WO2008088837A2 - Expression de protéines hla sur des cellules non humaines - Google Patents

Expression de protéines hla sur des cellules non humaines Download PDF

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Publication number
WO2008088837A2
WO2008088837A2 PCT/US2008/000595 US2008000595W WO2008088837A2 WO 2008088837 A2 WO2008088837 A2 WO 2008088837A2 US 2008000595 W US2008000595 W US 2008000595W WO 2008088837 A2 WO2008088837 A2 WO 2008088837A2
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WO
WIPO (PCT)
Prior art keywords
hla
trimolecular complex
individual
functionally active
biological sample
Prior art date
Application number
PCT/US2008/000595
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English (en)
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WO2008088837A3 (fr
Inventor
William H. Hildebrand
Original Assignee
The Board Of Regents Of The University Of Oklahoma
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by The Board Of Regents Of The University Of Oklahoma filed Critical The Board Of Regents Of The University Of Oklahoma
Priority to AU2008205526A priority Critical patent/AU2008205526A1/en
Priority to EP08724561A priority patent/EP2115122A4/fr
Publication of WO2008088837A2 publication Critical patent/WO2008088837A2/fr
Publication of WO2008088837A3 publication Critical patent/WO2008088837A3/fr
Priority to IL199861A priority patent/IL199861A0/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing

Definitions

  • MHC human major histocompatibility complex
  • HLA human leukocyte antigens
  • NK Natural Killer Cells
  • Display of peptide antigens on the MHC I and MHC Il molecules are the basis for the recognition of "self vs. non-self and the onset of important immune responses such as transplant rejection, graft-versus-host-disease, autoimmune disease, and healthy anti-viral and antibacterial immune responses.
  • Class Il MHC molecules designated HLA class Il in humans, also bind and display peptide antigen ligands upon the cell surface. Unlike class I MHC molecules which are expressed on virtually all nucleated cells, class Il MHC molecules are normally confined to specialized cells, such as B lymphocytes, macrophages, dendritic cells, and other antigen presenting cells which take up foreign antigens from the extracellular fluid via an endocytic pathway.
  • the peptide antigens bound and presented by class Il HLA are derived from extracellular foreign antigens, such as products of bacteria that multiply outside of cells, wherein such products include protein toxins secreted by the bacteria or any other bacterial protein to which the human immune system might respond in a protective manner.
  • Figure 1 illustrates expression of MHC Il on NS-1. Shaded area, untransfected NS-1 ; green line, human T2 cells, known not to express MHC II; Blue line and red line, THP- 1 and U937 respectively, human macrophage-like cell lines known to express MHC II; black line, NS-1 double transfected with DRA1*0101 and DRB1*0101.
  • Figure 8 illustrates a sera screen similar to Figure 7, except using a different clone of HLA-DRB * 0401.
  • the membrane-proximal ⁇ 2 and ⁇ 2 domains bear sequence homology to the immunoglobulin-fold domain structure.
  • the membrane-distal domain of a class Il molecule is composed of the Ct 1 and fa domains, which form an antigen-binding cleft for processed peptide antigen.
  • the peptides presented by class Il molecules are derived from extracellular proteins (not cytosolic intracellular peptide antigens as in class I); hence, the MHC class ll-dependent pathway of antigen presentation is called the endocytic or exogenous pathway.
  • trimolecular complex as used herein will be understood to refer to the MHC heterodimer associated with a peptide.
  • An "MHC class I trimolecular complex” or “HLA class I trimolecular complex” will be understood to include the class I heavy and light chains associated together and having a peptide displayed in an antigen binding groove thereof.
  • the terms "MHC class Il trimolecular complex” and “HLA class Il trimolecular complex” will be understood to include the class Il alpha and beta chains associated together and having a peptide displayed in an antigen binding groove thereof.
  • HLA human protein
  • Other systems that use human cells to produce HLA typically purify the HLA and then put the HLA on a surface meant to resemble the cell from which HLA was extracted. This allows the HLA to be the only human protein available, but these systems struggle to purify the desired HLA away from other HLA.
  • HLA will be the only human protein available on the cell. Therefore, the HLA may never need to be extracted from the non-human cell, purified, concentrated, and adhered to a cell-like surface. All of these extraction/purification/concentration/adherence steps can impact HLA protein yield and can alter the HLA protein conformation such that it loses some or all of its native structure and serologic recognition. Expressing an HLA protein on a non-human cell obviates extraction, purification, adherence, and the like.
  • non-human cells examples include, but are not limited to, mouse DC lines, macrophage lines, and B cell lines. Specific examples include, but are not limited to, murine B cell lines such as NS-
  • Affinity chromatography occupies a unique place in separation technology since it is the only technique which enables purification of almost any biomolecule on the basis of its biological function or individual chemical structure.
  • Affinity chromatography makes use of specific binding interactions that occur between molecules. It is a type of adsorption chromatography in which the molecule to be purified is specifically and reversibly adsorbed by a complementary binding substance (ligand) immobilized on an insoluble support (matrix).
  • ligand complementary binding substance
  • matrix insoluble support
  • a single pass through an affinity column can achieve a 1 ,000-10,000 fold purification of ligand from a crude mixture. It is possible to isolate a compound in a form pure enough to obtain a single band upon SDS-polyacrylamide gel electrophoresis. Any component that has an interacting counterpart can be attached to a support and used for affinity purification.
  • the present invention is also directed to methods of removing anti- HLA antibodies from a biological sample.
  • an HLA platform as described above is provided.
  • a biological sample is also provided and reacted with the HLA platform, whereby antibodies specific for the HLA trimolecular complex will bind to the HLA platform and are thus removed from the biological sample.
  • the method of detecting and/or removing anti-HLA antibodies discussed herein above may also be performed utilizing purified HLA.
  • the present invention also includes a method for detecting the presence of anti-HLA antibodies in a biological sample. Such method includes providing a substrate and a functionally active, individual HLA trimolecular complex purified substantially away from other proteins such that the individual HLA trimolecular complex maintains the physical, functional and antigenic integrity of a native HLA trimolecular complex.
  • the functionally active, individual HLA trimolecular complex comprises alpha and beta chains with an endogenously loaded peptide displayed in an antigen binding groove formed by the alpha and beta chains.
  • the functionally active, individual HLA trimolecular complex may be directly attached to the substrate, or the HLA trimolecular complex may be indirectly attached to the substrate via an anchoring moiety selected from the group consisting of an antibody to the functionally active, individual HLA trimolecular complex and a tail or tag attached to the functionally active, individual HLA trimolecular complex.
  • the tail or tag may be a histidine tag, a biotinylation signal peptide, a VLDLr tail or a FLAG tail.
  • HLA-DR alpha and beta chains next it was tested whether these HLA-DR expressing mouse cells were recognized by human sera specific for particular human class Il molecules. Sera that is specific for HLA-DRA1*0101/DRB1*0101 was obtained from a clinical laboratory. Using flow cytometry, this DRB1*0101 sera was tested against mouse cells expressing DRA1*0101/DRB1*0101 , DRA1 * 0101/DRB1*0401 , DRA1*0101/DRB1 * 0801 , and DRA1 * 0101/DRB1*1101.
  • EXAMPLE 2 Expression of human MHC Il on mouse cell line.
  • Figure 6 graphically represents the experimental procedure for the detection of specific Anti-HLA class Il antibodies in human sera, utilizing HLA class Il trimolecular complexes produced in accordance with the present invention.
  • the presented sandwich ELISA platform uses an anti-Class Il antibody (L243), capturing a class Il allele of interest and presenting it to the test sera.
  • Anti-HLA class Il antibodies with the sera will specifically bind to those MHC molecules and can then be detected by an anti-human IgG antibody.
  • Figure 7 demonstrates the typical reactivity patterns of specific human sera with the Class Il molecule DRB1*0401 (CL-017). Eight human sera containing anti-class Il antibodies with different amounts and specificity were tested.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une composition qui comprend un complexe trimoléculaire HLA individuel, fonctionnellement actif, exprimé sur la surface d'une lignée cellulaire non humaine immortalisée. Des procédés pour obtenir une expression élevée de la molécule HLA transférée, ainsi que des analyses qui utilisent une telle lignée cellulaire recombinante en tant que plate-forme, sont également décrits. Des procédés de purification du HLA à partir d'une telle lignée cellulaire recombinante et des procédés d'utilisation du HLA purifié pour détecter ou enlever des anticorps anti-HLA sont également décrits.
PCT/US2008/000595 2007-01-17 2008-01-17 Expression de protéines hla sur des cellules non humaines WO2008088837A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2008205526A AU2008205526A1 (en) 2007-01-17 2008-01-17 Expression of HLA proteins on non-human cells
EP08724561A EP2115122A4 (fr) 2007-01-17 2008-01-17 Expression de proteines hla sur des cellules non humaines
IL199861A IL199861A0 (en) 2007-01-17 2009-07-14 Expression of hla proteins on non-human cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US88083807P 2007-01-17 2007-01-17
US60/880,838 2007-01-17

Publications (2)

Publication Number Publication Date
WO2008088837A2 true WO2008088837A2 (fr) 2008-07-24
WO2008088837A3 WO2008088837A3 (fr) 2008-12-11

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PCT/US2008/000595 WO2008088837A2 (fr) 2007-01-17 2008-01-17 Expression de protéines hla sur des cellules non humaines

Country Status (4)

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EP (1) EP2115122A4 (fr)
AU (1) AU2008205526A1 (fr)
IL (1) IL199861A0 (fr)
WO (1) WO2008088837A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2702066A2 (fr) * 2011-04-29 2014-03-05 The Board of Regents of the University of Oklahoma Dispositif d'élimination d'anticorps anti-hla sélectifs et procédés d'obtention et d'utilisation associés

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PAJOT A. ET AL.: 'Comparison of HLA-DR1-restricted T cell response induced in HLA-DR1 transgenic mice deficient for murine MHC class II and HLA-DR1 transgenic mice expressing endogenous murine MHC class II molecules' INT. IMMUNOL. vol. 16, no. 9, September 2004, pages 1275 - 1282, XP002314835 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2702066A2 (fr) * 2011-04-29 2014-03-05 The Board of Regents of the University of Oklahoma Dispositif d'élimination d'anticorps anti-hla sélectifs et procédés d'obtention et d'utilisation associés
CN103857691A (zh) * 2011-04-29 2014-06-11 俄克拉何马大学董事会 选择性抗hla抗体去除装置及其生产方法和用途
EP2702066A4 (fr) * 2011-04-29 2014-12-31 Univ Oklahoma Dispositif d'élimination d'anticorps anti-hla sélectifs et procédés d'obtention et d'utilisation associés

Also Published As

Publication number Publication date
IL199861A0 (en) 2010-04-15
WO2008088837A3 (fr) 2008-12-11
AU2008205526A1 (en) 2008-07-24
EP2115122A4 (fr) 2010-12-29
EP2115122A2 (fr) 2009-11-11

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