Chimeric proteins

Abstract

Chimeric proteins possessing Kunitz-type domain 1 of TFPI-2 and Kunitz-type domain 2 of TFPI are disclosed, as are muteins of TFPI and TFPI-2. Nucleic acid sequences, expression vectors and transformed host cells encoding and capable of producing the disclosed chimeric proteins and muteins are also disclosed. Finally, methods for prevention and treatment of septic shock using the chimeric proteins and muteins are disclosed.

1 - 27 . (canceled)  
     
     
         28 . A TFPI mutein, said TFPI mutein having one or more amino acid substitutions, said one or more amino acid substitutions occurring exclusively in the P1-reactive site in one or more Kunitz-type domains.  
     
     
         29 . The TFPI mutein of  claim 28  wherein said one or more amino acid substitutions is one amino acid substitution.  
     
     
         30 . The TFPI mutein of  claim 28  wherein said mutein has the primary amino acid sequence of SEQ ID NO:9.  
     
     
         31 . A TFPI-2 mutein, said TFPI-2 mutein having one or more amino acid substitutions, said one or more amino acid substitutions occurring exclusively in the P1-reactive site in one or more Kunitz-type domains.  
     
     
         32 . The TFPI-2 mutein of  claim 31  wherein said one or more amino acid substitutions is one amino acid substitution.  
     
     
         33 . The TFPI-2 mutein of  claim 32  wherein said one amino acid substitution is a Glu to Arg substitution at the P1 reactive site of Kunitz-type domain 2 SEQ ID NO:4.  
     
     
         34 . An isolated nucleic acid molecule encoding a chimeric protein chimeric protein comprising: 
 (a) a Kunitz-type domain 1 of TFPI-2 or a mutein thereof and (b) a Kunitz-type domain 2 of TFPI or a mutein thereof; or    (c) a Kunitz-type domain 2 of TFPI-2 or a mutein thereof and (d) a Kunitz-type domain 1 of TFPI or a mutein thereof.    wherein the chimeric protein binds and inhibits factor VIIa/tissue factor complex and binds to and inhibits factor Xa.    
     
     
         35 . An isolated nucleic acid molecule encoding the TFPI mutein of  claim 28 .  
     
     
         36 . An isolated nucleic acid molecule encoding the TFPI-2 mutein of  claim 31 .  
     
     
         37 . An expression vector capable of producing a chimeric protein chimeric protein comprising: 
 (a) a Kunitz-type domain 1 of TFPI-2 or a mutein thereof and (b) a Kunitz-type domain 2 of TFPI or a mutein thereof, or    (c) a Kunitz-type domain 2 of TFPI-2 or a mutein thereof and (d) a Kunitz-type domain 1 of TFPI or a mutein thereof.    wherein the chimeric protein binds and inhibits factor VIIa/tissue factor complex and binds to and inhibits factor Xa,    said expression vector comprising the nucleic acid molecule of  claim 34  and expression control sequences compatible for expression of said chimeric protein.    
     
     
         38 . An expression vector capable of producing the TFPI mutein of  claim 28 , said expression vector comprising the nucleic acid molecule of  claim 35  and expression control sequences compatible for expression of the TFPI mutein of  claim 28 .  
     
     
         39 . An expression vector capable of producing the TFPI-2 mutein of  claim 31 , said expression vector comprising the nucleic acid molecule of  claim 36  and expression control sequences compatible for expression of the TFPI-2 mutein of  claim 31 .  
     
     
         40 . A transformed host cell capable of producing a chimeric protein, said transformed host cell comprising the expression vector of  claim 37  wherein said expression vector further comprises expression control sequences operably linked to an expression control sequence operable in said host cell.  
     
     
         41 . A transformed host cell capable of producing the TFPI mutein of  claim 28 , said transformed host cell comprising the expression vector of  claim 38  wherein said expression vector further comprises expression control sequences operably linked to an expression control sequence operable in said host cell.  
     
     
         42 . A transformed host cell capable of producing the TFPI-2 mutein of  claim 31 , said transformed host cell comprising the expression vector of  claim 39  wherein said expression vector further comprises expression control sequences operably linked to an expression control sequence operable in said host cell.  
     
     
         43 . A method of producing a chimeric protein chimeric protein comprising: 
 (a) a Kunitz-type domain 1 of TFPI-2 or a mutein thereof and (b) a Kunitz-type domain 2 of TFPI or a mutein thereof; or    (c) a Kunitz-type domain 2 of TFPI-2 or a mutein thereof and (d) a Kunitz-type domain 1 of TFPI or a mutein thereof,    wherein the chimeric protein binds and inhibits factor VIIa/tissue factor complex and binds to and inhibits factor Xa, said method comprising:    (a) providing a transformed host cell comprising an expression vector comprising (1) a polynucleotide sequence encoding the chimeric protein and (2) expression control sequences compatible for expression of the polynucleotide sequence encoding the chimeric protein; and    (b) incubating said transformed host cell under conditions that allow expression of said polynucleotide sequence encoding the chimeric protein.    
     
     
         44 . The method of  claim 43  wherein said host cell is a yeast cell.  
     
     
         45 . The method of  claim 44  wherein said yeast cell is selected from the group consisting of  Candida albicans; Candida maltosa; Hansenula polymorpha; Kluyveromyces fragilis; Kluyveromyces lactis; Pichia guillerimondii; Pichia pastoris; Saccharomyces cerevisiae; Schizosaccharomyces pombe ; and  Yarrowia lipolytica.    
     
     
         46 . The method of  claim 45  wherein said yeast cell is  Saccharomyces cerevisiae.    
     
     
         47 . The method of  claim 46  wherein said chimeric protein is retained within the yeast cell.  
     
     
         48 . The method of  claim 47  wherein said polynucleotide sequence encoding said chimeric protein is immediately preceded in frame by a polynucleotide sequence encoding ubiquitin.  
     
     
         49 . The method of  claim 43  wherein said chimeric protein is secreted from the yeast cell.  
     
     
         50 . The method of  claim 49  wherein said polynucleotide sequence encoding said chimeric protein is immediately preceded in frame by a polynucleotide sequence encoding yeast a factor.  
     
     
         51 . The method of  claim 43  wherein said yeast cell is prevented from producing α-1,6-polymannose terminal carbohydrate.  
     
     
         52 . The method of  claim 51  wherein said yeast cell carries och1, mnnI, and alg3 mutations.  
     
     
         53 . A method of producing the TFPI mutein of  claim 28 , said method comprising: 
 (a) providing a transformed host cell comprising an expression vector comprising (1) a polynucleotide sequence encoding the TFPI mutein of  claim 28  and (2) expression control sequences compatible for expression of said polynucleotide sequence encoding the TFPI mutein of  claim 28;  and    (b) incubating said transformed host cell under conditions that allow expression of said polynucleotide sequence encoding the TFPI mutein of  claim 28 .    
     
     
         54 . The method of  claim 53  wherein said host cell is a yeast cell.  
     
     
         55 . The method of  claim 54  wherein said yeast cell is selected from the group consisting of Candida albicans, Candida maltosa; Hansenula polymorpha; Kluyveromyces fragilis; Kluyveromyces lactis; Pichia guillerimondii; Pichia pastoris; Saccharomyces cerevisiae; Schizosaccharomyces pombe; and  Yarrowia lipolytica.    
     
     
         56 . The method of  claim 55  wherein said yeast cell is  Saccharomyces cerevisiae.    
     
     
         57 . The method of  claim 56  wherein said protein is retained within the yeast cell.  
     
     
         58 . The method of  claim 57  wherein said polynucleotide sequence encoding the TFPI mutein of  claim 28  is immediately preceded in frame by a polynucleotide sequence encoding ubiquitin.  
     
     
         59 . The method of  claim 53  wherein said protein is secreted from the yeast cell.  
     
     
         60 . The method of  claim 59  wherein said polynucleotide sequence encoding the TFPI mutein of  claim 28  is immediately preceded in frame by a polynucleotide sequence encoding yeast a factor.  
     
     
         61 . The method of  claim 53  wherein said yeast cell is prevented from producing α-1,6-polymannose terminal carbohydrate.  
     
     
         62 . The method of  claim 61  wherein said yeast cell carries och1, mnn1 and alg3 mutations.  
     
     
         63 . A method of producing the TFPI-2 mutein of  claim 31 , said method comprising: 
 (a) providing a transformed host cell comprising an expression vector comprising (1) a polynucleotide sequence encoding the TFPI-2 mutein of  claim 31  and (2) expression control sequences compatible for expression of said polynucleotide sequence encoding the TFPI-2 mutein of  claim 31;  and    (b) incubating said transformed host cell under conditions that allow expression of said polynucleotide sequence encoding encoding the TFPI-2 mutein of  claim 31 .    
     
     
         64 . The method of  claim 63  wherein said host cell is a yeast cell.  
     
     
         65 . The method of  claim 64  wherein said yeast cell is selected from the group consisting of Candida albicans, Candida maltosa; Hansenula polymorpha; Kluyveromyces fragilis; Kluyveromyces lactis; Pichia guillerimondii; Pichia pastoris, Saccharomyces cerevisiae; Schizosaccharomyces pombe; and  Yarrowia lipolytica.    
     
     
         66 . The method of clain  65  wherein said yeast cell is  Saccharomyces cerevisiae.    
     
     
         67 . The method of  claim 66  wherein said TFPI-2 mutein is retained within said yeast cell.  
     
     
         68 . The method of  claim 67  wherein said polynucleotide sequence encoding the TFPI-2 mutein of  claim 31  is immediately preceded in frame by a polynucleotide sequence encoding ubiquitin.  
     
     
         69 . The method of  claim 66  wherein said TFPI-2 mutein is secreted from the yeast cell.  
     
     
         70 . The method of  claim 69  wherein said polynucleotide sequence encoding said protein is immediately preceded in frame by a polynucleotide sequence encoding yeast a factor.  
     
     
         71 . The method of  claim 66  wherein said yeast cell is prevented from producing α-1,6-polymannose terminal carbohydrate.  
     
     
         72 . The method of  claim 71  wherein said yeast cell carries och1, mnn1 and alg3 mutations.  
     
     
         73 . (canceled)  
     
     
         74 . A pharmaceutical composition comprising the TFPI mutein of  claim 28  and a pharmaceutically acceptable carrier.  
     
     
         75 . A pharmaceutical composition comprising the TFPI-2 mutein of  claim 31  and a pharmaceutically acceptable carrier.  
     
     
         76 . A method of treating septic shock comprising administering to a mammal in need thereof an effective amount of a pharmaceutical composition comprising: 
 a chimeric protein comprising: 
 (a) a Kunitz-type domain 1 of TFPI-2 or a mutein thereof and (b) a Kunitz-type domain 2 of TFPI or a mutein thereof: or  
 (c) a Kunitz-type domain 2 of TFPI-2 or a mutein thereof and (d) a Kunitz-type domain 1 of TFPI or a mutein thereof,  
   wherein the chimeric protein binds and inhibits factor VIIa/tissue factor complex and binds to and inhibits factor Xa, and 
 a pharmaceutically acceptable carrier.  
   
     
     
         77 . A method of treating septic shock comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of  claim 74 .  
     
     
         78 . A method of treating septic shock comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of  claim 75 .  
     
     
         79 . A method of treating thrombosis disorders comprising administering to a mammal in need thereof an effective amount of a pharmaceutical composition comprising: 
 a chimeric protein comprising: 
 (a) a Kunitz-type domain 1 of TFPI-2 or a mutein thereof and (b) a Kunitz-type domain 2 of TFPI or a mutein thereof; or  
 (c) a Kunitz-type domain 2 of TFPI-2 or a mutein thereof and (d) a Kunitz-type domain 1 of TFPI or a mutein thereof  
   wherein the chimeric protein binds and inhibits factor VIIa/tissue factor complex and binds to and inhibits factor Xa; and 
 a pharmaceutically acceptable carrier.  
   
     
     
         80 . A method of treating thrombosis disorders comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of  claim 74 .  
     
     
         81 . A method of treating thrombosis disorders comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of  claim 75 .  
     
     
         82 . A monoclonal antibody, said monoclonal antibody capable of selectively binding to a chimeric protein comprising: 
 (a) a Kunitz-type domain 1 of TFPI-2 or a mutein thereof and (b) a Kunitz-type domain 2 of TFPI or a mutein thereof, or    (c) a Kunitz-type domain 2 of TFPI-2 or a mutein thereof and (d) a Kunitz-type domain 1 of TFPI or a mutein thereof,    wherein the chimeric protein binds and inhibits factor VIIa/tissue factor complex and binds to and inhibits factor Xa.    
     
     
         83 . The monoclonal antibody of  claim 82  wherein said antibody has an affinity constant of at least 10 −7 /M.  
     
     
         84 . A monoclonal antibody, said monoclonal antibody capable of selectively binding to the TFPI mutein of  claim 28 .  
     
     
         85 . The monoclonal antibody of  claim 84  wherein said antibody has an affinity constant of at least 10 −7 /M.  
     
     
         86 . A monoclonal antibody, said monoclonal antibody capable of selectively binding to the TFPI-2 mutein of  claim 31 .  
     
     
         87 . The monoclonal antibody of  claim 86  wherein said antibody has an affinity constant of at least 10 −7 /M.