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

• the present invention relates generally to a methodology of expression of human HLA proteins on non-human cells as well as to methods of utilizing such non-human cells having HLA proteins expressed thereon.
• 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 I and class Il HLA molecules differ from person to person. Each person expresses a different complement of class I and class Il on the surface of their cells. For transplant purposes it is important to determine which of the multiple HLA expressed on a cell are recognized by the antibodies of another individual. Anti-HLA antibodies can lead to hyperacute organ rejection. It is often difficult to determine which of many HLA are recognized by antibodies because sera can have antibodies to non-HLA proteins, multiple HLA molecules, and sera may crossreact among different HLA molecules.
• Antibodies to HLA proteins may also lead to problems during the transfusion of blood products, whereby antibodies in the blood of the blood donor may react with the HLA class I and class Il antigens of the recipient of the blood product. Antibodies in the blood product that recognize the recipient's HLA may lead to transfusion related acute lung injury (TRALI).
• TRALI transfusion related acute lung injury
• Class I MHC molecules designated HLA class I in humans, bind and display peptide antigen ligands upon the cell surface.
• the peptide antigen ligands presented by the class I MHC molecule are derived from either normal endogenous proteins ("self) or foreign proteins ("nonself ) introduced into the cell. Nonself proteins may be products of malignant transformation or intracellular pathogens such as viruses.
• class I MHC molecules convey information regarding the internal fitness of a cell to immune effector cells including but not limited to, CD8 + cytotoxic T lymphocytes (CTLs), which are activated upon interaction with "nonself peptides, thereby lysing or killing the cell presenting such "nonself peptides.
• CTLs cytotoxic T lymphocytes
• 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.
• class Il molecules convey information regarding the existence of pathogens in extracellular spaces that are accessible to the cell displaying the class Il molecule.
• Class Il HLA expressing cells then present peptide antigens derived from the extracellular antigen/bacteria to immune effector cells, including but not limited to, CD4 + helper T cells, thereby helping to eliminate such pathogens.
• the elimination of such pathogens is accomplished by both helping B cells make antibodies against microbes, as well as toxins produced by such microbes, and by activating macrophages to destroy ingested microbes.
• Class I and class Il HLA molecules exhibit extensive polymorphism generated by systematic recombinatorial and point mutation events; as such, hundreds of different HLA types exist throughout the world's population, resulting in substantial immunologic diversity. Such extensive HLA diversity throughout the population results in tissue or organ transplant rejection between individuals as well as differing susceptibilities and/or resistances to infectious diseases. HLA molecules also contribute significantly to autoimmunity and cancer. Because HLA molecules mediate most, if not all, adaptive immune responses, and because of their tremendous diversity, large quantities of individual HLA proteins are required in order to effectively study transplantation, autoimmunity disorders, and for vaccine development.
• HLA protein there has been no readily available source of individual HLA molecules.
• the quantities of HLA protein available have been small and typically consist of a mixture of different HLA molecules.
• Production of HLA molecules traditionally involves growth and lysis of cells expressing multiple HLA molecules. Ninety percent of the population is heterozygous at each of the HLA loci; codominant expression results in multiple HLA proteins expressed at each HLA locus.
• To purify native class I or class Il molecules from human cells requires time-consuming and cumbersome purification methods in order to separate individual class I or class Il HLA molecules away from other HLA proteins expressed by the cell, and since each cell typically expresses multiple surface- bound HLA class I or class Il molecules, HLA purification results in a mixture of many different HLA class I or class Il molecules.
• HLA class I contains three genes of interest: HLA-A, B, and C
• HLA class Il contains multiple gene products as well including, DRA, DRB, DPA, DPB, DQA, and DQB
• DRA, DRB, DPA, DPB, DQA, and DQB must be taken into consideration.
• these proteins are polymorphic and are expressed in a heterozygous fashion, meaning that each cell expresses one molecule form from the mother and another from the father, leading to the expression of many different MHC molecules on each cell surface.
• antibodies have a difficult time discriminating among various class Il - this complicates the serologic selection of cells that express large numbers of a particular HLA class II, and this complicates the purification of a given class Il HLA protein.
• 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 2 illustrates NS-1 transfectants expressing human MHC II. Shaded area, untransfected NS-1; black line, DRA1*0101/DRB1 * 0401; blue line, DRA1*0101/DRB1*0801 ; red line, DRA1 * 0101/DRB1 * 1101 ; and green line, DRA1*0101/DRB1*0102 [0016]
• Figure 3 illustrates plasma from 4 individuals pre-tested as DR1 , tested again against NS-1 transfected with human MHC II.
• Figure 4 illustrates the selection of high expression of NS-1 transfected with DRA1 *0101. NS-1 cells were analyzed by flow cytometry for their expression of DRA*0101. Column 1 , untransfected NS-1 cells; column 2, initial transfer and expression of DRA*0101 ; columns 3-5: NS-1 cells transfected with DRA1 * 0101 after one, two or three sorting steps, respectively.
• Figure 5 illustrates the selection of high expression of NS-1 transfected with
• NS-1 cells were analyzed by flow cytometry for their expression of
• Figure 6 graphically represents a sandwich ELISA platform for the detection of specific Anti-HLA class Il antibodies in human sera, in accordance with the present invention.
• Figure 7 show typical reactivity patterns of specific human sera with the Class Il molecule DRB1*0401 in the ELISA platform of Figure 6.
• Figure 8 illustrates a sera screen similar to Figure 7, except using a different clone of HLA-DRB * 0401.
• Figure 9 graphically represents a direct detection approach for the detection of specific Anti-HLA class Il antibodies in human sera, in accordance with the present invention.
• FIG. 10 illustrates ELISA results of HLA-DRB1 * 0401 directly coated in the
• Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
• the foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Coligan et al. Current Protocols in Immunology (Current Protocols, Wiley lnterscience (1994)), which are incorporated herein by reference.
• isolated polynucleotide and isolated nucleic acid segment shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide” or “isolated nucleic acid segment” (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide” or “isolated nucleic acid segment” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
• isolated protein means a protein of genomic, cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of murine proteins, (3) is expressed by a cell from a different species, or, (4) does not occur in nature.
• polypeptide as used herein is a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein, fragments, and analogs are species of the polypeptide genus.
• naturally-occurring refers to the fact that an object can be found in nature.
• a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.
• Antibody or “antibody peptide(s)” refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single- chain antibodies. An antibody other than a "bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical.
• An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay).
• MHC Major Histocompability Complex
• HLA Human Leukocyte Antigens, which is defined as the major histocompatibility antigens found in humans.
• HLA is the human form of "MHC”.
• MHC class I light chain and "MHC class I heavy chain” as used herein will be understood to refer to portions of the MHC class I molecule.
• class I molecules are heterodimers comprised of two noncovalently bound polypeptide chains, a larger "heavy” chain ( ⁇ ) and a smaller “light” chain ( ⁇ -2-microglobulin or ⁇ 2m).
• the polymorphic, polygenic heavy chain (45 kDa), encoded within the MHC on chromosome six, is subdivided into three extracellular domains (designated 1 , 2, and 3), one intracellular domain, and one transmembrane domain.
• the two outermost extracellular domains, 1 and 2 together form the groove that binds antigenic peptide.
• interaction with the TCR occurs at this region of the protein.
• the 3 rd extracellular domain of the molecule contains the recognition site for the CD8 protein on the CTL; this interaction serves to stabilize the contact between the T cell and the APC.
• the invariant light chain (12 kDa), encoded outside the MHC on chromosome 15, consists of a single, extracellular polypeptide.
• MHC class I light chain " ⁇ -2-microglobulin", and “ ⁇ 2m” may be used interchangeably herein. Association of the class I heavy and light chains is required for expression of class I molecules on cell membranes.
• class Il molecules are also heterodimers, but in this case consist of two nearly homologous ⁇ and ⁇ chains, both of which are encoded in the MHC.
• the class Il MHC molecules are membrane-bound glycoproteins, and both the ⁇ and ⁇ chains contain external domains, a transmembrane anchor segment, and a cytoplasmic segment.
• Each chain in a class Il molecule contains two external domains: the 33-kDa ⁇ chain contains O 1 and ⁇ 2 external domains, while the 28-kDa ⁇ chain contains ⁇ i and ⁇ 2 external domains.
• 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.
• class Il molecules Loading of class Il molecules must still occur inside the cell; extracellular proteins are endocytosed, digested in lysosomes, and bound by the class Il MHC molecule prior to the molecule's migration to the plasma membrane. Because the peptide-binding groove of MHC class Il molecules is open at both ends while the corresponding groove on class I molecules is closed at each end, the peptides presented by MHC class Il molecules are longer, generally between 13 and 24 amino acid residues long.
• class I HLA the peptides that bind to class Il molecules often have internal conserved "motifs', but unlike class l-binding peptides, they lack conserved motifs at the carboxyl- terminal end, since the open ended binding cleft allows a bound peptide to extend from both ends.
• 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.
• antibody is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, F(ab')2 and Fv) so long as they exhibit the desired biological activity.
• Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
• substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
• biological sample as used herein will be understood to include, but not be limited to, serum, tissue, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid, organ or tissue culture derived fluids, and fluids extracted from physiological tissues.
• biological sample as used herein will also be understood to include derivatives and fractions of such fluids, as well as combinations thereof.
• biological sample will also be understood to include complex mixtures.
• One advantage of the present invention over the prior art is the ability to select for non-human cells that express a high number of one specific surface-bound human HLA class I or class Il molecule.
• the one specific HLA molecule, or an alpha or beta chain thereof, will be transferred into the non-human cells. Because the non-human cells do not express other competing HLA, the present invention allows for the selection of high expression of a single human HLA protein without background interference from other human proteins that may be serologically crossreactive. This will facilitate the establishment of cell lines that express high amounts of a single class I or class Il HLA protein.
• Class I and class Il highly expressed HLA proteins can then be purified without interference from other human proteins that might interfere with the HLA purification due to crossreactivity or similarity to the human HLA protein to be characterized.
• the present invention allows for the utilization of a human-specific HLA purification scheme that will not be impacted by other human proteins because they are not found in the non-human cells that have been transfected to express HLA.
• the present invention provides control of the environment where the HLA molecule is expressed (i.e., the HLA molecule will be the only human molecule on a non- human mammalian background), many other experiments can be performed in order to better understand the biochemical and biophysical properties of the human MHC.
• the present invention essentially eliminates background noise that would otherwise interfere with studying how manipulations change expression and purification of an individual HLA protein. Changes can be made in the HLA protein, the gene promoter, etc., and the impact of these changes on the HLA protein in question can be assessed without interference from other non-modified HLA.
• Non-human cells expressing human HLA molecules can be used in cell arrays (Stephan et al., 2002), where human serum could be tested against a panel of non-human cells that are each expressing different HLA molecules. Thus, non-human cells that are not reactive to serum can be identified, and then a single HLA protein can be expressed in these non-human cells, thus facilitating the clear and precise serologic characterization of this HLA protein.
• the non-human cells will only express a single human molecule, the background given by the binding antibody to a given molecule will be low, and could be completely eliminated by subtracting the background of the HLA non-transfected non- human cells, from the binding observed when using the non-human cell expressing the given HLA molecule.
• 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 expressing the HLA protein may be used as an HLA platform themselves rather than requiring extraction of the HLA protein. Therefore, one embodiment of the present invention is a composition comprising a recombinant, non- human cell having at least one functionally active, individual HLA trimolecular complex on a surface thereof.
• HLA protein as used herein will be understood to refer to any HLA molecule, complex thereof or fragment thereof that is capable of being expressed on a surface of a non-human cell.
• HLA proteins that may be utilized in accordance with the present invention include, but are not limited to, an HLA class I trimolecular complex, an HLA class Il trimolecular complex, an HLA class Il ⁇ chain and an HLA class Il ⁇ chain.
• HLA class Il ⁇ and/or ⁇ proteins that may be utilized in accordance with the present invention include, but are not limited to, those encoded at the following gene loci: HLA-DRA; HLA-DRB1 ; HLA-DRB3,4,5; HLA-DQA; HLA-DQB; HLA- DPA; and HLA-DPB.
• non-human cell as used herein will be understood to refer to any cell capable of expressing a recombinant HLA protein (as defined herein above) on a surface thereof. Therefore, any "non-human cell” utilized in accordance with the present invention must contain the necessary machinery and transport proteins required for expression of MHC/HLA proteins and/or MHC/HLA trimolecular complexes on a surface of such cell. "Non-human cells” utilized in accordance with the present invention must have (A) machinery for chaperoning and loading MHC/HLA proteins, such as class I and class Il proteins; and (B) such machinery must be able to interact and work with human HLA proteins, such as class I and class Il proteins.
• class Il MHC protein not all cells express class Il MHC protein; only professional immune cells such as but not limited to dendritic cells (DC), macrophages, B cells, and the like express class Il proteins. Therefore, when it is desired to express class Il HLA protein in a non-human cell, such non-human cell must express class Il MHC for that species and contain the appropriate machinery for interacting and working with both that species' class Il MHC as well as human class Il HLA.
• the present invention also includes the use of cells of other lineages that have been induced to express class Il MHC, such as but not limited to, cytokines, cells that have been subjected to mutagenesis, and the like.
• non-human cells utilized in accordance with the present invention must not only express class Il MHC for that species and be capable of expressing human class Il HLA, but the endogenous class Il MHC present in such cell must also not cross react with antibodies against the human class Il HLA. This allows for high human HLA class
• non-human cell refers to immortalized non-human cell lines and does not include animals or primary cells.
• 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-
• the non-human cells expressing HLA can be repeatedly selected for high expression of the HLA protein put into the cell without interference from other human proteins.
• the non-human cells can be selected for high expression of the selected HLA protein, and then the HLA protein can be extracted in large quantities due to high expression.
• the present invention therefore provides a method of purifying HLA, wherein the method comprises providing a recombinant vector comprising at least one nucleic acid segment encoding alpha and beta chains of at least one HLA molecule (as defined herein above).
• An immortalized, non-human cell line is transformed with the recombinant vector to produce a non-human cell line having at least one functionally active, individual HLA trimolecular complex expressed on a surface thereof, wherein the functionally active, individual HLA trimolecular complex comprises alpha and beta chains having an endogenously loaded peptide displayed in an antigen binding groove formed by the alpha and beta chains.
• the functionally active, individual HLA trimolecular complex is then 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.
• 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.
• biospecific ligand antibody, enzyme, receptor protein or other affinity reagent
• Methods must also include removing the bound material in active form with low pH, high pH, or high salt.
• the selection of the ligand for affinity chromatography is influenced by two factors. Firstly, the ligand should exhibit specific and reversible binding affinity for the substance to be purified. Secondly, it should have chemically modifiable groups, which allow it to be attached to the matrix without destroying its binding activity. The ligand should ideally have an affinity for the binding substance in the range 10-4 to 10-8 M in free solution.
• the purification step of the methods of the present invention may comprise affinity chromatography utilizing, for example but not by way of limitation, an antibody specific for the HLA trimolecular complex, or an anchoring moiety attached to the HLA trimolecular complex.
• anchoring moieties and chromatography affinity reagents examples include, but are not limited to, (i) a hisitidine tag, utilized with affinity reagents such as nickel, copper and/or combinations thereof; (ii) a biotinylation signal peptide, utilized with affinity reagents such as avidin or streptavidin; and (iii) a VLDLr tail or FLAG tail, utilized with affinity reagents that recognize the VLDLr or FLAG tails.
• affinity reagents such as nickel, copper and/or combinations thereof
• a biotinylation signal peptide utilized with affinity reagents such as avidin or streptavidin
• VLDLr tail or FLAG tail utilized with affinity reagents that recognize the VLDLr or FLAG tails.
• the present invention is not limited to the specific combinations of anchoring moieties and chromatography affinity reagents described above.
• the cell surface is the natural environment for HLA, and using the transfected non-human cell as an HLA platform will provide the most natural environment for assays that require or benefit from HLA in its native conformation.
• the native conformation of HLA expressed on non-human cells is especially useful for screening antibodies in serologic assays because anti-HLA antibodies are especially sensitive to changes in the native conformation of the HLA protein.
• the expression of HLA on non-human cells provides a "conformationally natural platform" for antibody screening assays.
• the present invention is also directed to a method for detecting the presence of anti-HLA antibodies in a biological sample.
• an HLA platform is provided; the HLA platform comprises a recombinant, non-human cell having at least one functionally active, individual HLA trimolecular complex on a surface thereof, wherein the non-human cell is from an immortalized, non-human cell line and contains the necessary machinery and transport proteins required for expression of MHC proteins on a surface of the non-human cell.
• the individual HLA trimolecular complex comprises alpha and beta chains having an endogenously loaded peptide in an antigen binding groove formed by the alpha and beta chains.
• a biological sample is also provided and reacted with the HLA platform, whereby antibodies specific for the HLA protein will bind to the HLA platform.
• the HLA platform is then washed to remove unbound portions of the biological sample, followed by reacting the HLA platform with means for detecting anti-HLA antibodies. It is then determined that anti-HLA antibodies specific for the functionally active, individual HLA trimolecular complex are present in the biological sample if the means for detecting anti-HLA antibodies is positive.
• the means for detecting an anti-HLA antibody may be a labeled antibody that recognizes at least one of anti-human IgG, IgM and IgA antibodies.
• Non-human cells expressing HLA can also be utilized for antibody removal assays.
• 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.
• HLA expressing non-human cells could be "fixed” such that they are no longer alive.
• fixed cells would express HLA in a native conformation at the surface of the fixed cells.
• Fixed cells could be immobilized on any number of platforms (see for example but not by way of limitation, Stephan et al., 2002), and sera or antibodies could be tested for their recognition of the fixed and immobilized cells.
• HLA expressing non-human cells could be fixed and tested for antibody reactivity in a liquid phase assay such as flow cytometry.
• HLA proteins present peptide ligands that provoke anti-viral, anti-bacterial, autoimmune, anti-transplant (minor histocompatibility antigens), regulatory, and anti-cancer immune responses
• individual HLA could be characterized for their ability to present cancer related, virus related, bacterial, and self (autoimmune and regulatory) peptides in non- human cells.
• HLA proteins expressed in mice are known to present peptide antigens as they do in humans (Rohrlich et al., 2003), and HLA molecules expressed in mice could be used to discover therapeutic and diagnostic peptide epitopes for cancer and for bacterial antigens taken into the non-human cell expressing the human HLA.
• HLA trimolecular complexes purified away from the non-human cells may be utilized.
• the HLA trimolecular complexes may be purified as described herein above or by any other methods known to a person having ordinary skill in the art.
• the present invention also includes a method in which a substrate is provided, wherein the substrate is selected from the group consisting of a well, a bead, a membrane, a microtiter plate, a matrix, a pore, plastic, glass, a polymer, a polysaccharide, nylon, nitrocellulose, a paramagnetic compound, and combinations thereof.
• 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 is provided.
• the functionally active, individual HLA trimolecular complex may be purified as described above or by any other method known in the art.
• 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 is then directly or indirectly linked to the substrate, wherein the conformation of the functionally active, individual HLA trimolecular complex is maintained when the functionally active, individual HLA trimolecular complex is linked to the substrate.
• 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.
• 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 is then directly or indirectly linked to the substrate, wherein the conformation of the functionally active, individual HLA trimolecular complex is maintained when the functionally active, individual HLA trimolecular complex is linked to the substrate.
• a biological sample is then reacted with the substrate having the functionally active, individual HLA trimolecular complex linked thereto, and the substrate is washed to remove unbound portions of the biological sample.
• the substrate having the functionally active, individual HLA trimolecular complex linked thereto is then reacted with means for detecting anti-HLA antibodies, and it is determined that anti-HLA antibodies specific for the individual HLA trimolecular complex are present in the biological sample if the means for detecting anti-HLA antibodies is positive.
• 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.
• the substrate may be a solid support selected from the group consisting of a well, a bead, a membrane, an ELISA plate, and a matrix.
• the present invention also includes a method for removing anti-HLA antibodies from a biological sample.
• the 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 is then directly or indirectly linked to the substrate, wherein the conformation of the functionally active, individual HLA trimolecular complex is maintained when the functionally active, individual HLA trimolecular complex is linked to the substrate.
• a biological sample is then reacted with the substrate having the functionally active, individual HLA trimolecular complex linked thereto, whereby antibodies specific for the HLA trimolecular complex are removed from the biological sample.
• 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.
• the substrate may be a solid support selected from the group consisting of a well, a bead, a membrane, an ELISA plate, and a matrix.
• human HLA molecules on non-human cells can be a powerful tool in facilitating the identification and characterization of serologic antibodies directed towards HLA molecules.
• Serologic background reactivity to other human proteins, especially to other HLA can be greatly reduced and eliminated.
• Such anti-HLA antibodies are naturally occurring in all individuals and must be identified and characterized prior to allogeneic grafting, transplantation and the transfer of blood products.
• the lack of human HLA on non-human cells allows for the selection of non-human cells expressing high levels of human HLA. High levels of human HLA facilitate specificity and sensitivity in downstream diagnostic and therapeutic applications.
• EXAMPLE 1 Transfection of Class Il HLA-DR molecules on mouse NS-1 cells.
• the methods began by transfecting either the HLA-DR alpha chain (DRA * 0101) or the HLA-DR beta chain (DRB * 0101) into NS-1 cells. It was then shown by flow cytometry that the transfected alpha or beta chain could be expressed on the cell surface. Once it was known that an alpha or beta chain could be expressed alone, these alpha and beta chains were selected by flow cytometry for high expression. Next, the corresponding chain was then transfected so that DRB*0101 and DRA * 0101 were expressed together. Flow cytometry was then utilized to show that the class Il alpha and beta chains were being co- expressed on the surface of NS-1.
• HLA DR alpha and beta chains can be expressed on mouse cells and that HLA alpha/beta coexpression greatly enhances HLA class Il expression as measured by antibodies specific for human class Il molecules.
• 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.
• Murine cell line NS-1 was first transfected with DRA1*0101 expressed in the pcDNA3.1/G418 vector (Invitrogen). After proper antibiotic selection, transfectants were identified by flow cytometry using mAb mouse anti-HLA-DR L243 (BioLegends) F(ab) 2 fragment PE-goat anti-mouse IgG. High expressers for DRA1 were single cell cloned and then double transfected with the human MHC Il beta chain expressed in the pcDNA3.1/Zeocin vector (Invitrogen).
• the transfectants were stained with mAb mouse anti-HLA-DRB TDR31.1 (ABCAM) and then with F(ab) 2 fragment PE-goat anti-mouse IgG, and compared to untransfected NS-1 and to different human cell lines ( Figure 1).
• Figure 4 illustrates analysis of NS-1 transfected with DRA1 * 0101.
• NS-1 cells were analyzed by flow cytometry for their expression of DRA1 * 0101.
• column 1 the untransfected NS-1 cells show no DRA1 * 0101 class Il expression.
• column 2 the initial transfer and expression of DRA1*0101 showed that 22% of the cells were positive.
• column three after one sort, 42% were positive for DRA1 * 0101 class Il expression; 74% were positive after two sorts in column 4, and 88% were positive after 3 sorts in column five.
• Figure 5 illustrates analysis of NS-1 transfected with DRB1 * 0101.
• NS-1 cells were analyzed by flow cytometry for their expression of DRB1 * 0401/DRA1 * 0101.
• column 1 the untransfected NS-1 cells show no class Il expression.
• column 2 the expression of the previously transfected DRA1 * 0101 is used to establish a threshold.
• class Il alpha/beta/peptide expression on the human cell line 721.221 known to express plentiful class II, is demonstrated in column three.
• column four the initial transfection of DRB1*0101 into the DRA1 * 0101 positive cells results in 18% of the NS-1 cells rising above the DRA1*0101-only threshold.
• EXAMPLE 4 Affinity Purification of HLA Class Il from Recombinant, Non-Human Cell Lines
• the Class Il molecules produced in accordance with the present invention were subjected to affinity purification.
• the affinity purification protocol is provided herein below.
• [0087] Upon completing a roller-bottle run generating approximately 10 10 cells, cells were washed in PBS followed by the extraction of HLA class Il complexes in MHC lysis buffer consisting of 1% NP-40 in PBS and a protease inhibitor cocktail (Sigma). To increase the efficiency of extraction, the samples were sonicated by applying ten 5-second bursts over a time period of 30 minutes.
• the crude extract was affinity purified using the DR class Il antibody L243 coupled to a Sepharose 4B matrix (Amersham; Piscataway, NJ). Extracts were applied to the column using a peristaltic pump system (Amersham) with a speed of 1 ml/min at 4°C. After the column was extensively washed with phosphate-buffered saline (PBS) 1 bound HLA molecules were eluted with 0.1 M glycine (pH 11.0) and immediately neutralized by addition of 1 M Tris-HCI, pH 7.0 to preserve the activity of the eluted molecules. Under these conditions, HLA molecules were recovered with a 60-70% yield. Purified molecules were buffer exchanged with PBS at pH
• 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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