one or more covalently modified natural or non-naturally encoded variant amino acids. 
     
     
         29 . The method according to  claim 28 , wherein said one or more covalently modified natural or non-naturally encoded variant amino acids is covalently modified to include a moiety selected from the group consisting of glycosaminoglycans, polysialic acids, dextran (−1, 6 polyglucose), dextran (−1, 4 polyglucose), hyaluronic acid, chitosans, linear or branched polyethylene glycols, polyether polyols, N-(2-hydroxypropyl) methacrylamide copolymers, poly(vinylpyrrolidone), poly(ethyleneimine), linear polyamidoamines, poly(L-lysine), poly(glutamic acid), poly(malic acid) and poly(aspartamides). 
     
     
         30 . The method according to  claim 21 , wherein protein encoded by the codon-optimized nucleic acid sequence is conjugated with a chemical moiety or chemical moieties, wherein the moiety or moieties improve the biotherapeutic properties of the codon-optimized nucleic acid sequence. 
     
     
         31 . The method according to  claim 21 , wherein the deletion variant of the encoded protein comprises a deletion of two or more domains that is between 2 domains and 14 domains. 
     
     
         32 . The method according to  claim 21 , wherein the codon-optimized nucleic acid sequence comprises a sequence at least 90% identical to any one of SEQ ID NO: 1-13 or at least 90% identical to a deletion variant of any one of SEQ ID NO: 1-13, wherein the deletion variant comprises a deletion of one or more domains. 
     
     
         33 . The method according to  claim 21 , wherein the protein encoded by the codon-optimized nucleic acid sequence retains mammalian FH-like biological activity. 
     
     
         34 . The method according to  claim 33 , wherein the mammalian FH-like biological activity is assessed according to a biological activity assay, wherein the biological activity assay is selected from the group consisting of:
 (i) ability of the protein encoded by the codon-optimized nucleic acid sequence to act as a cofactor for factor I-catalysed cleavage of C3b;   (ii) ability of the protein encoded by the codon-optimized nucleic acid sequence to promote acceleration of decay of C3bBb assembled on a surface plasmon resonance (SPR) sensor chip;   (iii) affinity of the protein encoded by the codon-optimized nucleic acid sequence for C3b immobilized on a sensor chip;   (iv) affinity of the protein encoded by the codon-optimized nucleic acid sequence for glycosaminoglycans (GAGs); or   (v) ability of the protein encoded by the codon-optimized nucleic acid sequence to protect sheep erythrocytes from complement-mediated hemolysis by FH-depleted human sera.   
     
     
         35 . A nucleic acid sequence capable of expressing a FH polypeptide or variant thereof, wherein the nucleic acid sequence is codon optimized for expression in a cell in culture in an amount greater than 1 mg per liter of culture, and
 wherein the nucleic acid sequence comprises a sequence at least 80% identical to any one of SEQ ID NO: 1-13 or at least 80% identical to a deletion variant of any one of SEQ ID NO: 1-13, wherein the deletion variant comprises a deletion of one or more domains.   
     
     
         36 . The nucleic acid sequence of  claim 35  wherein nucleic acid sequence comprises a sequence at least 90% identical to any one of SEQ ID NO: 1-13 or at least 90% identical to a deletion variant of any one of SEQ ID NO: 1-13, wherein the deletion variant comprises a deletion of one or more domains. 
     
     
         37 . A vector comprising the nucleic acid sequence according to  claim 35 . 
     
     
         38 . A recombinant version of mammalian FH produced as a result of the expression of the nucleic acid sequence of  claim 35 . 
     
     
         39 . A method of treating or preventing a disease in a subject in need thereof comprising the step of administering the recombinant version of mammalian FH according to  claim 38  to the subject. 
     
     
         40 . The method according to  claim 39  for use in treating or preventing a disease selected from the group consisting of AMD, atypical haemolytic uraemic syndrome (aHUS), or dense deposit disease (DDD).