EP0325224A2 — Methods of producing secreted receptor analogs an…

Claim 1

Independent Claim 95 words

A method for producing a secreted human PDGF receptor analog, comprising: introducing into a host cell a DNA construct capable of directing the expression and secretion of a human PDGF receptor analog, said DNA construct containing a transcriptional promoter operatively linked to a secretory signal sequence followed downstream in proper reading frame by a DNA sequence encoding at least a portion of the extracellular domain of a human PDGF receptor, said portion including a ligand-binding domain; growing said host cell in an appropriate growth medium; and isolating said PDGF receptor analog from said host cell.

Claim 2

Independent Claim 95 words

A method for producing a secreted human PDGF receptor analog, comprising: introducing into a host cell a DNA construct capable of directing the expression and secretion of a human PDGF receptor analog, said DNA construct containing a transcriptional promoter operatively linked to a secretory signal sequence followed downstream in proper reading frame by a DNA sequence encoding at least a portion of the extracellular domain of a human PDGF receptor, said portion including a ligand-binding domain; growing said host cell in an appropriate growth medium; and isolating said PDGF receptor analog from said host cell.

Claim 3

Dependent Claim 55 words

The method of claim 1 wherein said DNA sequence encodes a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to methionine, number 441 and a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to lysine, number 531.

Claim 4

Independent Claim 102 words

A method for producing a secreted, biologically active peptide dimer, comprising: introducing into a host cell a DNA construct capable of directing the expression and secretion of a biologically active peptide dimer, said DNA construct containing a transcriptional promoter operatively linked to at least one secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a peptide requiring dimerization for biological activity fused to a dimerizing protein; growing said host cell in an appropriate growth medium under conditions allowing the dimerization and secretion of the peptide dimer; and isolating said peptide dimer from said host cell.

Claim 5

Independent Claim 102 words

A method for producing a secreted, biologically active peptide dimer, comprising: introducing into a host cell a DNA construct capable of directing the expression and secretion of a biologically active peptide dimer, said DNA construct containing a transcriptional promoter operatively linked to at least one secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a peptide requiring dimerization for biological activity fused to a dimerizing protein; growing said host cell in an appropriate growth medium under conditions allowing the dimerization and secretion of the peptide dimer; and isolating said peptide dimer from said host cell.

Claim 6

Dependent Claim 36 words

The method of claim 1 or claim 3 wherein said host cell is a yeast cell carrying a defect in a gene whose product is required for the addition of outer chain oligosaccharide moieties to glycoproteins.

Claim 7

Dependent Claim 31 words

The method of claim 1 or claim 3 wherein said host cell is a yeast cell containing a genetic defect in the MNN9 gene or a disruption of the MNN9 gene.

Claim 8

Dependent Claim 51 words

A method according to claim 1 or claim 3 wherein said secretory signal sequence is selected from the group consisting of the MF α1 pre-pro sequence, the PHO5 signal sequence, the BAR1 signal sequence, the SUC2 signal sequence, the PDGF receptor signal sequence and the mouse immunoglobulin V H signal sequence.

Claim 9

Dependent Claim 48 words

The method of claim 3 wherein said dimerizing protein comprises at least a portion of a protein selected from the group consisting of an immunoglobulin light chain, an immunoglobulin heavy chain and yeast invertase, wherein said portion associates as a dimer in a covalent or a noncovalent manner.

Claim 10

Dependent Claim 25 words

The method of claim 3 wherein said dimerizing protein is selected from the group consisting of an immunoglobulin heavy chain hinge region and yeast invertase.

Claim 11

Independent Claim 179 words

A method for producing a secreted, biologically active peptide dimer, comprising: introducing into a host cell a first DNA construct containing a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a peptide requiring dimerization for biological activity joined to a dimerizing protein, said dimerizing protein comprising an immunoglobulin light chain constant region; introducing into said host cell a second DNA construct containing a transcriptional promoter operatively linked to a second secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin hinge region; growing said host cell in an appropriate growth medium under conditions that allow the dimerization and secretion of said biologically active peptide dimer; and isolating said biologically active peptide dimer from said host cell.

Claim 12

Independent Claim 179 words

A method for producing a secreted, biologically active peptide dimer, comprising: introducing into a host cell a first DNA construct containing a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a peptide requiring dimerization for biological activity joined to a dimerizing protein, said dimerizing protein comprising an immunoglobulin light chain constant region; introducing into said host cell a second DNA construct containing a transcriptional promoter operatively linked to a second secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin hinge region; growing said host cell in an appropriate growth medium under conditions that allow the dimerization and secretion of said biologically active peptide dimer; and isolating said biologically active peptide dimer from said host cell.

Claim 13

Independent Claim 183 words

A method for producing a secreted, biologically active peptide dimer, comprising: introducing into a host cell a first DNA construct containing a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a peptide requiring dimerization for biological activity joined to a dimerizing protein, said dimerizing protein comprising at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin heavy chain hinge region; introducing into said host cell a second DNA construct containing a transcriptional promoter operatively linked to a second secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding at least an immunoglobulin light chain constant region; growing said host cell in an appropriate growth medium under conditions that allow the dimerization and secretion of said biologically active peptide dimer; and isolating said biologically acitve peptide dimer from said host cell.

Claim 14

Independent Claim 183 words

A method for producing a secreted, biologically active peptide dimer, comprising: introducing into a host cell a first DNA construct containing a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a peptide requiring dimerization for biological activity joined to a dimerizing protein, said dimerizing protein comprising at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin heavy chain hinge region; introducing into said host cell a second DNA construct containing a transcriptional promoter operatively linked to a second secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding at least an immunoglobulin light chain constant region; growing said host cell in an appropriate growth medium under conditions that allow the dimerization and secretion of said biologically active peptide dimer; and isolating said biologically acitve peptide dimer from said host cell.

Claim 15

Independent Claim 63 words

A method according to any of claims 3, 9 or 10 wherein said peptide requiring dimerization for biological activity is selected from the group consisting of a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to lysine, number 531 and a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to methionine, number 441.

Claim 16

Independent Claim 175 words

A method for producing a secreted, ligand-binding receptor analog, comprising: introducing into a host cell a first DNA construct containing a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a ligand-binding receptor analog joined to a dimerizing protein, said dimerizing protein comprising an immunoglobulin light chain constant region; introducing into said host cell a second DNA construct containing a transcriptional promoter operatively linked to a second secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin heavy chain hinge region; growing said host cell in an appropriate growth medium under conditions that allow the dimerization and secretion of said ligand-binding receptor analog; and isolating said ligand-binding receptor analog from said host cell.

Claim 17

Independent Claim 175 words

A method for producing a secreted, ligand-binding receptor analog, comprising: introducing into a host cell a first DNA construct containing a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a ligand-binding receptor analog joined to a dimerizing protein, said dimerizing protein comprising an immunoglobulin light chain constant region; introducing into said host cell a second DNA construct containing a transcriptional promoter operatively linked to a second secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin heavy chain hinge region; growing said host cell in an appropriate growth medium under conditions that allow the dimerization and secretion of said ligand-binding receptor analog; and isolating said ligand-binding receptor analog from said host cell.

Claim 18

Independent Claim 175 words

A method for producing a secreted, ligand-binding receptor analog, comprising: introducing into a host cell a first DNA construct containing a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a ligand-binding receptor analog joined to a dimerizing protein, said dimerizing protein comprising at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin hinge region; introducing into said host cell a second DNA construct containing a transcriptional promoter operatively linked to a second secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding at least an immunoglobulin light chain constant region; growing said host cell in an appropriate growth medium under conditions that allow the dimerization and secretion of said ligand-binding receptor analog; and isolating said ligand-binding receptor analog from said host cell.

Claim 19

Independent Claim 175 words

A method for producing a secreted, ligand-binding receptor analog, comprising: introducing into a host cell a first DNA construct containing a transcriptional promoter operatively linked to a first secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding a ligand-binding receptor analog joined to a dimerizing protein, said dimerizing protein comprising at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin hinge region; introducing into said host cell a second DNA construct containing a transcriptional promoter operatively linked to a second secretory signal sequence followed downstream by and in proper reading frame with a DNA sequence encoding at least an immunoglobulin light chain constant region; growing said host cell in an appropriate growth medium under conditions that allow the dimerization and secretion of said ligand-binding receptor analog; and isolating said ligand-binding receptor analog from said host cell.

Claim 20

Dependent Claim 57 words

The method of claim 12 or claim 13 wherein said ligand-binding receptor analog is selected from the group consisting of a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to lysine, number 531 and a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to methionine, number 441.

Claim 21

Independent Claim 57 words

A method according to any of claims 9, 10, 12 or 13 wherein said first and second secretory signal sequences are selected from the group consisting of the MF α1 pre-pro sequence, the PHO5 signal sequence, the BAR1 signal sequence, the SUC2 signal sequence, the PDGF receptor signal sequence and the mouse immunoglobulin V H signal sequence.

Claim 22

Independent Claim 27 words

A method according to any of claims 1, 3, 8, 9, 11 or 12 wherein the host cell is a yeast cell or a cultured mammalian cell.

Claim 23

Dependent Claim 27 words

A human PDGF receptor analog consisting essentially of the extracellular domain of a human PDGF receptor, said portion being capable of binding human PDGF or isoforms thereof.

Claim 24

Dependent Claim 27 words

A human PDGF receptor analog consisting essentially of the extracellular domain of a human PDGF receptor, said portion being capable of binding human PDGF or isoforms thereof.

Claim 25

Dependent Claim 56 words

The human PDGF receptor analog of claim 17 wherein said receptor analog is selected from the group consisting of a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to methionine, number 441 and a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to lysine, number 531.

Claim 26

Independent Claim 74 words

A method for determining the presence of human PDGF or isoforms thereof in a biological sample, comprising: incubating a polypeptide comprising a human PDGF receptor analog fused to a dimerizing protein with a biological sample suspected of containing human PDGF or an isoform thereof under conditions that allow the formation of receptor/ligand complexes; and detecting the presence of the receptor/ligand complexes as an indication of the presence of human PDGF or an isoform thereof.

Claim 27

Independent Claim 74 words

A method for determining the presence of human PDGF or isoforms thereof in a biological sample, comprising: incubating a polypeptide comprising a human PDGF receptor analog fused to a dimerizing protein with a biological sample suspected of containing human PDGF or an isoform thereof under conditions that allow the formation of receptor/ligand complexes; and detecting the presence of the receptor/ligand complexes as an indication of the presence of human PDGF or an isoform thereof.

Claim 28

Dependent Claim 62 words

The method of claim 19 wherein said human PDGF receptor analog comprises a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to methionine, number 441, and a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to lysine, number 531, fused to a dimerizing protein.

Claim 29

Dependent Claim 77 words

The method of claim 19 wherein said dimerizing protein is selected from the group consisting of an immunoglobulin light chain constant region, an immunoglobulin heavy chain hinge region, at least one immunoglobulin heavy chain constant region domain selected from the group consisting of C H 1, C H 2, C H 3, C γ 1, C γ 2, C γ 3, C γ 4, and µ joined to an immunoglobulin heavy chain hinge region and yeast invertase.

Claim 30

Dependent Claim 23 words

A pharmaceutical composition comprising a human PDGF receptor analog fused to a dimerizing protein in combination with a physiologically acceptable carrier or diluent.

Claim 31

Dependent Claim 23 words

A pharmaceutical composition comprising a human PDGF receptor analog fused to a dimerizing protein in combination with a physiologically acceptable carrier or diluent.

Claim 32

Dependent Claim 55 words

The pharmaceutical composition of claim 22 wherein said PDGF receptor analog is selected from the group consisting of a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to methionine, number 441 and a polypeptide comprising the amino acid sequence of Figure 1 from isoleucine, number 29, to lysine, number 531.

Claim 33

Dependent Claim 29 words

A diagnostic composition comprising a polypeptide comprising a human PDGF receptor analog fused to a dimerizing protein, said polypeptide tagged with a label capable of providing a detectable signal.

Claim 34

Dependent Claim 29 words

A diagnostic composition comprising a polypeptide comprising a human PDGF receptor analog fused to a dimerizing protein, said polypeptide tagged with a label capable of providing a detectable signal.

Claim 35

Independent Claim 69 words

A method for purifying human PDGF of an isoform thereof from a sample comprising: immobilizing a polypeptide comprising a PDGF receptor analog fused to a dimerizing protein on a substrate; contacting a sample containing human PDGF or an isoform thereof with the immobilized polypeptide under conditions such that the human PDGF or isoform thereof binds to the polypeptide; and eluting the human PDGF or isoform thereof from the polypeptide.

Claim 36

Independent Claim 69 words

A method for purifying human PDGF of an isoform thereof from a sample comprising: immobilizing a polypeptide comprising a PDGF receptor analog fused to a dimerizing protein on a substrate; contacting a sample containing human PDGF or an isoform thereof with the immobilized polypeptide under conditions such that the human PDGF or isoform thereof binds to the polypeptide; and eluting the human PDGF or isoform thereof from the polypeptide.

Methods of producing secreted receptor analogs and biologically active peptide dimers.

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