US 2003/0191286 A1 · Purification and characterization of soluble human HLA proteins

Abstract

The present invention relates generally to the production and use of functionally active soluble HLA molecules that are isolated and purified substantially away from other proteins, and methods of purifying same.

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Claims25

Dependent claims

A functionally active, individual soluble HLA molecule purified substantially away from other proteins such that the individual soluble HLA molecule maintains the physical, functional and antigenic integrity of the native HLA molecule, the functionally active, individual soluble HLA molecule produced by the method comprising the steps of:

isolating HLA allele mRNA from a source and reverse transcribing the mRNA to obtain allelic cDNA;
amplifying the allelic cDNA by PCR, wherein the amplification utilizes at least one locus-specific primer that truncates the allelic cDNA, thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic cDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
inoculating a cell pharm or a large scale mammalian tissue culture system with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules, wherein the soluble HLA molecules are folded naturally and are trafficked through the cell in such a way that they are identical in functional properties to an HLA molecule expressed from the HLA allele mRNA and thereby bind peptide ligands in an identical manner as full-length, cell-surface-expressed HLA molecules;
harvesting the soluble HLA molecules from the cell pharm or large scale tissue culture system;
and
purifying the individual, soluble HLA molecules substantially away from other proteins, wherein the individual soluble HLA molecules maintain the physical, functional and antigenic integrity of the native HLA molecule.

5 more elements

obtaining gDNA encoding a HLA allele;
amplifying the allelic gDNA by PCR, wherein the amplification utilizes at least one locus-specific primer that truncates the allelic gDNA, thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic gDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
inoculating a cell pharm with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules, wherein the soluble HLA molecules are folded naturally and are trafficked through the cell in such a way that they are identical in functional properties to an HLA molecule expressed from the HLA allele mRNA and thereby bind peptide ligands in an identical manner as full-length, cell-surface-expressed HLA molecules;
harvesting the soluble HLA molecules from the cell pharm;
and
purifying the individual, soluble HLA molecules substantially away from other proteins, wherein the individual soluble HLA molecules maintain the physical, functional and antigenic integrity of the native HLA molecule.

5 more elements

Dependencies are inferred from the claim text.

Abstract

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Drawings

Claims25

Dependent claims

isolating HLA allele mRNA from a source and reverse transcribing the mRNA to obtain allelic cDNA;
amplifying the allelic cDNA by PCR, wherein the amplification utilizes at least one locus-specific primer that truncates the allelic cDNA, thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic cDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
inoculating a cell pharm or a large scale mammalian tissue culture system with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules, wherein the soluble HLA molecules are folded naturally and are trafficked through the cell in such a way that they are identical in functional properties to an HLA molecule expressed from the HLA allele mRNA and thereby bind peptide ligands in an identical manner as full-length, cell-surface-expressed HLA molecules;
harvesting the soluble HLA molecules from the cell pharm or large scale tissue culture system;
and
purifying the individual, soluble HLA molecules substantially away from other proteins, wherein the individual soluble HLA molecules maintain the physical, functional and antigenic integrity of the native HLA molecule.

5 more elements

obtaining gDNA encoding a HLA allele;
amplifying the allelic gDNA by PCR, wherein the amplification utilizes at least one locus-specific primer that truncates the allelic gDNA, thereby resulting in a truncated PCR product having the coding regions encoding cytoplasmic and transmembrane domains of the allelic gDNA removed such that the truncated PCR product has a coding region encoding a soluble HLA molecule;
inserting the truncated PCR product into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule;
electroporating the plasmid containing the truncated PCR product into at least one suitable host cell;
inoculating a cell pharm with the at least one suitable host cell containing the plasmid containing the truncated PCR product such that the cell pharm produces soluble HLA molecules, wherein the soluble HLA molecules are folded naturally and are trafficked through the cell in such a way that they are identical in functional properties to an HLA molecule expressed from the HLA allele mRNA and thereby bind peptide ligands in an identical manner as full-length, cell-surface-expressed HLA molecules;
harvesting the soluble HLA molecules from the cell pharm;
and
purifying the individual, soluble HLA molecules substantially away from other proteins, wherein the individual soluble HLA molecules maintain the physical, functional and antigenic integrity of the native HLA molecule.

5 more elements

Dependencies are inferred from the claim text.

Description23,564 words

Summary1,863 words

#Not Applicable.

#1. Field of the Invention

#The present invention relates generally to the production and use of functionally active soluble HLA molecules that are isolated and purified substantially away from other proteins, and methods of purifying same.

Brief description of the drawings54 figures

#FIG. 1 is a graphical representation of a Class I location and sHLA class I construction strategy. (A) Simple map of the human MHC region with the class I HLA-B, -C, and -A loci noted. Genetic distances are in kilobases. (B) The basic exon structure of HLA class I gene transcripts. Seven exons encode the class I heavy chain. (C) PCR strategy for truncating the class I molecule so that it is secreted rather than surface bound.

#FIG. 2 is a pictorial representation of native and recombined truncated form of sHLA which differ in the presence of a transmembrane and cytosolic region in the native molecule. Both forms show no differences in their ambiguity and peptide presenting properties.

#FIG. 3 is a three dimensional pictorial representation of a truncated molecule. The bp view is visualizing the α1 and α2 domains harboring the peptide. The side view shows the full molecule with a detailed view of α3 and β2m domains.

Detailed description20,428 words

#Before explaining at least one embodiment of the invention in detail by way of exemplary drawings, experimentation, results, and laboratory procedures, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings, experimentation and/or results. The invention is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

#The present invention combines methodologies for the production of individual, soluble MHC molecules with novel and nonobvious methodologies for the isolation and purification of individual, soluble MHC molecules substantially away from other proteins. The method of production of individual, soluble MHC molecules has previously been described in detail in parent application U.S. Ser. No. 10/022,066, filed Dec. 18, 2001, entitled “METHOD AND APPARATUS FOR THE PRODUCTION OF SOLUBLE MHC ANTIGENS AND USES THEREOF,” the contents of which are hereby expressly incorporated in their entirety by reference herein. A brief description of this methodology is included herein below for the purpose of exemplification and should not be considered as limiting. One of ordinary skill in the art, given the disclosure in the U.S. Ser. No. 10/022,066 application would be truly capable of producing individual soluble MHC molecules to be used with the presently disclosed and claimed isolation and purification methodologies. It should be preliminary noted, however, that the presently claimed and disclosed isolation and purification methodologies can be used with HLA molecules (soluble or non-soluble) obtained by any means and should not be regarded as being limited to soluble HLA molecules produced according to the methodologies claimed and disclosed in the U.S. Ser. No. 10/022,066 application. In the event HLA molecules produced according to methodologies other than those produced according to methodologies disclosed and claimed in the U.S. Ser. No. 10/022,066 application are used in the isolation and purification methodologies disclosed and claimed herein, one of ordinary skill in the art (given in the present specification, drawings and claims) would be capable of making any necessary modifications or derivations to such HLA molecules such that they may be used in the isolation and purification methodologies presently claimed and disclosed herein in an efficient and accurate manner.

#Exemplary Production of Individual, Soluble MHC Molecules

Purification and characterization of soluble human HLA proteins

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Abstract

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Claims25

Purification and characterization of soluble human HLA proteins

Life of this patent

Life of this patent

Abstract

Drawings

Drawings

Claims25

Description23,564 words

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Classification26 codes

Life of this patent

Description23,564 words