WO2003057852A2 - Purification et caracterisation de proteines hla - Google Patents

Purification et caracterisation de proteines hla Download PDF

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Publication number
WO2003057852A2
WO2003057852A2 PCT/US2003/000243 US0300243W WO03057852A2 WO 2003057852 A2 WO2003057852 A2 WO 2003057852A2 US 0300243 W US0300243 W US 0300243W WO 03057852 A2 WO03057852 A2 WO 03057852A2
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Prior art keywords
hla molecule
hla
functionally active
molecules
molecule
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PCT/US2003/000243
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English (en)
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WO2003057852A3 (fr
Inventor
William H. Hildebrand
Rico Buchli
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Hildebrand William H
Rico Buchli
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Priority claimed from US10/022,066 external-priority patent/US20030166057A1/en
Application filed by Hildebrand William H, Rico Buchli filed Critical Hildebrand William H
Priority to IL16284503A priority Critical patent/IL162845A0/xx
Priority to AU2003202892A priority patent/AU2003202892A1/en
Priority to CA002514872A priority patent/CA2514872A1/fr
Publication of WO2003057852A2 publication Critical patent/WO2003057852A2/fr
Priority to AU2003270876A priority patent/AU2003270876A1/en
Priority to CA002539622A priority patent/CA2539622A1/fr
Priority to PCT/US2003/030096 priority patent/WO2004029280A2/fr
Publication of WO2003057852A3 publication Critical patent/WO2003057852A3/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

  • the present invention relates generally to the production and use of
  • MHC major histocompatibility complex
  • 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 presented by the class I MHC molecule
  • Nonself proteins introduced into the cell.
  • Nonself proteins may be any proteins
  • class I MHC molecules convey information
  • CTLs cytotoxic T lymphocytes
  • Class II MHC molecules designated HLA class II in humans, also bind and display peptide antigen ligands upon the cell surface. Unlike class I MHC
  • class II 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 ' peptides they bind and present 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 that often times have deleterious and even lethal effects on the host (e.g. human).
  • class II molecules convey information regarding the fitness of the extracellular space in the vicinity of the cell displaying the class II molecule to immune effector cells, including but not limited to, CD4 + helper T cells, thereby helping to eliminate such pathogens.
  • the examination 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 II HL.A 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 a large immunological 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, large quantities of pure isolated HLA proteins are required in order to effectively study transplantation, autoimmunity disorders, and for vaccine development.
  • MHC-peptide multimers as immunodiagnostic reagents for disease resistance/autoimmunity; assessing the binding of potentially therapeutic peptides; elution of peptides from MHC molecules to identify vaccine candidates; screening transplant patients for preformed MHC specific antibodies; and removal of anti-HLA antibodies from a patient. Since every individual has differing MHC molecules, the testing of numerous individual MHC molecules is a prerequisite for understanding the differences in disease susceptibility between individuals. Therefore, isolated and purified MHC molecules that are representative of the hundreds of different HLA types existing throughout the world's population are highly desirable for unraveling disease susceptibilities and resistances, as well as for designing therapeutics such as vaccines.
  • Class I HLA molecules alert the immune response to disorders within host cells.
  • Peptides which are derived from viral- and tumor-specific proteins within the cell, are loaded into the class I molecule's antigen binding groove in the endoplasmic reticulum of the cell and subsequently carried to the cell surface. Once the class I HLA molecule and its loaded peptide ligand are on the cell surface, the class I molecule and its peptide ligand are accessible to cytotoxic T lymphocytes (CTL). CTL survey the peptides presented by the class I molecule and destroy those cells harboring ligands derived from infectious or neoplastic agents within that cell.
  • CTL cytotoxic T lymphocytes
  • Discerning virus- and tumor-specific ligands for CTL recognition is an important component of vaccine design.
  • Ligands unique to tumorigenic or infected cells can be tested and incorporated into vaccines designed to evoke a protective CTL response.
  • Several methodologies are currently employed to identify potentially protective peptide ligands.
  • One approach uses T cell lines or clones to screen for biologically active ligands among chromatographic fractions of eluted peptides (Cox et al., Science, vol 264, 1994, pages 716-719, which is expressly incorporated herein by reference in its entirety). This approach has been employed to identify peptides ligands specific to cancerous cells.
  • a second technique utilizes predictive algorithms to identify peptides capable of binding to a particular class I molecule based upon previously determined motif and/or individual ligand sequences (De Groot et al., Emerging Infectious Diseases, (7) 4, 2001, which is expressly incorporated herein by reference in its entirety). Peptides having high predicted probability of binding from a pathogen of interest can then be synthesized and tested for T cell reactivity in precursor, tetramer or ELISpot assays.
  • HLA protein previously 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 II molecules from mammalian cells requires time-consuming and cumbersome purification methods, and since each cell typically expresses multiple surface-bound HLA class I or class II molecules, HLA purification results in a mixture of many different HLA class I or class II molecules.
  • the present invention solves
  • the present invention is directed to 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 term "physical, functional and antigenic integrity of the native HLA molecule”, as used herein, will be understood to mean that the soluble HLA molecules exhibit the same structure (including primary, secondary, tertiary and quaternary) as the extracellular portion of the native HLA molecules, that they are identical in functional properties to an HLA molecule expressed from the HLA allele mRNA or gDNA and thereby bind peptide ligands in an identical manner as full-length, cell-surface-expressed HLA molecules, and that they are recognized by the cellular machinery responsible for responses to
  • HLA-peptide complexes that is NK and T cells.
  • the functionally active, individual soluble HLA molecule is a Class I HLA molecule or a Class II HLA molecule, and may have an endogenous peptide
  • the peptide may be produced by several methods, including but not limited to the following.
  • HLA allele mRNA from a source is isolated and reverse transcribed to obtain allelic cDNA.
  • gDNA encoding a HLA allele is obtained.
  • the allelic cDNA or gDNA is amplified by PCR utilizing at least one locus-specific primer that truncates the allelic cDNA or gDNA, 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.
  • the at least one locus-specific primer may include a stop codon incorporated into a 3' primer, or the at least one locus-specific primer may include a sequence encoding a tail such that the soluble HLA molecule encoded by the truncated PCR product contains a tail attached thereto that facilitates in purification of the soluble HLA molecules produced therefrom.
  • the truncated PCR product is then inserted into a mammalian expression vector to form a plasmid containing the truncated PCR product having the coding region encoding a soluble HLA molecule, and the plasmid is electroporated into at least one suitable host cell.
  • the mammalian expression vector contains a promoter that facilitates increased expression of the truncated PCR product.
  • the host cell may lack expression of Class I HLA molecules.
  • a cell pharrn is inoculated 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.
  • the individual, soluble HLA molecules are then harvested from the cell pharm and purified substantially away from other proteins.
  • the purification process involves affinity column purification and filtration.
  • the purified individual soluble HLA molecules maintain the physical, functional and antigenic integrity of the native HLA molecule.
  • the source is selected from the group consisting of mammalian DNA and an immortalized cell line.
  • the gDNA is obtained from blood, saliva, hair, semen, or sweat.
  • 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 ⁇ i and ⁇ 2 domains harboring the
  • the side view shows the full molecule with a detailed view of ⁇ X 3 and
  • FIG. 4 is a pictorial representation showing the peptide binding platform in more detail where two helices form the rim and seven ⁇ sheets
  • FIG. 5 is a graphical representation of an ELISA procedure demonstrating that W6/32-coupled affinity column can be saturated with crude harvest containing sHLA-B*0702His.
  • FIG. 6 is a graphical representation of an ELISA procedure
  • FIG. 7 is a graphical representation of an EUSA procedure demonstrating the elution of sHLA-B*0702His from the W6/32-co ⁇ pled affinity column of FIG. 5.
  • FIG. 8 is a chart showing the buffer exchange and concentration procedure using MACROSEPTM filters. ELISA performed during the filtration steps confirm minimal loss of protein.
  • FIG. 9 is a chart showing the final sterile filtration step optimized to remove remaining particles within the filtrate.
  • FIG. 10 is a tabular representation showing a summary of values measured during the purification procedure directly related to the efficiency.
  • FIG. 11 is a pictorial representation illustrating the protein Sequence Data for MH ⁇ Class I-HLA-A*0201T.
  • FIG. 12 is a pictorial representation showing the protein Sequence Data for MHC Class I-HLA-B*0702T.
  • FIG. 13 is a pictorial representation illustrating the Protein Sequence Data for MHC Class I-HLA-B*1512T.
  • FIG. 14 is a tabular representation illustrating the amino acid analysis of B*1512 following proteolysis of whole molecule.
  • FIG. 15 is a graphical representation showing SuperdexTM chromatography to demonstrate sample purity of sHLA-B*1512T.
  • FIG. 16 is a graphical representation illustrating a Triple analysis of B*15l2T. It shows a separation of sHLA under denaturing and under native conditions.
  • FIG. 17 is a graphical representation showing a SuperdexTM profile of A*0201T.
  • FIG. 18 is a. pictorial representation of an SDS-PAGE gel analysis of several purified sHLA samples confirming the purity with this procedure.
  • FIG. 19 is a pictorial representation of a Western blot analysis to follow the HC and ⁇ 2m subunits of sHLA.
  • FIG. 20 is a chart depicting an activity confirmation of sHLA using standard sandwich ELISA procedure.
  • FIG. 21 is a pictorial scheme of antibody binding scenarios for the direct ELISA procedure.
  • Several antibodies were tested on intact as well as denatured sHLA. Direct finding of sHLA molecules causes partial denaturization of the molecules and thus no specific denaturation step is necessary.
  • FIGS. 22-27 are charts showing reaction panels for conformation- specific Ab binding assays using the direct ELISA procedure.
  • FIG. 28 is a pictorial scheme of the two antibody binding scenarios using W6/32 or anti-b2m as capturing antibodies in a sandwich ELISA procure. Several detection antibodies were used.
  • FIGS. 29-32 are charts showing reaction panels for conformation- specific Ab binding assays using several Pan-Class I monoclonal antibodies in the sandwich ELISA procedure.
  • FIGS. 33-34 are charts illustrating various antibody combinations to test for artificial structural forms such as aggregation or dimeric structures showing A, B, and C alleles.
  • FIGS. 35-36 are charts illustrating neutralization experiments to verify antigenic integrity using sHLA-A*0201T and A2 alloantiserum 102 as well as Ab MA2.1.
  • FIG. 37 is a pictorial representation illustrating anti-calreticulin blot of full-length HLA-B27 (+), HLA negative cell line 721.221 (-) and various constructs of soluble HLA-B15 molecules immunoprecipitated with the HLA- specific antibody HC-10.
  • FIGS. 38-51 are charts showing ELISA reactions testing a panel of selected sHLA alleles using different commercially available single specificity monoclonal antibodies.
  • FIGS. 52-53 are charts illustrating ELISA Reaction panels testing antibodies Bw6 and Bw4.
  • FIG. 54 is a pictorial representation depicting a motif comparison between sHLA-B*1501 and membrane bound B*1501 from another laboratory.
  • FIG. 55 is a pictorial representation showing a fluorescence polarization scheme allowing the detection of bound and free peptides to the sHLA complex in solution without separation using radiometric measurements of parallel and perpendicular fluorescent intensities. Free peptides create a low FP signal where bound peptides show high FP values.
  • FIGS. 56-57 are graphical representations showing a one phase exponential association curve using the sHLA allele A*0201T combined with the FITC-labeled peptide P5 (A*0201).
  • FIGS. 58-59 are graphical representations showing saturation experiments generating saturation curve wherein sHLA (binder) is held constant to determine the dissociation constant (K & ).
  • FIGS. 60-61 are graphical representations showing competition experiments of fixed concentration of fluorescent-labeled synthetic peptide in the presence of various concentrations of ⁇ nlabeled test competitor-peptides to determine the IC50 value.
  • FIG. 62 is a graphical representation showing an ELISA procedure demonstrating the binding of a HBV peptide to sHLA molecules and successful replacement of the endogenous peptide with the HBV peptide.
  • FIGS. 63-66 are charts showing ELISA procedures demonstrating stability of sHLA-B*1512T in different buffers and solutions during different days with a summary given in FIG. 66.
  • FIG. 67 is a graphical representation showing an ELISA procedure demonstrating the influence of temperature on stability of sHLA complex.
  • FIG. 68 is a graphical representation showing the influence of freeze- thaw cycle on stability.
  • FIG. 69 is a pictorial representation showing the experimental procedure for determining loss of complex reactivity due to nonspecific adhesion to surfaces of tubes,
  • FIG. 70 is a chart showing the effects of different microcentrifuge tubes or cryo vials on reactivity of sHLA.
  • FIG. 71 is a chart showing the effects of larger tubes on reactivity of sHLA.
  • FIG. 72-73 are charts depicting the effects of blocking agents on reactivity of sHLA, including PVP and PEG.
  • FIG, 74 is a chart showing the effects of non-ionic detergents on reactivity of s HLA.
  • FIG. 75 is a chart showing the effect of different BSA concentrations on reactivity of sHLA.
  • FIG. 76 is a chart showing the effect of different StabilguardTM concentrations on reactivity of sHLA.
  • FIG. 77 is a chart showing the effect of PEG concentrations on reactivity of sHLA.
  • FIG. 78 is a chart showing the effect of PVP concentrations on reactivity of sHLA.
  • FIGS. 79-85 are charts illustrating a sera screen assay that utilizes HLA to identify antigen-specific antibodies in human sera.
  • FIG. 86 is a chart showing SHLA A*0201T reactivity on beads sampled through the EDC method.
  • FIG. 87 is a graphical representation depicting the screening of test competitors for ability to inhibit FITC-labeled standard peptide from binding to sHLA.
  • FIG. 88 is a graphical representation showing constructed IC 50 binding curves using a single inhibition value obtained at 100 ⁇ M competitor concentration.
  • FIG. 89 is a graphical representation showing IC 50 values obtained during the single value procedure as well as the more accurate 9 point procedure sorted according to their measured affinities.
  • FIGS. 90-91 are graphical representations illustrating the improvement of binding of modified peptides to sHLA-A2 as compared to the native test-peptides Vac 104 and Vac 105.
  • FIG. 92 is a graphical representation summarizing the purification and characterization procedures for soluble human HLA proteins of the present invention.
  • the present invention combines methodologies for the production of individual, soluble MHC molecules with novel and nonobvious methodologies
  • HLA molecules soluble or non-soluble
  • 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 10/022,066 application.
  • the methods of the present invention may, in one embodiment, utiHze a method of producing MHC molecules (from genomic DNA or cDNA) that are secreted from mammalian cells in a bioreactor unit.
  • MHC molecules from genomic DNA or cDNA
  • Substantial quantities of individual MHC molecules may be obtained in the manner by more particularly modifying class I or class II MHC molecules so that they are capable of being secreted, isolated, and purified.
  • Secretion of soluble MHC molecules overcomes the disadvantages and defects of the prior art in relation to the quantity and purity of MHC molecules produced. Problems of quantity are overcome because the cells producing the MHC do not need to be detergent lysed or killed in order to obtain the MHC molecule.
  • Production of the MHC molecules in a hollow fiber bioreactor unit allows cells to be cultured at a density substantially greater than conventional liquid phase tissue culture permits. Dense culturing of cells secreting MHC molecules further amplifies the ability to continuously harvest the transfected MHC molecules. Dense bioreactor cultures of MHC secreting cell lines allow for high concentrations of individual MHC proteins to be obtained. Highly concentrated individual MHC proteins provide an advantage in that most downstream protein purification strategies perform better as the concentration of the protein to be purified increases. Thus, the culturing of MHC secreting cells in bioreactors allows For a continuous production of individual MHC proteins in a concentrated form.
  • hollow fiber bioreactor units or cell pharms have been described herein for utilization in the culturing methods of the present invention, it is to be understood that any large scale mammalian tissue culture system evident to a person having ordinary skill in the art may be utilized in the methods of the present invention, and therefore the present invention is not specifically limited to the use of a hollow fiber bioreactor unit or a cell pharm.
  • the method of producing MHC molecules utilized in the present invention and described in detail in parent application U.S. Serial No. 10/022,066 begins by obtaining genomic or complementary DNA which encodes the desired MHC class I or class II molecule. Alleles at the locus which encode the desired MHC molecule are PCR amplified in a locus specific manner. These locus specific PCR products may include the entire coding region of the MHC molecule or a portion thereof. In one embodiment a nested or hemi-nested PCR is applied to produce a truncated form of the class I or class II gene so that it will be secreted rather than anchored to the cell surface. FIG. 1 illustrates the PCR products resulting from such nested PCR reactions. In another embodiment the PCR will directly truncate the MHC molecule.
  • Locus specific PCR products are cloned into a mammalian expression vector and screened with a variety of methods to identify a clone encoding the desired MHC molecule.
  • the cloned MHC molecules are DNA sequenced to ensure fidelity of the PCR, Faithful truncated clones of the desired MHC molecule are then transfected into a mammalian cell line.
  • Such cell line When such cell line is transfected with a vector encoding a recombinant class I molecule, such cell line may either lack endogenous class I MHC molecule expression or express endogenous class I MHC molecules.
  • the transfected class I MHC molecule can be "tagged" such that it can be specifically purified away from spontaneously released endogenous class I molecules in cells that express class I molecules.
  • a DNA fragment encoding a HIS tail may be attached to the protein by the PCR reaction or may be encoded by the vector into which the PCR fragment is cloned, and such HIS tail, therefore, further aids in the purification of the class I MHC molecules away from endogenous class I molecules.
  • Tags beside a histidine tail have also been demonstrated to work, and one of ordinary skill in the art of tagging proteins for downstream purification would appreciate and know how to tag a MHC molecule in such a manner so as to increase the ease by which the MHC molecule may be purified.
  • genomic DNA fragments contain both exons and introns as well as other non-translated regions at the 5' and 3' termini of the gene.
  • gDNA genomic DNA
  • mRNA messenger RNA
  • Transfection of MHC molecules encoded by gDNA therefore facilitates reisolation of the gDNA, mRNA/cDNA, and protein.
  • MHC molecules in non-mammalian cell lines such as insect and bacterial cells require cDNA clones, as these lower cell types do not have the ability to splice introns out of RNA transcribed from a gDNA clone.
  • the mammalian gDNA transfectants of the present invention provide a valuable source of RNA which can be reverse transcribed to form MHC cDNA.
  • the cDNA can then be cloned, transferred into cells, and then translated into protein.
  • such gDNA transfectants therefore provide a ready source of mRNA, and therefore cDNA clones, which can then be transfected into non-mammalian cells for production of MHC.
  • the present invention which starts with MHC genomic DNA clones allows for the production of MHC in cells from various species.
  • a key advantage of starting from gDNA is that viable cells containing the MHC molecule of interest are not needed. Since all individuals in the population have a different MHC repertoire, one would need to search more than 500,000 individuals to find someone with the same MHC complement as a desired individual - such a practical example of this principle is observed when trying to find a donor to match a recipient for bone marrow transplantation. Thus, if it is desired to produce a particular MHC molecule for use in an experiment or diagnostic, a person or cell expressing the MHC allele of interest would first need to be identified.
  • the MHC molecule of interest could be obtained via a gDNA clone as described herein, and following transfection of such clone into mammalian cells, the desired protein could be produced directly in mammalian cells or from cDNA in several species of cells using the methods described herein.
  • RNA is inherently unstable and is not as easily obtained as is gDNA. Therefore, if production of a particular MHC molecule starting from a cDNA clone is desired, a person or cell line that is expressing the allele of interest must traditionally first be identified in order to obtain RNA. Then a fresh sample of blood or cells must be obtained; experiments using the methodology of the present invention show that > .
  • cDNA may be substituted for genomic DNA as the starting material
  • production of cDNA for each of the desired HLA class I types will require hundreds of different, HLA typed, viable cell lines, each expressing a different HLA class I type.
  • fresh samples are required from individuals with the various desired MHC types.
  • genomic DNA as the starting material allows for the production of clones for many HLA molecules from a single genomic DNA sequence, as the amplification process can be manipulated to mimic recombinatorial and gene conversion events.
  • Several mutagenesis strategies exist whereby a given class I gDNA clone could be modified at either the level of gDNA or at the cDNA resulting from this gDNA clone.
  • the process of producing MHC molecules utilized in the present invention does not require viable cells, and therefore the degradation which plagues RNA is not a problem.
  • Soluble MHC Molecules The ability to produce large quantities of single specificity sHLA molecules allows for assay procedures to be quantitative and resistant to interferences encountered in biological matrices as well as also being reliable, highly reproducible, sensitive, and therefore applicable for high- throughput systems.
  • Alternative economical methodologies for obtaining large quantities of sHLA molecules do not currently exist since: (1) there is no readily available source of individual HLA molecules; (2) purification of native class I molecules from mammalian cells requires time-consuming and cumbersome purification methods and does not deliver sufficient quantities; and (3) native molecules from mammalian cells typically consist of a mixture of different HLA molecules. Such a mixture of specificities is not useful and/or applicable for single specificity studies.
  • HLA class I molecules are antigen-presenting glycoproteins expressed universally in nucleated cells.
  • heavy chains are encoded at 3 loci (B, C, and A) within the MHC on the short arm of chromosome 6 (FIG. IA).
  • RG. IB illustrates each a-chain comprised of Oi, ⁇ 2 , and ⁇ a domains, as well as a transmembrane domain, which tethers the molecule to the cell surface and a short C-terminal cytoplasmic domain.
  • the light chain is encoded outside of the MHC (on chromosome 15 in humans) and bears no such anchoring domain; it instead associates noncovalently with the 0 3 domain of the heavy chain.
  • IC illustrates the approach for creating sHLA class I transcripts.
  • the PCR primers truncate the class I heavy chain following exon 4, just before the transmembrane domain and cytoplasmic domains.
  • Using this PCR truncation strategy we have successfully created sHLA class I gene products for a series of fifty divergent HLA-molecules.
  • Class I sHLA gene constructs created as in FIG. IC are cloned and DNA sequenced to insure fidelity of each clone. The individual class I constructs are then subcloned into a suitable protein expression vector.
  • sHLA molecules have close to identical primary structures as papain solubilized HLAs. Truncated molecules have been shown by the present inventors to maintain their structural integrity.
  • HLA-Aw68 from which the complete alpha 3 domain has been proteolytically removed, shows no gross morphological changes compared to the intact protein. A decameric peptide complexed with the intact HLA-Aw68 is seen to bind to the proteolized molecule in the conventional manner, demonstrating that the alpha 3 domain is not required for the structural integrity of the molecule or for peptide binding.
  • Pictures of sHLA graphics FIG. 2) and 3D structures (FIG. 3) more clearly visualize how the molecules look like.
  • HLA/MHC genes are the most polymorphic system in mammals, generated by systematic recombinatorial and- point mutation events; as such, hundreds of different HLA types exist throughout the world's population, resulting in a large immunological diversity. Individuals inherit a set of three class I genes from each parent, and since their expression is codominant, a single person may therefore display up to six different HLA class I molecules upon his or her nucleated cells. Such extensive HLA diversity results in differing susceptibilities and/or resistances between individuals in infectious diseases. Depending upon allelic composition, two individuals' molecules may not necessarily bind the same peptides with equal affinity or even at all.
  • a binding platform is shown in FIG. 4.
  • the first two domains (alpha l, alpha 2) of the heavy chain create the peptide binding cleft and the surface that contacts the T-cell receptor.
  • X-ray crystallographic analysis indicates that a processed antigen is presented as a peptide bound in a cleft between the two o-helices of the heavy chain of the HLA complex (Bjorkman P.J., 1987; Nature 329: 506-512 & 512-518 / Garett TJ.
  • the third domain (alpha 3) associates with the T-cell co-receptor, CD8, during T-cell recognition.
  • Availability of a wide spectrum of recombinant sHLA molecules overcomes the current art limitations on population coverage imposed by the rules of MHC restriction. In most cases, a single-peptide epitope will be useful only for treating a small subset of patients who express the MHC allele product that is capable of binding that specific peptide. Since every individual has differing MHC molecules, the testing of numerous individual MHC molecules is a prerequisite for understanding the difference in disease susceptibility between individuals.
  • 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, or receptor protein
  • 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 figand 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
  • the protocol herein discussed provides a method to couple protein to a commercially available CNBr-activated Sepharose 4B (APB #17-0430-01).
  • An alternative option would be running the procedure with Sepharose 4 Fast flow (APB #17-0981-01).
  • Sepharose Fast Flow is more highly crosslinked than Sepharose 4B.
  • Fast Flow * beads are more stable and can withstand higher flow rates than the 4B beads.
  • CNBr-activated Sepharose 4B is better suited for batch chromatography and small columns with gravity flow.
  • Another difference is in coupling capacities.
  • the coupling reaction proceeds most efficiently in the pH range 8-10 where the amino groups on the ligand are predominantly in the unprotonated form.
  • a buffer at pH 8.3 is most frequently used for coupling proteins.
  • IgGs are often coupled at a slightly higher pH, for example in a NaHCO 3 buffer (0.2-0.25 M) containing
  • the coupling buffer solution should have a high salt content (about 0.5 M NaCI) to minimize protein- protein adsorption caused by the polyelectrolyte nature of proteins. Coupling at low pH is less efficient but may be advantageous if the ligand loses biological activity when it is fixed too firmly, e.g. by multi-point attachment, or because of steric hindrance between binding sites which occurs when a large amount of high molecular weight ligand is immobilized. A buffer of approximately pH 6 is used. Tris and other buffers containing amino groups must not be used at this stage since these buffers will couple to the gel.
  • Protein coupled to CNBr-activated SepharoseTM 4B is usually more stable to denaturation than the protein in free solution, but reasonable care in the choice of storage conditions should be exercised. Suspensions should be stored in a refrigerator below 4°C in the presence of a suitable bacteriostatic agent. The choice of buffer solution depends on the properties of he particular coupled protein.
  • high-affinity antibodies will be significantly more efficient at removing the antigen from solution than low-affinity antibodies.
  • Several small-scale columns can be used to determine the best conditions for binding and collecting the antigen. Although the exact affinity of an antibody for an antigen can be calculated, for most work the crucial criterion is whether the antibodies will remove the antigen from solution quantitatively. The easiest method to test this is to set up small-scale reactions and examine the first wash buffer for the presence of the antigen. The amount of bound antigen may be increased by using higher amounts of antibodies on the beads, by increasing the number of beads, or by increasing the amount of time for binding. Unfortunately, all of these conditions will raise the nonspecific background, so a compromise normally will result in the highest yields with the lowest acceptable background.
  • Use of high-affinity antibodies solves the problem of efficiently collecting the antigen. Consequently, they can be used in dilute solutions, at relatively lower antigen. Consequently, they can be used in dilute solutions, at relatively lower concentrations, and for shorter times.
  • a titration can be performed as a first step in estimating the ratio of column matrix needed to bind a given amount of antigen. This can be handled where an equal volume of the antibody/Sepharose 4B matrix is added to samples containing increasing concentrations of the antigen. The slurry is mixed at 4°C for 1 hr and then processed. This will yield a rough idea of the volume of column matrix needed to collect the desired amount of antigen. If the supematants from the binding reaction are assayed for the
  • the extent of antigen depletion also can be determined.
  • Developing the best elution conditions is an empirical task determined by testing a series of buffers. Three types of elution are possible.
  • the antigen-antibody interaction ' s can be broken by (1) treating with harsh conditions, (2) adding a saturating amount of a small compound that mimics the binding site, and/or (3) treating with an agent that induces an alloster ⁇ c change that releases the antigen.
  • the most commonly used elution procedure relies on breaking the bonds between the antibody and antigen.
  • the elutions may be harsh, denaturing the antibody and the antigen, or mild, leaving both the antigen and antibody in active states.
  • the mildest elution conditions are required if the protein of interest is labile. Avoid dithiothreitol and other reducing agents, as they will break disulfide linkages . Any buffers that fail to elute the antigen should be considered as good candidates for wash buffers. Some noneluting buffers may, in fact, drive the antibody-antigen equilibrium toward complex formation.
  • the usual procedure when elution conditions have not been defined is to try the mildest elution conditions first and proceed to harsher treatments. If trying for the gentlest elution conditions, start with acid conditions first, then check basic elution buffers. If these conditions do not elute the antigen, try others. A general order to check the various conditions would be:
  • Microconcentrators are used primarily for removal of excess salts in protein purification or analysis.
  • a variety of materials have been used to fabricate these semipermeable membranes, ranging from cellulose and cellulose esters to polyethersulfone (PES) or polyvinylidene difluoride (PVDF).
  • All membranes are characterized by their molecular-weight cutoff (MWCO) value. This is usually defined as the molecular weight of a solute that is 90% prevented from penetrating the membrane under a chosen set of conditions. How readily a particular protein is rejected by the membrane is a function of the shape, hydration state, and charge of the protein molecule.
  • MWCO values are not sharp; rather, there is a gradual increase in retention as the size of solute molecules approaches and exceeds the average membrane pore size. Only at the point where all pores are smaller than a particular solute molecule is that molecule completely excluded.
  • the advantage of desalting processes based on ultrafiltration over those based on simple dialysis is that the rate of low-molecular-weight solute removal is not determined by a concentration differential, but rather by the flow rate of solvent and the rejection of the solute by the ultrafiltration membrane employed.
  • Membranes for ultrafiltration are generally selected on the basis of the MWCO needed to retain the protein of interest but allow the maximum amount of other materials to pass through. It is usually best to choose an MWCO value that is roughly one-half the molecular weight of the species to be retained. This provides a reasonable margin of retention whereby almost none of the protein of interest should be lost, but at the same time provides the largest difference between the MWCO value and the molecular weight of the salts to be removed, thereby maximizing filtration rate.
  • the flow rate is a function of the filter area and the degree to which concentration polarization can be avoided.
  • Buildup of protein on the surface will result in slow filtration, even when the protein concentration of the sample is relatively low. Filtration rates at 4°C are often only one-half those seen at 25 °C because of the influence of viscosity.
  • monomorphic monoclonal antibodies are particularly useful for identification of HLA locus products and their subtypes.
  • W6/32 is one of the most common monoclonal antibodies (mAb) used to characterize human class I major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • This antibody recognizes only mature complexed class I molecules. It is directed against a conformational epitope on the intact MHC molecule that includes both residue 3 of beta2m and residue 121 of the heavy chain (Ladasky JJ, Shum BP, Canavez F, Seuanez HN, Parham P. Residue 3 of beta2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody. Immunogenetics 1999 Apr;49(4):312-20, the contents of which is expressly incorporated herein by reference in its entirety.).
  • the constant portion of the molecule W6/32 binds to is recognized by CTLs and thus can inhibit cytotoxicity.
  • the reactivity of W6/32 is sensitive to the amino terminus of human beta - microglobulin (Shields MJ, Ribaudo RK. Mapping of the monoclonal antibody W6/32: sensitivity to the amino terminus of beta2- microglobulin. Tissue Antigens 1998 May;51(5): 567-70, the contents of which is expressly incorporated herein by reference in its entirety.).
  • HLA-C could not be clearly identified in immunoprecipitations with W6/32 suggesting that HLA-C locus products may be associated only weakly with ⁇ 2m, explaining some of the difficulties encountered in biochemical studies of HLA-C antigens.
  • the polypeptides correlating with the C-locus products are recognized far better by HC-10 than by W6/32 which seems to confirm that at least some of the C products may be associated with ⁇ 2m more weakly than HLA-A
  • HC-10 is reactive with almost all HLA-B locus free heavy chains.
  • the A2 heavy chains are only very weakly recognized by HC-10.
  • HC- 10 reacts only with a few HLA-A locus heavy chains.
  • HC-10 seems to react well with free heavy chains of HLA-C types.
  • No evidence for reactivity of HC-10 with heavy-chain/ ⁇ 2m complex was obtained. None of the immunoprecipitates obtained with HC-10 contained ⁇ 2m. This suggests that HC-10 is directed against a site of the HLA class I heavy chain that might include the portion involved in interaction with the ⁇ 2m.
  • the pattern of HC-10 precipitated material is qualitatively different from that isolated with W6/32. '
  • TP25.99 detects a determinant in the alpha3 domain of HLA-ABC. It is found on denatured HLA-B (in Western) as well as partially or fully folded HLA-A, B,& C. It doesn't require a peptide or ⁇ 2m, i.e. it works with the alpha 3 domain which folds without peptide. This makes it useful for HC determination.
  • Anti-human ⁇ 2m (HRP) (DAKO P0174) recognizes denatured as well as complexed ⁇ 2m. Although in principle anti- ⁇ 2m reagents could be used for the purpose of identification of HLA molecules, they are less suitable when association of heavy chain and ⁇ 2m is weak. The patterns of class I molecules precipitated with W6/32 and anti- ⁇ 2m are usually indistinguishable.
  • the present invention is directed to a unique method for producing, isolating, and purifying class I molecules in substantial quantities.
  • the following graphs show that the test allele B*0702His produced in static culture can be purified to homogeneity and eluted as intact molecule.
  • RG- 5 demonstrates that a W6/32-coupled affinity column can be saturated with crude harvest containing sHLA. Individual values were determined through a standardized sandwich ELISA procedure using W6/32 as capturing antibody and anti- ⁇ 2m as detecting antibody. This ELISA procedure allows only the detection of intact sHLA molecules. After successful loading, the column is washed with PBS.
  • FIG. 6 shows the washing step.
  • FIG. 8 shows two rounds of buffer-exchange and confirms minimal loss of protein after the last step. All wash flow- through's (WFt's) have minimal sHLA content and are usually discarded after the procedure. The sHLA content was elaborated using the standard ELISA technique. To remove possible particles or bacterial growth, filtration through a 0.2 micron filter is standard procedure.
  • FIG. 10 shows the efficiency of the procedure measured at each step. A 100% was defined as the sHLA content directly bound to the column after loading and wash. All Flow-through's and washes having substantial amounts of sHLA are recovered and can be reused as loading material for a second round of purification. With this purification run, a total efficiency of 75% was achieved.
  • FIGS. 11-13 illustrate protein sequence data for MHC Class I HLA-A*0201T, HLA-B*0702T, and HLA-B*1512T, respectively.
  • the comparison clearly shows that the sHLA's are correctly translated at the amino terminal end. It is also evidence that no other major impurity was present in those samples.
  • Pan class I antibodies give conclusive results about the conformational status of the sHLA molecules.
  • sHLA activity tests using Pan-class I antibodies such as W6/32, TP25.99, and Pan class I (One Lambda) were performed.
  • W6/32 only recognizes conformationally intact molecules;
  • TP25.99 recognizes the complexed sHLA molecule as well as free HC and
  • the Pan class I One Lambda which has equal recognition patterns as seen with W6/32.
  • the antibody HC10 is useful in distinguishing free from bound heavy chain (HC) since this antibody only recognizes the HC of denatured sHLA molecules.
  • Anti- ⁇ 2m recognizes the ⁇ 2m subunit in both cases, complexed to the HC as well as free in solution and gives complementary information in addition to the other antibodies.
  • FIG. 21 Illustrated in FIG. 21 is a scheme of antibody binding scenarios, while FIGS. 22-27 each illustrate reaction panels for conformation-specific Ab binding assays using Sandwich EUSA assays.
  • the Sandwich EUSA assays include six steps: (1) choice of appropriate support; (2) coating with pan HLA specific antibodies; (3) blocking procedure to reduce non-specific protein binding; (4) capturing of single specificity sHLA molecules at different epitopes; (5) positive (or negative) SERA binding to presented sHLA alleles; and (6) detection of reactive SERA antibodies using secondary anti-human
  • IgG (IgM) antibody IgG (IgM) antibody.
  • Sandwich assays can be used to study a number of aspects of protein complexes. If antibodies are available to different components of a heteropolymer, a two-antibody assay can be designed to test for the presence of the complex. Using a variation of these assays, monoclonal antibodies can be used to test whether a given antigen is multimeric. If the same monoclonal antibody is used for both the solid phase and the label, monomeric antigens cannot be detected. Such combinations, however, may detect multimeric forms of the antigen.
  • the W6/32 - anti- ⁇ 2m antibody sandwich assay is one of the best techniques for determining the presence and quantity of sHLA.
  • Two antibody sandwich assays are quick and accurate, and if a source of pure antigen is available, the assay can be used to determine the absolute amounts of antigen in unknown samples.
  • the assay requires two antibodies that bind to non-overlapping epitopes on the antigen. This assay is particularly useful to study a number of aspects of protein complexes.
  • the wells of microtiter plates are coated with the specific (capture) antibody W6/32 followed by the incubation with test solutions containing antigen. Unbound antigen is washed out and a different antigen-specific antibody (anti- ⁇ 2m) conjugated to HRP is added, followed by another incubation. Unbound conjugate is washed out and substrate is added. After another incubation, the degree of substrate hydrolysis is measured. The amount of substrate hydrolyzed is proportional to the amount of antigen in the test solution.
  • the major advantages of this technique are that the antigen does not need to be purified prior to use and that the assays are very specific.
  • the sensitivity of the assay depends on four factors: (1) the number of capture antibodies; (2) the avidity of the capture antibody for the antigen; (3) the avidity of the second antibody for the antigen; and (4) the specific activity of the labeled second antibody.
  • 35-36 demonstrate that the sHLA molecule A0201T highly competes with the A2 alloantiserum M102 as well as with the monoclonal Ab MA2.1 confirming the correct behavior of the molecule in this neutralization experiment. This indicates the presence of a native conformationally correct molecule within the samples. Particularly, the MA2.1 (1:600) monoclonal Ab recognizing specific epitopes on A0201T was 93% blocked. Different buffer supplements do not appear to have any influence on the capability to block. The recognition by conformation- sensitive mAbs indicates that the recombinant complex contains native epitopes, consistent with the presence of a correctly folded molecular complex. 3. Chaperone interaction experiments
  • the class I molecule interacts with several chaperones as it traffics through the cell on its way to the cell surface.
  • These chaperones include, but are not limited to, calnexin, calreticulin, Tapasin, and Erp 94.
  • 35 S pulse chase/immunoprec ⁇ pitation experiments were performed to demonstrate that the sHLA class I proteins produced and purified by the method of the present invention interact with chaperones normally. Interaction with calreticulin, calnexin, and tapasin has been demonstrated, and interaction with calreticulin is shown in FIG. 37.
  • a panel of selected sHLA alleles was tested using commercially available single specificity monoclonal antibodies (FIGS. 38-51). All experiments performed resulted in the recognition of the allele corresponding to the chosen antibody.
  • the single specificity monoclonal antibodies act as detecting antibodies. Soluble HLA is presented to the detecting antibodies through W6/32 as well as anti- ⁇ 2m capturing to ELISA plates. In single cases, no purified sHLA was readily available to be tested. Thus, crude material marked with (C) was used. Because crude material does have excess amounts of free ⁇ 2m which neutralize binding to anti- ⁇ 2m, no signal was expected. In addition, Bw6 and Bw4 Abs were tested (FIGS. 52-53).
  • FIG. 54 shows a motif comparison between sHLA-B*1501 purified by the methods of the present invention and a membrane bound B*1501 motif from another laboratory. The motifs are nearly identical. The same result has been seen with six sHLA class I molecules analyzed.
  • sHLA proteins of the present invention appear to traffic and bind peptides as do membrane bound class I.
  • Fluorescence polarization allows the direct measurement of the ratio between free and bound labeled ligand in solution without any separation steps (FIG. 55). Ratiometric measurements are an advantage as these types of measurements can self-correct for variations caused by lamp intensity fluctuations or interferences caused by quenching of the fluorescence. In the move towards a wider adoption of fluorescence technologies, there is the added benefit of abandoning radioactive tracers, which are increasingly becoming liabilities because of their cost and safety profile. Most important, FP allows real time measurements of single reactions to determine binding kinetics as well as equilibriums. Furthermore, since no biological system can show polarization below 0 mP or greater than 500 mP, FP automatically checks assay validity. Considered a negative point in using FP is that detected values often result in the loss of about 10-90% of fluorescence intensity. This in itself may reduce the sensitivity of fluorescence polarization assay as opposed to assays with direct intensity measurements.
  • FP The technique of FP is based on the fact that if excited with plane- polarized light, the light emitted by a fluorophore Is polarized as well. The angle between the planes of exciting and emitted light is highly dependent on the molecular motion of the fluorophore. FP values are defined by the equation:
  • I ⁇ is the intensity of the fluorescence measured in the parallel (
  • binding assay was developed to demonstrate that the labeled probe will bind to the molecule of interest. The following criteria, however, must be met in order to validate the binding assay: (1) binding should be saturable, indicating a finite number of binding sites; (2) the binding should have the requisite specificity, where the binding affinity, defined as the dissociation constant (Kd), should be consistent with values determined for physiological molecules; and (3) ligand binding should be reversible, reflecting the dynamic nature of the chemical transmission process and reaching equilibrium when the ligand association rate is equal to the dissociation rate.
  • Kd dissociation constant
  • a saturation curve is generated by holding the sHLA (binder) constant. Varying the tracer concentration (dose range: 0.1 nM - 1 mM) in case of constant binder (concentration of sHLA determined above) was tested in order to determine the affinity constant (K d ) of the labeled peptide and to obtain a smooth saturation curve. The lower the K d value, the higher the affinity of the peptide for the sHLA molecule. Only values that have reached equilibrium (Ymax) can be used for saturation experiments.
  • the IC 50 is determined by three factors: (1) the affinity of sHLA for the competitor peptide - if the affinity Is high, the IC 50 will be low; (2) the concentration of fluorescent-labeled tracer peptide -choosing a higher concentration of tracer will take a larger concentration of unlabeled peptide to compete for half the binding sites; and (3) the affinity of tracer peptide for sHLA (Kd). It takes more unlabeled competitor peptide to compete for a tightly bound tracer peptide (low K d ) than for a loosely bound tracer peptide (high K d ).
  • the parameters identified will be optimized to the point where the lowest concentration of a competitor test peptide results in a clearly distinguishable, positive response. No competition should be detected in the case of using an irrelevant unlabeled competitor peptide.
  • an HBV peptide known to bind strongly to A*0201T was used to replace the endogenous peptide in solution.
  • the sHLA complexes were immobilized on a solid support through the HLA specific antibody W6/32.
  • the HBV peptide/A*0201T complex was then detected using a highly specific antibody only recognizing this particular conformation. Saturation of the W6/32 coated ELISA plate could be achieved, demonstrating the binding of the HBV peptide to' sHLA molecules and a successful replacement of the endogenous peptides with the HBV peptide. No saturation was detected using the irrelevant peptide p53, indicating that peptide p53 as well as endogenous peptides do not contribute to the specific signal obtained by the HBV peptide/A*0201T complex selective antibody.
  • Each protein may have specific requirements once it is extracted from its normal biological milieu. If these requirements are not satisfied, the protein can rapidly lose its ability to carry out specific functions, and an already limited lifetime may be drastically reduced. Thus, failure to determine and manage these requirements has often been a major hurdle in obtaining successful protein characterization. In some cases, the difficulty has been to stabilize the protein against external proteolysis, while in other cases the problem has been to maintain ligand-binding or enzymatic activity. Solutions to these problems are highly specific.
  • a buffer is defined as a mixture of an acid and its conjugate base which can reduce changes in solution pH when acid or alkali are added.
  • the selection of an appropriate buffer is important in order to maintain a protein at the desired pH and to ensure reproducible results. Buffers are often present at the highest concentration of all components in a protein solution and may have significant effects on a protein or enzyme.
  • Triton X-100 at four days appears to be the highest value achieved during the whole assay. However, it also shows a high standard deviation value. It appears to be more likely to be an outsider result due to a dilution mistake rather than increased stability of sHLA after 4 days. This value was not considered in calculating the average.
  • Kinetic stability is usually measured at elevated temperatures, but the inactivating event(s) at high temperatures may not mirror those at the much lower temperatures used for storage. It is not feasible, however, to monitor stability in real time at the actual storage temperature. Fortunately, there is a methodology that can in many cases overcome these difficulties, namely accelerated degradation testing. This involves the periodic assay of samples incubated at different temperatures and use of the Arrhenius equation to predict shelf lives at temperatures of interest.
  • Ink -Ea/RT
  • k is the first-order rate constant of activity decay
  • Ea is the activation energy
  • R is the gas constant
  • T is the temperature in Kelvin.
  • This log form of the Arrhenius equation yields a straight-line plot of Ink against 1 T with slope -Ea/R. Extrapolation of this plot can give the rate constant (and hence the useful life) at a particular temperature. Accelerated storage testing has been used as a practical means of quality assurance for biological standards (Jerne, N. K. and Perry, W. L. M. (1956) The stability of biological standards.. Bull. Wld. Hlth. Org. 14, 167-182, the contents of which are hereby expressly incorporated herein in their entirety.).
  • FIG. 69 demonstrates the experimental procedure. From a protein stock, a dilution of 300 ng/ml, was mixed in PBS. To equilibrate the diluted sample, it was mixed 16 hours before starting the experiment and stored at 4°C . After this time, liquid was removed from one tube to another every 30
  • Tubes with larger volume capacity performed no better than the Fisher microcentrifuge tube (FIG. 71).
  • an exception was borosilicated glass tubes, which did not bind protein and only caused a loss of reactivity of 20%.
  • the tubes need either to be coated with a blocking agent or the blocker should be added directly to any molecule dilution.
  • Dilute protein solutions are highly prone to inactivation and lose activity quickly, possibly via denaturation at surfaces such as glass and plasticware.
  • High protein concentrations provide some auto-buffering capacity. Where the usage of high concentrations is not possible, inactivation may be prevented by addition of an exogenous compound.
  • Blocking agents used to coat Fisher (05-402-25) microcentrifuge tubes were tested for their ability to prevent inactivation and/or adhesion to the surface (FIGS. 72-73). The tubes were incubated with the blocker overnight at 4°C, extensively washed with PBS and finally air dried to remove any traces of liquid. 10% BSA, 3% gelatine or 5% Blotto (milk) worked best and did not result in any loss of protein or activity compared to the tube preincubated with PBS. Usage of StabilGuard Biomolecule Stabilizer (Sur odics, Eden Prairie, MN; SG01-0125) coated to the tube walls highly protected the protein against tube surfaces. However, the EUSA resulted in higher concentrations than actually put into the tube.
  • Stabilguard is a possible candidate to be used in reactions of HLA with allosera. (The optimal % of Stabilguard needs to be established first).
  • agents such as PVP (RG. 72) or PEG (FIG. 73) also showed good results .
  • crowding agents they push proteins out of solutions in the mechanical/physical sense and in the thermodynamic sense. The crowding action, aided by any degree of affinity of protein molecules for one another promote protein-to-protein association. Conformationally loose protein molecules are "squeezed" on by these agents, promoting protein molecule tightening and sometimes promoting an ordered protein conformation. Thus, these are the most potential candidates to be used in solution.
  • 2% BSA and 10% FBS also worked, however with lesser intensity.
  • nonionic detergents did not greatly help preventing the loss of sHLA compared to 10% BSA (FIG. 74).
  • these agents should not be excluded to be considered as supportive compounds since many proteins retain their activity in 1 - 3%.
  • a 10 times lower concentration was used, and the trend of better performance can be seen (FIG. 74).
  • 0.1 % Tween 20 performed better than 0.05%.
  • Stabilguard seems to work better with lower percentages (FIG. 76). A steady decrease in signal is observed using higher concentrated samples indicating an interference in protein-protein interaction rather then inefficiency in blocking. PEG can be used at concentrations up to 15% (FIG. 77). After that, PEG seems to highly interfere with the recognition of sHLA. PVP seems to be a great blocker at 5% (FIG. 78). However, it is absolutely not usable at higher concentrations, as it completely abolishes any interaction with sHLA.
  • Coupling of sHLA molecules to Lum ⁇ exTM beads to detect HLA antibodies in human sera can also be used with the individual, isolated, and purified sHLA molecules of the present invention.
  • Disclosed herein is the information used to bind various sHLA alleles produced to a solid support in order to obtain specific recognition of the alleles by human sera. Binding to a solid bead support was accomplished via the EDC method, coupled sHLA to l-ethyl-3-(3-dimethylam ⁇ noproplyl) carbodiimide-HCI (EDC) activated beads (FIG. 86). The results shown indicate that the isolated and purified sHLA of the present invention is indeed of high value in such assays.
  • Test Competitors were pre-screened for their ability to inhibit a FITC- labeled standard peptide from binding to the sHLA molecule at a competitor concentration of 100 ⁇ M (RG. 87). After obtaining equilibrium values for each test-peptide, IC50 values are calculated. A single measurement obtained at 100 ⁇ M competitor concentration can be used to construct such an IC50 value without support of additional data (RG. 88). This constructed graph allows us to sort all competitors and easily categorize them into high, medium, low and no binders (FIG. 89). Additionally, full scale IC50 determinations are performed on all candidates identified showing binding capacity to the allele tested. Usually, both methods are coming very close as seen in RG. 89 in which one point IC50 determinations (bottom) are shown together with 8 point IC50 determinations (boxed, top).
  • Appropriate modification of the sequence of a peptide epitope can increase the affinity for the MHC molecule(s) without interfering with recognition by the TCR of T cells specific for the natural ligand sequence. Therefore, by this process of epitope enhancement or optimization, one should be able to create a more potent vaccine.
  • the first step towards a successful epitope alteration approach is to increase the binding affinity and HLA-A2 stabilization capacity of HLA-A2-bound peptides. Since many immunodominant epitopes are high affinity MHC binders (Sette, 1994), one strategy is to increase the binding affinity of 'intermediate to low' binding peptides and therefore increase their potential as immunogens.
  • the second step is that these substitutions preserve the antigenic specificity and do not interfere with the peptide/TCR interaction. It is particularly noteworthy that the CTL responses raised against the modified peptide do cross-react with the naturally occurring epitope. This will depend upon the nature and position of the modification. Cross-recognition of native peptides and their modified variants by specific CTL is the most important issue in the design of optimized vaccines.
  • FIGS. 90 and 91 show improvement of modified peptides compared to the native test-peptide.
  • RG. 90 shows the IC50 of a native peptide VaclOS (ITNSRPPAV) to A*0201T whose binding capacity was improved by changing position 2T to 2L or 2M. The addition of an amino acid residue at the end did not result in a several fold improvement of binding (Vacl04/105).
  • RG. 91 shows a much higher binding of the decamer Vacl04/105 (KITNSRPPAV) than the two ninemers Vacl04 (KITNSRPPA) or Vacl05 (ITNSRPPAV). In summary, shown in RG.
  • the first step involves purification of soluble HLA, beginning with cell pharm run-large scale production of sHLA followed by production analysis.
  • the sHLA is then purified by affinity column purification (which includes the steps of loading, washing and elution) and buffer exchange and concentration of purified allele using Macrocep concentration filters.
  • the pure protein is then sterile filtered, aliquoted and stored, and the concentration of the stored pure protein is estimated.
  • quality control demonstrating the extent of chemical purification is performed using techniques known to those of ordinary skill in the art, including but not limited to, SDS-PAGE, Western blot analysis, SuperdexTM chromatography to demonstrate sample purity, and the like.
  • the second step in the method of the present invention involves characterization of the purified sHLA-peptide complex. Physical purity of the
  • ⁇ complex can be demonstrated by one or more of the following: sequence analysis to demonstrate the presence of all components of the complex; protein visualization procedures to demonstrate not only presence of all components but also formation of complex (including, but not limited to, SDS-PAGE, Western, SuperdexTM chromatography, and the like); and Mass Spectrometry data for use in peptide motif comparisons.
  • Functional purity of the complex can be demonstrated by one or more of the following: demonstration of antigenic integrity of sHLA using EUSA assays and neutralization experiments; demonstration of structural integrity using Chaperone interaction experiments; and demonstration of specificity, peptide binding capacity, and structural integrity using fluorescence polarization based association and saturation experiments.
  • the sHLA produced by the method of the present invention is feasible for use in the following various applications: sera screen assay that utilizes HLA to identify antigen-specific antibodies in human sera; Luminex bead approach to identify antigen-specific antibodies in human sera; competition assays, such as screening of test competitors for the ability to inhibit FITC- labeled standard peptide from binding to sHLA; and procedures to improve binding of modified peptides to sHLA as compared to native test-peptides.
  • the final step in the method of the present invention involves determining the optimum storage and handling conditions for soluble HLA.
  • the following factors in storage and handling have been described herein previously: stability testing in different buffers; thermodynamic stability of sHLA complexes; the influence of freeze-thaw cycles on stability; determination of loss of complex reactivity due to nonspecific adhesion to surfaces of storage vessels; and identification of appropriate blocking agents to maintain reactivity of sHLA.
  • RG. 92 has provided a general outline that indicates how each of the individual experiments described herein previously are interrelated to each other in the methods of purification, characterization, storage and handling of the present invention.
  • a very high ligand content can have three adverse effects on affinity chromatography. Firstly the binding efficiency of the adsorbent may be reduced due to steric hindrance between the active sites; this is particularly important when large molecules such as antibodies, antigens and enzymes are immobilized. Secondly, substances are more strongly bound to the immobilized ligand which may result in difficult elution. Thirdly, the extent of non-specific binding increases at very high ligand concentrations which can reduce the selectivity of the adsorbent.
  • start-value ts should be as accurate as possible to allow an estimation of the coupling efficiency (ligand binding efficiency).
  • ligand binding efficiency With the knowledge of total amount of antibody bound, a maximal antigen loading capacity can be calculated. However, this is only possible when the molecular weight of all interactive compounds is known.
  • the reading is performed at A280. Because stray light can affect the linearity of absorbance versus concentration, absorbance values >2.0 should not be used for any sample of proteins measured by the A280 method.
  • ⁇ material should be stored below 4°C. Under these conditions the shelf life is approximately 18 months, although further storage is not usually accompanied by rapid loss of activity.
  • the opened package should be stored dry below 4°C).
  • Coupling a ligand to the activated matrix involves first swelling and washing the gel in 1 mN HCl.
  • the protein binding activity of the gel is preserved better by washing at low pH than by washing at pH's above 7.
  • the use of HCl preserves the activity of the reactive groups which hydrolyze at high pH.
  • a sintered glass filter is a glass funnel with a built-in glass frit.
  • the glass frit is used instead of a membrane filter.
  • the filter unit is placed on top of a side- arm vacuum flask and filtration occurs using suction/vacuum.
  • the glass frit is available in different porosities. Medium porosity (porosity G3) is recommended for Sepharose.
  • freeze-dried powder is suspended in 1 mN HCl.
  • the gel swells immediately and should be washed during a time period of 15 minutes on the sintered glass filter with the same solution. Let the mixture equilibrate a few minutes during each washing step. Approximately 210 ml solution is added in several aliquots for each gram of dry gel. Suck off the supernatant between successive additions.
  • Coupling occurs very fast under our chosen conditions, and is usually complete after 20-30 minutes at room temperature (20-25 °C). If cold temperatures are necessary, coupling can also be performed overnight at 4°C.
  • the amount of protein which couples under a given set of conditions depends mainly on the ratio of protein to gel volume, the pH of the reaction and the protein itself as well as the duration and temperature of the reaction. A number of conditions can lead to poor coupling: low ligand concentration, suboptimal pH, impure ligand, improperly prepared matrix, inaccessibility of ligand or improperly prepared buffers.
  • the coupling reaction may be conveniently followed by observing the decrease in the absorbance of the supernatant solution at 280 nm.
  • si mechanism is very fast, the starting values are more important than the later ones.
  • the next step is to wash away the excess ligand with coupling buffer. Most efficient way to wash the gel is to use the sintered glass filter.
  • Block remaining active groups by transfering the gel to a vessel with 15 gel volumes of 0.1 M Tris-HCl, pH 8.0. Shake in an Erienmayer flask at 180 rpm at room temperature for 2 hours. (Alternatively, active groups can also be blocked using 0.2 mM glycine, pH 8.0 or 1 M ethanolamine, pH 8.0).
  • the final product is then washed alternately with 10 gel volumes of low pH wash buffer (0.1 M sodium acetate containing 0.5 M NaCI, pH 4.0) and high pH wash buffer (0.1 M Tris-HCl containing 0.5 M NaCI, pH 8.0) for 4 times. Thorough washing of the coupled product is necessary to remove traces of non-covalently adsorbed materials. The washing-cycle of low and high pH is essential for the best results. This procedure ensures that no free ligand remains ionically bound to the immobilized ligand. Let the mixture equilibrate a few minutes d ⁇ rihg each washing step.
  • the total amount of antibody bound per ml of gel is directly proportional to the antigen loading capacity which will give an estimate of how much protein maximally can bind per ml gel. To take into consideration is the Mw of the IgG molecule of 150 kDa as well as its capability to bind 2 antigens.
  • class I complex (57 kDa): heavy chain; 45 kDa, ⁇ 2-microglobulin; 12 kDa, peptide).
  • XK columns are jacketed and available in different dimensions with diameters of 26 mm (XK26) and 50 mm (XK50). These columns are only used with adaptors.
  • the column can be used in aqueous and nearly all organic solvents (exceptions: acetone, chloroform, phenol). Solutions containing more than 10% NaOH, 10% HCl or 5% acetic acids should not be used. Kontes Flex- columns are a more simpler version of columns but as effective.
  • AKTATM prime is a compact, automated liquid chromatography system. It is designed for standard separation applications. Flow rates up to 50 ml/min and pressures up to 1000 kPa can be applied. The system includes components for measuring UV, conductivity, generating gradients and collecting fractions. The AKTATM prime system may be utilized in the large scale purification procedure of the present invention in accordance with manufacturer's recommendations. Large scale purification procedure
  • the protein concentration in the sample solution should be determined using a quantitative EUSA procedure.
  • the sample volume loaded will depend on the size and loading capacity of the column and the concentration of the sample. Calculate the volume of the sample solution maximally saturating the column according to the columns capacity to bind the antigen,
  • the simplest method to bind the antigen to the antibody/Sepharose 4B matrix is to apply the sample through the system pump and pass the protein solution down the column.
  • Human MHC class I (sHLA) molecules are best eluted from a W6/32 column by 0.1 M glycine, pH 11.0. Absorbance is used for generating a protein elution profile.
  • MACROSEPTM centrifugal concentrators (Pall Filtron; Northborough, MA; MACROSEP 10K; OD010C37). Keep the protein on ice at all times and centrifuge at 4°C.
  • the purity of the eluted sHLA can be assessed by SDS-PAGE, Western blotting or performing a Superdex column analysis.
  • MACROSEPT centrifugal concentrators (Pall Filtron; Northborough, MA; MACROSEP 10K; OD010C37) provide rapid and convenient concentration, purification, and desalting of 5 ml to 15 ml biological samples. A starting sample of 15 ml can be concentrated to 0.5 ml in 30 to 60 minutes without multiple decanting steps. The MACROSEP's ease of use saves valuable lab time.
  • Each centrifugal concentrator is constructed of polypropylene and
  • OMEGA low-protein-binding OMEGATM membrane, two factors which significantly reduce non-specific adsorption and enable the device to yield the highest recoveries.
  • OMEGA membranes are made from polyethersulfone (PES) specifically modified to minimize protein binding. These membranes provide equivalent or higher recoveries than comparable regenerated cellulose membranes. MACROSEP centrifugal devices are ideal for
  • Centrifugation up to 5,000 x g provides the driving force for filtration, moving sample towards the encapsulated OMEGA membrane. Biomolecules larger than the nominal molecular weight cutoff of the membrane are retained in the sample reservoir. Solvent and low molecular weight molecules pass through the membrane into the filtrate receiver.
  • the MACROSEP centrifugal concentrator is available with 9 different molecular weight cutoffs (MWCO): IK, 3K, 10K, 30K, 50K, 100K, 300K, 1000K, and 0.3 ⁇ m. For maximum retention, select a MACROSEP device with a molecular weight cutoff that is 3 to 5 times smaller than the weight of the molecule to be retained.
  • a 10K MACROSEPTM centrifugal concentrator is utilized in accordance with manufacturer's recommendations.
  • OMEGA T M membranes in the MACROSEP devices contain trace amounts of glycerine and sodium azide. If these chemicals interfere with an assay, they may be removed. Filter 15 ml of deionized water or buffer through the membrane.
  • the experiment is designed using an ELISA protocol template, and a clear 96-well polystyrene assay plate is labeled.
  • Polystyrene is normally used as a microtiter plate. (Because it is not translucent, enzyme assays that will be quantitated by a plate reader should be performed in polystyrene and not PVC plates).
  • Coating of the W6/32 should be performed in Tris buffered saline (TBS); pH 8.5. Prepare a coating solution of 8.0 ⁇ g/ml of specific W6/32 antibody in TBS (pH 8.5). (Use the blue tube preparation stored at -20°C with a concentration of 0.2 mg/ml and a volume of 1 ml giving 0.2 mg per tube).
  • Blocked plates may be stored for at least 5 days at 4°C.
  • the pipette working position is always vertical: Non-vertical positions may cause too much liquid to be drawn in.
  • the immersion depth should be only a few millimeters.
  • pre-wetting should be used at all times. To do this, the required set volume is first drawn in one or two times using the same tip and then returned. Pre-wetting is absolutely necessary on the more difficult liquids such as 3% BSA.
  • the tip In order to reduce the effects of surface tension, the tip should be in contact with the side of the container when the liquid is dispensed.
  • a stock solution of 1 ⁇ g/ml should be prepared, aliquoted in volumes of 300 ⁇ l and stored at 4 ⁇ C. Prepare a 50 ml batch of standard at the time. (New batches need to be compared to the old batch before used in quantitation).
  • test solutions containing sHLA should be assayed over a number of at least 4 dilutions to assure to be within the range of the standard curve.
  • OPD o-Phenylenediamine
  • the substrate produces a soluble end product that is yellow in color.
  • the OPD reaction is stopped with 3 N H2SO4, producing an orange-brown product and read at 492 nm.
  • the background should be around 0.1. If the background is higher, the substrate may have been contaminated with a peroxidase. If the subtrate background is low and the background in you're the assay is high, this may be due to insufficient blocking.
  • proteins were obtained by denaturating with a solution containing 4% SDS, 20% glycerol, 0.02% bromophenol blue, and 200 mM dithiothreitol in 0.5 M Tris-HCl (pH 6.8).
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis
  • proteins were obtained by denaturating with a solution containing 4% SDS, 20% glycerol, 0.02% bromophenol blue, and 200 mM dithiothreitol in 0.5 M Tris-HCl (pH 6.8).
  • Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) was performed by using the procedures described previously by [Laemmli, 1970]. Briefly, the proteins were separated on a 12.5% gel, electroblotted onto an Immobilon-P mem-branes (Millipore, Bedford, MA), and blocked
  • the invention illustratively disclosed or claimed herein suitably may be practiced in the absence of any element which is not specifically disclosed or claimed herein.
  • the invention may comprise, consist of, or consist essentially of the elements disclosed or claimed herein.

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Abstract

De manière générale, cette invention concerne la production et l'utilisation de molécules HLA solubles et fonctionnellement actives qui sont isolées et purifiées sensiblement à l'écart d'autres protéines. L'invention concerne également des procédés de purification de ces molécules.
PCT/US2003/000243 2001-12-18 2003-01-02 Purification et caracterisation de proteines hla WO2003057852A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
IL16284503A IL162845A0 (en) 2002-01-02 2003-01-02 Purification and characterization of hla proteins
AU2003202892A AU2003202892A1 (en) 2002-01-02 2003-01-02 Purification and characterization of hla proteins
CA002514872A CA2514872A1 (fr) 2002-01-02 2003-01-02 Purification et caracterisation de proteines hla
AU2003270876A AU2003270876A1 (en) 2002-09-24 2003-09-24 Anti-hla assay and methods
CA002539622A CA2539622A1 (fr) 2002-09-24 2003-09-24 Dosage biologique anti-hla et procedes
PCT/US2003/030096 WO2004029280A2 (fr) 2002-09-24 2003-09-24 Dosage biologique anti-hla et procedes

Applications Claiming Priority (3)

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US10/022,066 US20030166057A1 (en) 1999-12-17 2001-12-18 Method and apparatus for the production of soluble MHC antigens and uses thereof
US34790602P 2002-01-02 2002-01-02
US60/347,906 2002-01-02

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012056233A1 (fr) * 2010-10-27 2012-05-03 The Binding Site Group Limited Billes revêtues

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6255073B1 (en) * 1995-03-08 2001-07-03 The Scripps Research Institute Antigen presenting system and methods for activation of T-cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6255073B1 (en) * 1995-03-08 2001-07-03 The Scripps Research Institute Antigen presenting system and methods for activation of T-cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PRILLIMAN ET AL.: 'HLA-B15 peptide ligands are preferentially anchored at their C termini' THE JOURNAL OF IMMUNOL. vol. 162, 1999, pages 7277 - 7284, XP002966691 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012056233A1 (fr) * 2010-10-27 2012-05-03 The Binding Site Group Limited Billes revêtues
CN103168237A (zh) * 2010-10-27 2013-06-19 结合点集团有限公司 包被的珠
US9389224B2 (en) 2010-10-27 2016-07-12 The Binding Site Group Limited Coated beads

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