Multimeric proteins and methods of making and using same

Abstract

The invention provides multimerization polypeptides capable of conferring formation of multimers when the multimerization polypeptide is linked to a molecule, such as a heterologous polypeptide sequence.

What is claimed is:  
     
         1 . A multimerization polypeptide selected from the amino acid sequences set forth in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163.  
     
     
         2 . A polypeptide having 70% or greater identity to a multimerization polypeptide of  claim 1 , said polypeptide capable of multimerization.  
     
     
         3 . A polypeptide having 75% or greater identity to a multimerization polypeptide of  claim 1 , said polypeptide capable of multimerization.  
     
     
         4 . A polypeptide having 80% or greater identity to a multimerization polypeptide of  claim 1 , said polypeptide capable of multimerization.  
     
     
         5 . A polypeptide having 85% or greater identity to a multimerization polypeptide of  claim 1 , said polypeptide capable of multimerization.  
     
     
         6 . A polypeptide having 90% or greater identity to a multimerization polypeptide of  claim 1 , said polypeptide capable of multimerization.  
     
     
         7 . A polypeptide having 95% or greater identity to a multimerization polypeptide of  claim 1 , said polypeptide capable of multimerization.  
     
     
         8 . A polypeptide subsequence of the multimerization polypeptide of  claim 1 , said polypeptide subsequence capable of multimerization.  
     
     
         9 . The multimerization polypeptide of  claim 2 , said polypeptide having one or more amino acid substitutions, provided that all of positions a or d of a seven residue zipper repeat sequence (a.b.c.d.e.f.g), are either leucine, isoleucine or valine, said substituted polypeptide capable of conferring multimerization.  
     
     
         10 . The multimerization polypeptide of  claim 2 , said polypeptide having one or more amino acid substitutions, provided that one or more positions a or d of a seven residue zipper repeat sequence (a.b.c.d.e.f.g), are either leucine, isoleucine or valine, said substituted polypeptide capable of conferring multimerization.  
     
     
         11 . The multimerization polypeptide of  claim 10 , wherein at least one of positions a or d of a seven residue zipper repeat sequence (a.b,c.d.e.f.g), are an amino acid other than leucine, isoleucine or valine.  
     
     
         12 . The multimerization polypeptide of  claim 10 , said polypeptide having 1 to 5 amino acid substitutions.  
     
     
         13 . The multimerization polypeptide of  claim 2 , said polypeptide having one or more amino acid substitutions in positions b, c, e, f or g, provided that said substituted polypeptide is capable of conferring multimerization.  
     
     
         14 . The multimerization polypeptide of  claim 13 , said polypeptide having 1 to 5 amino acid substitutions.  
     
     
         15 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence at least 11 amino acids in length.  
     
     
         16 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence at least 15 amino acids in length.  
     
     
         17 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence at least 18 amino acids in length.  
     
     
         18 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence at least 22 amino acids in length.  
     
     
         19 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence at least 27 amino acids in length.  
     
     
         20 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence at least 31 amino acids in length.  
     
     
         21 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence less than about 125 amino acids in length.  
     
     
         22 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence less than about 100 amino acids in length.  
     
     
         23 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence less than about 75 amino acids in length.  
     
     
         24 . The multimerization polypeptide of  claim 1 , said polypeptide having a sequence less than about 50 amino acids in length.  
     
     
         25 . A chimeric polypeptide comprising the multimerization polypeptide of  claim 1 , fused to a heterologous polypeptide.  
     
     
         26 . The chimeric polypeptide of  claim 25 , wherein the multimerization polypeptide is fused to the amino terminus of the heterologous polypeptide.  
     
     
         27 . The chimeric polypeptide of  claim 25 , wherein the multimerization polypeptide is fused to the carboxy terminus of the heterologous polypeptide.  
     
     
         28 . The chimeric polypeptide of  claim 25 , wherein the multimerization polypeptide has 50% or greater identity to a multimerization polypeptide of  claim 1 , said chimeric polypeptide capable of conferring multimerization.  
     
     
         29 . The chimeric polypeptide of  claim 25 , wherein the multimerization polypeptide is a subsequence of the multimerization polypeptide of  claim 1 , said chimeric polypeptide capable of conferring multimerization.  
     
     
         30 . The chimeric polypeptide of  claim 25 , wherein the multimerization polypeptide has one or more amino acid substitutions, provided that one or more of positions a and d of a seven residue zipper repeat sequence (a.b.c.d.e.f.g) are either leucine, isoleucine or valine, and said chimeric polypeptide is capable of multimerization.  
     
     
         31 . The chimeric polypeptide of  claim 25 , wherein the chimeric polypeptide has a sequence length from about 18-30, 30-50, 50-75, 75-100, 100-150, 150-200, 200-250, 250-500 or 500-1000 amino acids.  
     
     
         32 . The chimeric polypeptide of  claim 25 , wherein the heterologous polypeptide is selected from: a binding protein, enzyme, receptor, ligand, nucleic acid binding protein, growth regulatory factor, differentiative factor, and chemotactic factor.  
     
     
         33 . The chimeric polypeptide of  claim 32 , wherein the binding protein comprises an antigen binding polypeptide.  
     
     
         34 . The chimeric polypeptide of  claim 33 , wherein the antigen binding polypeptide comprises at least one antibody variable domain.  
     
     
         35 . The chimeric polypeptide of  claim 34 , wherein the antibody variable domain is human or humanized.  
     
     
         36 . The chimeric polypeptide of  claim 33 , wherein the antigen binding polypeptide comprises a single chain antibody, Fab, Fab′, (Fab′) 2 , or Fv antibody subsequence.  
     
     
         37 . The chimeric polypeptide of  claim 33 , wherein the antigen binding polypeptide comprises a multispecific or multifunctional antibody.  
     
     
         38 . The chimeric polypeptide of  claim 33 , wherein the antigen binding polypeptide binds to ICAM-1 or an epitope thereof.  
     
     
         39 . The chimeric polypeptide of  claim 33 , wherein the antigen binding polypeptide 30 inhibits human rhinovirus infection of a cell that expresses ICAM-1.  
     
     
         40 . The chimeric polypeptide of  claim 33 , wherein the antigen binding polypeptide forms a hetero- or homo-dimer, -trimer, -tetramer or higher order oligomer.  
     
     
         41 . The chimeric polypeptide of  claim 40 , wherein the K D  of the monomers comprising the homo-dimer, -trimer, -tetramer or higher order oligomer is 1×10 −7  or less.  
     
     
         42 . The chimeric polypeptide of  claim 40 , wherein the K D  of the monomers comprising the homo-dimer, -trimer, -tetramer or higher order oligomer is 1×10 −8  or less.  
     
     
         43 . A homo- or hetero-dimer, trimer, tetramer or higher order polypeptide oligomer comprising at least one chimeric polypeptide of  claim 25 .  
     
     
         44 . The chimeric polypeptide of  claim 25 , wherein the polypeptide contains a linker between the multimerization polypeptide and the heterologous polypeptide.  
     
     
         45 . The chimeric polypeptide of  claim 44 , wherein the linker comprises a human or humanized amino acid sequence.  
     
     
         46 . The chimeric polypeptide of  claim 44 , wherein the linker comprises an amino acid sequence from about 5 to 20 amino acids.  
     
     
         47 . The chimeric polypeptide of  claim 44 , wherein the linker comprises an amino acid sequence from about 10 to 30 amino acids.  
     
     
         48 . The chimeric polypeptide of  claim 44 , wherein the linker comprises an amino acid sequence from about 25 to 50 amino acids.  
     
     
         49 . The chimeric polypeptide of  claim 44 , wherein the linker comprises an amino acid sequence from about 30 to 60 amino acids.  
     
     
         50 . The chimeric polypeptide of  claim 44 , wherein the linker comprises an amino acid sequence from about 50 to 75 amino acids.  
     
     
         51 . The chimeric polypeptide of  claim 44 , wherein the linker comprises an amino acid sequence set forth in any of SEQ ID NO:43 (D30), SEQ ID NO:44 (D35), SEQ ID NO:45 (ED), SEQ ID NO:46 (EDC) or SEQ ID NO:47 (D63).  
     
     
         52 . A chimeric polypeptide comprising a linker polypeptide sequence set forth in any of SEQ ID NO:43 (D30), SEQ ID NO:44 (D35), SEQ ID NO:45 (ED), SEQ ID NO:46 (EDC) or SEQ ID NO:47 (D63), fused to a heterologous polypeptide.  
     
     
         53 . The chimeric polypeptide of  claim 52 , wherein the linker polypeptide sequence is a subsequence set forth in any of SEQ ID NO:43 (D30), SEQ ID NO:44 (D35), SEQ ID NO:45 (ED), SEQ ID NO:46 (EDC) or SEQ ID NO:47 (D63).  
     
     
         54 . A pharmaceutical formulation comprising the chimeric polypeptide of  claim 23 .  
     
     
         55 . A nucleic acid encoding the polypeptide of  claim 25  or  52 .  
     
     
         56 . An expression cassette comprising the nucleic acid of  claim 55 , operably linked to an expression control element.  
     
     
         57 . A vector comprising the nucleic acid of  claim 56 .  
     
     
         58 . A cell comprising the nucleic acid of  claim 56 .  
     
     
         59 . The cell of  claim 58 , wherein the cell is selected from the group of: bacterial, fungal, animal, plant, and insect cells.  
     
     
         60 . The cell of  claim 57 , wherein the animal cell is mammalian.  
     
     
         61 . A nucleic acid comprising a sequence that encodes a multimerization polypeptide of  claim 1 .  
     
     
         62 . The nucleic acid of  claim 61 , operably linked to an expression control element.  
     
     
         63 . The nucleic acid of  claim 61 , fused in frame to a nucleic acid encoding a heterologous polypeptide.  
     
     
         64 . The nucleic acid of  claim 63 , wherein a nucleic acid encoding a linker sequence is located between the nucleic acid of  claim 61  and the nucleic acid encoding the heterologous polypeptide.  
     
     
         65 . A vector comprising the nucleic acid sequence of  claim 61 .  
     
     
         66 . The vector of  claim 65 , wherein the vector is an expression vector.  
     
     
         67 . A cell comprising the nucleic acid of  claim 61 .  
     
     
         68 . The cell of  claim 67 , wherein the cell is selected from the group of: bacterial, fungal, animal, plant, and insect cells.  
     
     
         69 . The cell of  claim 68 , wherein the animal cell is mammalian.  
     
     
         70 . A method of producing a multimerization polypeptide comprising one or more seven residue repeat sequence, (a.b.c.d.e.f.g), that confers formation of a multimer, comprising modifying a polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a or d are replaced with either leucine or isoleucine, thereby producing a multimerization polypeptide that confers multimer formation.  
     
     
         71 . The method of  claim 70 , wherein the modified polypeptide forms a trimer, tetramer or pentamer, whereas the unmodified polypeptide forms a dimer.  
     
     
         72 . The method of  claim 70 , wherein the modified polypeptide has increased or decreased stability in a multimer in comparison to the unmodified polypeptide.  
     
     
         73 . The method of  claim 70 , wherein the modified polypeptide forms a tetramer or pentamer, whereas the unmodified polypeptide forms a dimer or trimer.  
     
     
         74 . A method of producing a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), that confers formation of a dimer, comprising modifying a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein positions a or d are replaced with valine and either of leucine or isoleucine, thereby producing a multimerization polypeptide that confers dimer formation.  
     
     
         75 . The method of  claim 74 , wherein the unmodified polypeptide forms a trimer or tetramer or pentamer.  
     
     
         76 . The method of  claim 74 , wherein the modified polypeptide has increased or decreased stability in a multimer in comparison to unmodified polypeptide.  
     
     
         77 . A method of producing a chimeric polypeptide that forms a trimer, comprising producing a chimeric polypeptide comprising a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a and d are either leucine or isoleucine, fused to a heterologous polypeptide thereby producing chimeric polypeptide that forms a trimer.  
     
     
         78 . The method of  claim 77 , wherein the unmodified polypeptide forms a dimer or tetramer or pentamer.  
     
     
         79 . The method of  claim 77 , wherein the modified polypeptide has increased or decreased stability in a multimer in comparison to unmodified polypeptide.  
     
     
         80 . A method of producing a chimeric polypeptide that forms a tetramer, comprising producing a chimeric polypeptide comprising a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a and d are either leucine or isoleucine, fused to a heterologous polypeptide thereby producing chimeric polypeptide that forms a tetramer.  
     
     
         81 . The method of  claim 80 , wherein the unmodified polypeptide forms a dimer or trimer or pentamer.  
     
     
         82 . The method of  claim 80 , wherein the modified polypeptide has increased or decreased stability in a multimer in comparison to the unmodified polypeptide.  
     
     
         83 . A method of producing a chimeric polypeptide that forms a pentamer, comprising producing a chimeric polypeptide comprising a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a and d are either leucine or isoleucine, fused to a heterologous polypeptide thereby producing chimeric polypeptide that forms a pentamer.  
     
     
         84 . The method of  claim 83 , wherein the unmodified polypeptide forms a dimer or trimer or tetramer.  
     
     
         85 . The method of  claim 83 , wherein the modified polypeptide has increased or decreased stability in a multimer in comparison to the unmodified polypeptide.  
     
     
         86 . A method of producing a molecule that forms a multimer, comprising producing a molecule including a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein positions a or d are valine and either of leucine or isoleucine, fused to the molecule thereby producing a molecule that forms a multimer.  
     
     
         87 . The method of  claim 86 , wherein the molecule is a polypeptide.  
     
     
         88 . A method of producing a molecule that forms a trimer or tetramer or pentamer, comprising linking a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a and d are either leucine or isoleucine, to a molecule thereby producing a molecule that forms a trimer or tetramer or pentamer.  
     
     
         89 . The method of  claim 88 , wherein the molecule is a polypeptide.  
     
     
         90 . A method of identifying a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), comprising: 
 a) incubating a polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a and d are either leucine, isoleucine or valine, under conditions allowing formation of homo- or hetero-multimers; and    b) assaying for the presence of homo- or hetero-multimers of the polypeptide, wherein formation of a homo- or hetero-multimer identifies a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g).    
     
     
         91 . The method of  claim 90 , wherein the polypeptide of step a) comprises a heterologous polypeptide fused to a polypeptide comprising the seven residue repeat sequence, (a.b.c.d.e.f.g).  
     
     
         92 . The method of  claim 91 , wherein the polypeptide contains a linker between the heterologous polypeptide and the polypeptide comprising the seven residue repeat sequence, (a.b.c.d.e.f.g).  
     
     
         93 . A method of inhibiting RSV infection of a cell comprising contacting RSV or a cell susceptible to RSV infection with an amount of a hetero- or homo-dimer, -trimer, -tetramer, -pentamer or higher order oligomer of the antibody of  claim 36  effective to inhibit RSV infection of the cell.  
     
     
         94 . The method of  claim 87 , wherein the cell is present in a subject.  
     
     
         95 . The method of  claim 88 , wherein the subject has or is at risk of having asthma.  
     
     
         96 . The method of  claim 87 , wherein the cell is an epithelial cell.  
     
     
         97 . The method of  claim 87 , wherein the antibody is humanized.  
     
     
         98 . The method of  claim 87 , wherein the antibody is administered locally.  
     
     
         99 . The method of  claim 87 , wherein the antibody is administered via inhalation or intranasaly.  
     
     
         100 . A method of inhibiting RSV infection, inhibiting RSV progression or treating RSV infection of a subject comprising administering to a subject having or at risk of having RSV infection an amount of an antibody of  claim 38 , or a hetero- or homo-dimer, -trimer, -tetramer or higher order oligomer of the antibody of  claim 38 , effective to inhibit, inhibit progression or treat RSV infection of the subject.  
     
     
         101 . The method of  claim 100 , wherein the antibody is humanized.  
     
     
         102 . The method of  claim 100 , wherein the antibody is administered locally.  
     
     
         103 . The method of  claim 100 , wherein the antibody is administered via inhalation or intranasaly.  
     
     
         104 . The method of  claim 100 , wherein the subject has or is at risk of having asthma.  
     
     
         105 . The method of  claim 100 , wherein the subject is a new born or between the ages of 1 to 5, 5 to 10 or 10 to 18.  
     
     
         106 . A method of treating the common cold comprising administering to a subject having or at risk of having a common cold an amount of an antibody of  claim 38 , or a hetero- or homo-dimer, -trimer, -tetramer or higher order oligomer of the antibody of  claim 38 , effective to treat the common cold in the subject.  
     
     
         107 . The method of  claim 106 , wherein the treatment comprises inhibiting infection by HRV, progression of HRV infection or a symptom of HRV infection.  
     
     
         108 . The method of  claim 106 , wherein the antibody is humanized.  
     
     
         109 . The method of  claim 106 , wherein the antibody is administered locally.  
     
     
         110 . The method of  claim 106 , wherein the antibody is administered via inhalation or intranasaly.  
     
     
         111 . The method of  claim 106 , wherein the subject has or is at risk of having asthma.  
     
     
         112 . The method of  claim 106 , wherein the subject is a new born or between the ages of 1 to 5, 5 to 10 or 10 to 18.