EP1353950A2 - Procede et dispositif de production d'antigenes et utilisation de ceux-ci - Google Patents

Procede et dispositif de production d'antigenes et utilisation de ceux-ci

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Publication number
EP1353950A2
EP1353950A2 EP01994366A EP01994366A EP1353950A2 EP 1353950 A2 EP1353950 A2 EP 1353950A2 EP 01994366 A EP01994366 A EP 01994366A EP 01994366 A EP01994366 A EP 01994366A EP 1353950 A2 EP1353950 A2 EP 1353950A2
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EP
European Patent Office
Prior art keywords
hla
truncated
pcr product
class
molecules
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP01994366A
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German (de)
English (en)
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William H. Hildebrand
Kiley Rae Prilliman
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University of Oklahoma
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Individual
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Priority claimed from US09/974,366 external-priority patent/US7541429B2/en
Application filed by Individual filed Critical Individual
Publication of EP1353950A2 publication Critical patent/EP1353950A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding

Definitions

  • apparatus for the production of soluble MHC antigens and more particularly, but not by way of limitation, to at least one method and apparatus for the
  • the soluble Class I and II HLA molecules can be produced from either gDNA or cDNA starting material.
  • One such exemplary, but non-limiting, use is the formation of a tetrameric sHLA (or other multimeric complex) complex which may be used to test immunogenicity of peptide ligands of interest - i.e. will a peptide ligand of interest provoke a CTL response and/or preferentially bind a CTL.
  • Class I HLA molecules are polymorphic human glycoproteins that endogenously bind and then extracellularly present peptide ligands to CD8 + T lymphocytes. Polymorphisms within the class I peptide binding groove are positioned to moderate ligand binding and presentation to such immune system cells. To date, the small quantities of natural ligands available to those of skill
  • peptide ligands were extracted from five different HLA-B15 allotypes and subsequently examined. Mapping and characterizing the ligands obtained from these allotypes demonstrated that they: (i) vary in length from 7 to 12 residues; (ii) are more conserved at their C termini than at their N- proximal residues; and (iii) are presented as overlaps contingent on C-terminal preferences. These results provide insight into class I and class II ligand loading not available via other methods, demonstrating that an elemental role is played by a peptide ligand's C terminus during endogenous binding and provides the starting material for a multimeric complex to be used to test the functionality of a peptide ligand of interest.
  • the data obtained, and disclosed herein validates, and illustrates the unique methods disclosed herein for the production of sHLA from either gDNA or DNA starting material and the uses to which this sHLA material may be put.
  • Class I and class II MHC molecules bind and display peptide antigens upon the cell surface.
  • the peptides they present are derived from either normal endogenous proteins ("self") or foreign proteins ("nonself”).
  • Nonself proteins include items such as the products of malignant transformation or intracellular pathogens.
  • class I and class II molecules convey information regarding the internal fitness of a cell to CD8 + CTLs which are activated upon interaction with "nonself" peptides. Such activation may lead the CD8+ CTLs to kill and/or suppress a cell which is malignant or contains intracellular pathogens.
  • HLA-A, B, and C molecules are encoded by the most polymorphic genes in mammals.
  • Translating class I polymorphism into the tertiary structure of the class I molecule indicates that residues positioned to affect class I peptide presentation to T lymphocytes are most frequently affected by the mutagenic events which diversify class I loci.
  • HLA class I molecules exhibit a high degree of polymorphism that is generated by systematic recombinatorial events and collectively allows for the presentation of a vast array of different peptides. Depending upon allelic composition, two individuals' molecules may not necessarily bind the same peptides with equal affinity or even at all.
  • MHC class I molecules While the general structure and function of MHC class I molecules has been reasonably well studied and established, their polymorphic nature and how they specifically influence the capacity of class I in peptide binding and presentation remains an issue of persistent inquiry by those of ordinary skill in the art.
  • the nature of precise overlaps in peptide binding specificity to HLA class I is particularly ill-defined at the current time due to the complexity of peptides bound. For example, this and other issues must be clarified in order to effectively pursue vaccines capable of eliciting protective CTL responses across an extensive population range. Unraveling the functional significance of class I polymorphism is an important issue that requires an understanding of how the mutagenic events diversifying the class I binding groove differentially moderate the presentation of peptide ligands.
  • the heavy chains of class I molecules are encoded within the MHC and, upon assembling into heterodimers with the light chain, ⁇ 2 m, are responsible for selectively gathering endogenously processed peptides. Once peptides are collected, mature class I molecules transport the bound peptides to the cell surface where receptors on CD8 + T lymphocytes engage the class I molecules to inspect the ligands. CTLs may then be triggered by class I molecules bearing virus or tumor-derived peptides.
  • the class I molecules expressed upon the nucleated cells of all vertebrate systems studied to date are heterodimers composed of a glycosylated 45 kDa heavy chain ( -chain) and a 12 kDa light chain ( ⁇ 2 m).
  • heavy chains are encoded at 3 loci (B, C, and A) within the MHC on the short arm of chromosome 6 (FIG. 1A).
  • IB illustrates each ⁇ -chain comprised of cx lf ⁇ 2 , and ⁇ 3 domains, as well as a transmembrane domain, which tethers the molecule to the cell surface and a short C-terminal cytoplasmic domain (Bj ⁇ rkman and class I location and heavy chain coding region).
  • X-ray crystallography (Bj ⁇ rkman etal. 1987a; Madden et al. 1991; Saper et al. 1991; Madden et al. 1992; Collins et al. 1995; Reid et al. 1996; Smith et al. 1996a; Smith et al. 1996b; Glithero et al. 1999) has illustrated details of the structural relationship of the extracellular ⁇ -chain domains and ⁇ 2 m (FIG. 2). The membrane-proximal domains, ⁇ 3 and ⁇ 2 m, associate in an immunoglobulin fold structure.
  • the membrane-distal ⁇ x and ⁇ 2 domains together create a closed basket-like structure that sits atop the ⁇ 3 and ⁇ 2 m structure (FIG. 2A). It consists of two ⁇ -helical "walls" with a "floor” created by eight anti-parallel ⁇ -sheets (Bj ⁇ rkman et al. 1987a; Madden 1995; and FIG. 2, B and C).
  • detection of electron density situated in the ⁇ t / ⁇ 2 groove helped to clarify the experimentally-suspected occupancy of peptide fragments 8-10 residues long and thus the function of class I molecules in presenting such peptides upon the cell surface (Bj ⁇ rkman et al. 1987b).
  • thermodynamic stability studies indicate that networks of hydrogen bonds to structural residues lining the A- and F-pockets, which lie at opposite ends of the groove, serve to fasten a peptide by its N and C termini, respectively (Bouvier and Wiley 1994); these two pockets are thus implicated in the fixed orientation of a peptide within the binding groove.
  • Class I molecules primarily associate with peptide fragments, thus forming ⁇ -chain/ ⁇ 2 m/peptide trimolecular complexes, via an endogenous processing pathway during their assembly (Germain 1994; Heemels and Ploegh 1995; Lehner and Cresswell 1996; York and Rock 1996; Pamer and Cresswell 1998); in fact, the very binding of peptides is essential for the stabilization and expression of these molecules (Ljunggren et al. 1990; Townsend et al. 1990; Elliott 1991).
  • the class I ⁇ -chain and ⁇ 2 m are cotranslationally translocated into the ER lumen (Townsend et al. 1990; Germain and Margulies 1993; Neefjes et al.
  • Proteins in the cytoplasmic compartment are first enzymatically degraded into peptides of relatively uniform length by an ATP-dependent proteasome complex (Coux et al. 1996).
  • Some proteasome components include the IFN- ⁇ inducible subunits LMP2 and LMP7; these are themselves encoded within the MHC (Gaczynska et al. 1994).
  • the typically nonameric fragments produced are then actively conveyed across the ER membrane via a dimer of TAP1/TAP2, an MHC-encoded ATP-binding cassette transporter (Monaco et al. 1990; Parham 1990; Grandea III et al. 1995).
  • class I molecules figuratively serve as external banners that advertise the inner contents of the cells. Indeed, these antigens of peptide ligands indicate to the immune system as a whole which cells are to be eliminated and/or protected. Malignancies and/or pathogens effectively use this system to camouflage their existence and thereby escape detection and elimination by CD8+ CTLs, for example.
  • Thymic education of lymphocytes prevents activation in response to characteristic cell-derived peptides (Robey and Fowlkes 1994), but peptides acquired through the degradation of atypical proteins are recognized and induce cytolysis (Townsend et al. 1985; Gotch et al. 1988; Walker et al. 1988; Clark et al. 1995). Therefore, it is not surprising that CD8 + T lymphocytes play a critical role in controlling and/or eliminating infected and neoplastic cells.
  • CD8+T lymphocytes are implicated in immunity to pathogens such as viruses, which are intracellular invaders that utilize the host cell's biosynthetic machinery to produce their own foreign proteins (Yap et al. 1978; Lin and Askonas 1981; Jamieson et al. 1987; Harty and Bevan 1992; Riddell et al. 1992; Kulkami et al. 1995; Heslop et al. 1996; Schmitz et al. 1999).
  • CTL responses are likewise extended to include stimulation by aberrant proteins such as those associated with malignancy (Vose and Bonnard 1982; Muul et al. 1987; Coulie et al. 1992; Melief 1992; Kittlesen et al.
  • class I molecules are capable of binding and presenting to CTLs any protein introduced into the endogenous processing pathway by either natural or artificial means (Gooding and O'Connell 1983; Moore et al. 1988; Yewdell and Bennink 1990; Bertoletti et al. 1991; Donnelly et al. 1993; Ikonomidis et al. 1994; Ballard et al. 1996; Day et al. 1997; Goletz et al. 1997; Kim et al. 1997).
  • This knowledge serves as a motivating factor behind the development of both protein/peptide-based vaccines and other therapeutics intended to elicit protective CTL responses to microbial pathogens and other abnormalities, which otherwise remain cytoplasmically concealed from detection.
  • class I molecules in ligand presentation as described above is complicated by ⁇ -chain polymorphism. Class I structural differences resulting from genetic variation confer extreme heterogeneity upon regions of the molecule that interact with peptides. The knowledge of how polymorphism specifically impacts the natural presentation of peptide epitopes upon the cell surface is consequently limited.
  • class I molecules The characteristic polymorphism observed among class I molecules is thought to originate primarily through recombination and gene conversion (Kuhner et al. 1991; Parham et al. 1995); point mutations are believed to contribute more rarely to the pool of new alleles continually arising (Parham et al. 1989).
  • 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. From these alleles, new forms evolving progressively within populations can be passed on to subsequent generations and likewise serve as templates upon which yet further diversity may be introduced. This occurs through events such as single or double recombination (Parham et al.
  • HLA class I polymorphism While both inter- and intra-locus genetic events may give rise to polymorphism, the latter is most commonly observed; alleles at a locus generally tend to be more closely related to one another than to those present at other loci (Parham et al. 1988; Parham et al. 1995).
  • the forces driving HLA class I polymorphism are believed to be those of overdominant or balancing selection (Hughes and Nei 1988; Hughes and Yeager 1998); this is based upon values of d N >d s within the coding regions of ⁇ x and ⁇ 2 for specificity determining pocket residues positioned to interact with the peptide binding groove, while the contrary (d s >d N ) is observed among the remaining ⁇ x / ⁇ 2 residues.
  • class I structural polymorphisms are generated and maintained demonstrates that HLA genetic variability affords both advantages and disadvantages. It is beneficial in ensuring that at least a small portion of the human population will possess class I molecules capable of: (i) binding immunogenic peptides derived from any given pathogen; (ii) presenting those peptides to CTLs; and (iii) evoking a protective imune response. In short, annihilation of the species is guarded against by molecular diversity (Parham 1992).
  • heterozygote advantage through polymorphism as a mechanism for effectively allowing broader peptide binding abilities and thus broader CTL recognition of pathogenic peptides has been strongly emphasized from a statistical perspective (Hughes and Nei 1988; Nei and Hughes 1992).
  • HLA heterozygosity has been correlated with diminished progression to disease following HIV infection (Carrington et al. 1999).
  • the "nonrandomness" with which certain polymorphisms are maintained within populations following their evolution supports positive natural selection. This might occur in response to specific pathogenic pressures, as seen in the strong association of the West African allele HLA-B*5301 with resistance to malaria (Hill et al. 1991; Hill et al.
  • characterizing functional overlaps would provide an advantage not only to explore the general effects of binding groove architecture but more specifically to understand the similarities and/or differences of what is presented to CTLs by the class I molecules of genetically diverse individuals.
  • methods including pooled Edman sequencing, mass spectrometric analysis, and binding/reconstitution assays have been employed.
  • each approach bears its own strengths and limitations and none so far has been significantly successful in comparatively evaluating levels of functional overlap across class I polymorphisms.
  • the resultant motifs from N-Terminal Edman degradation are invariably nine amino acids in length and describe, as based upon relative yield increases per cycle, conserved "anchor" residues, or sites of stereochemical preferences, for peptides that are bound by a class I molecule.
  • These anchors typically appearing to involve both P2 and P9 of the ligands, are considered to be allele- specific and thus common among nearly all of the peptides bound by a given class I allotype.
  • the remaining positions demonstrate no overriding amino acid preferences, although the motifs of a few molecules demonstrate anchors at P3 or P5 (Rammensee et al. 1997).
  • the P2 anchor is assumed to associate with the B-pocket of the binding groove, while P9, the C-terminal residue assignment, associates with the F-pocket.
  • "Auxiliary" or “secondary” anchors are additionally included in the motifs of some class I molecules (Rammensee et al. 1997).
  • endogenous peptide binding and/or loading requires a nonamer with particular P2 and P9 anchors.
  • many searches (and consequent failed attempts) for putative viral or tumor class I-presented epitopes have subsequently been predicated upon nonameric templates with P2 and P9 anchor assignments.
  • de novo reconstitution assays can be performed by incubating synthetic peptides with free ⁇ -chains and ⁇ 2 m and comparing the resultant quantities of free versus complexed ⁇ -chains (Tanigaki 1992; Fruci et al. 1993).
  • Other methods of reconstitution have also been applied (Silver et al. 1991; Parker et al. 1994; Gnjatic et al. 1995; Robbins et al. 1995; Tan et al. 1997).
  • Ruppert and colleagues and Drijfout and colleagues each employed competitive binding assays; however, the former involved assessing the ability of synthetic peptides to inhibit binding of a radioiodinated standard peptide to membrane-extracted class I complexes, while the latter involved stripping peptides from class I complexes on B-LCLs and determining by FACS analysis the ability of synthetic peptides to inhibit binding of a different fluorescence- labelled standard.
  • Parker and colleagues employed a reconstitution assay which measured the incorporation of radioiodinated ⁇ 2 m into complexes refolded using synthetic peptides and ⁇ -chains prepared from Escherichia coli inclusion bodies.
  • the present invention(s) aim to provide a methodology for the production of soluble MHC Class I and II molecules from either gDNA or cDNA starting material such that the structural and functional impact of HLA class I polymorphism on peptide binding can be assessed and, in particular, to test how natural ligand presentation overlaps exist in varying degrees across the polymorphisms of divergent class I binding grooves.
  • the soluble MHC molecules can be used in the functional testing of CTL binding assays and vaccine deveopment.
  • FIG. 1 is a graphical representation of MHC class I location and heavy chain coding region.
  • A Simple map of the human MHC region, specifically highlighting the B, C, and A loci that encode the class I heavy chains as simplfied from (Janeway and Travers 1994). Genetic distances are estimated in kilobases. Other genes (not shown) including heavy chains and transporter/chaperone proteins (class II) and complement proteins and cytokines (class III) are encoded within the remaining MHC regions.
  • B The basic exon structure of MHC class I transcripts.
  • a total of seven exons encode the leader peptide (1), a t domain (2), ⁇ 2 domain (3), ⁇ 3 domain (4), transmembrane domain (5), and cytoplasmic domains (6-7).
  • Specific exon regions are indicated in parentheses for HLA-B*15011 (accession number U03859, Hildebrand et al. 1994), which encodes the HLA-B*1501 heavy chain.
  • FIG. 2 is a three-dimensional graphical representation of the configuration of the MHC class I extracellular domains. (A) The complete extracellular portion
  • FIG. 3 is a graphical representation of the specificity determining pockets of the MHC class I ligand binding groove.
  • the structural residues contributing to the six pockets (A through F) of the antigen binding groove formed by x and ⁇ 2 are indicated in black and numbered from the N terminus of the mature class I ⁇ -chain.
  • the residues are as collectively defined in the literature (Saper et al. 1991; Matsumura et al. 1992; Chelvanayagam 1996).
  • the pockets are assumed to accommodate the amino acids occuring at given ligand positions as follows: A-pocket, PI; B-pocket, P2; C- pocket, P6; D-pocket, P3; E-pocket, P7; and F-pocket, P9.
  • the majority of residues that contribute to these binding pockets are oriented such that they are solvent-inaccessible in mature tr ⁇ mers.
  • FIG. 4 is a pictorial representation summerizing MHC class I trimer assembly and transport.
  • the typical endogenous processing pathway shown is simplified from the description disclosed herein.
  • Major proteins participating in the processes are specifically illustrated and labelled.
  • Some alternative processing and/or transport mechanisms currently under investigation are designated in the diagram by question marks.
  • Assembled trimers travel through the Golgi complex prior to vesicular delivery to the surface membrane of the cell (not shown).
  • FIG. 5 is a schematic representation of the deduced contact region between the MHC class I Ck a 2 and TCR V ⁇ /V ⁇ interfaces.
  • FIG. 6 is a flow diagram of the overall strategy for comparatively mapping and characterizing peptide ligands presented by class I HLA.
  • the approach taken to address the presence of overlapping ligands across diverse class I molecules consisted of three basic parts: (A) sHLA-producing transfectant establishment and culture; (B) extraction, purification, and separation of ligands; and (C) ion map generation/comparison and characterization of individual peptides. Though only two molecules are indicated for simplification, numerous additional molecules (as indicated in the specification) were simultaneously carried through the steps shown.
  • FIG. 7 Hypothesized evolutionary relationships of HLA-B15 allotypes according to expressed ⁇ -chain polymorphisms. Shown in this scheme are the 46 allotypes whose amino acid sequences are provided in herein. Solid lines indicate single mutagenic events separating given allotypes. Dashed lines indicate that greater than one mutagenic event separates given allotypes; intermediaries are indicated by question marks unless more specifically suspected (as in the case of a probable B*1539/B17 recombinant). None of the line lengths are reflective of mutational rates.
  • FIG. 8 is a graphical representation of the localization of antigen binding groove substitutions distinguishing the B*1512, B*1508, B*1501, B*1503, B*1518, and B*1510 allotypes.
  • the structural residues of the antigen binding groove formed by cx t and ⁇ 2 which differ among the six alleles according to Table 1 are indicated on the ribbon diagram in black and numbered from the N terminus of the mature class I ⁇ -chain.
  • FIG. 9 is a graphical representation summarizing of the Unisyn Technologies CP-3000 basic flow path. The system was assembled and operated as described herein. Arrows indicate unidirectional media flow.
  • FIG. 10 is two graphs showing sHLA production during bioreactor runs with two different B*1501 transfectants. Constructs using PSR ⁇ . neo and pcDNA3 for producing soluble B*1501 were separately transfected into B-LCL 721.221; subclones were then cultured in CP-3000 systems as described herein. Harvests samples drawn on.the days indicated along the x-axis during each run were subjected to ELISA, yielding the representative sHLA production data shown.
  • FIG. 11 is a graph showing RP-HPLC fractionation of peptides extracted from B*1510.
  • the UV trace obtained during separation as described herein of approximately 400 ⁇ g of peptides extracted from B*1510 reveals the bulk of absorbance to occur along the gradient (black line) between 10-40% buffer B (indicated at the right).
  • FIG. 12 is a schematic showing the generalized components of a triple quadrupole mass spectrometer.
  • the basic constituents of the system described herein include: (A) an electrospray source/ionization interface for sample introduction; (B) three quadrupoles for ion manipulation, which includes mass filtration (Ql or Q3), transmission (all quadrupoles), and/or collision (q2); and (C) a detector for amplifying transmitted ion signals so that they can be recorded and analyzed.
  • FIG. 13 includes two graphs (A and B) and chart C that show the identification and verification of a ligand overlap across divergent HLA-B15 molecules.
  • NanoES-MS spectral ion maps obtained individually from RP-HPLC fraction 8 for each of B*1501, B*1503, B*1508, and B*1510 were aligned for comparison; an expanded view of the range 495-555 m/z, or amu, is shown in (A).
  • the ion mass centered at 517.2 m/z matched across the spectra of B*1501, B*1503, and B*1508 (top three panels) but not B*1510 (bottom panel). This ion was subsequently selected for NanoES-MS/MS from fraction 8 of the first three class I MHC molecules.
  • the homologous spectra resulting from fragmentation of this [M+2H] 2+ ion (B) classified the peptide as a positive match, or ligand overlap, occurring across B*1501, B*1503, and B*1508 and allowed for primary sequence derivation (C).
  • N- and C-terminal peptide fragments present in all three NanoES-MS/MS spectra are labelled according to standard nomenclature (Roepstorff and Fohlman 1984) in the top panel of (B) and underlined in (C); immonium ions are indicated by their single-letter amino acid codes in (B), and the sequences of internal cleavage products are also specified.
  • FIG. 14 is a graphical representation showing the reproducibility of the class I HLA ligand mapping and characterization strategy disclosed herein.
  • NanoES-MS ion mapping and comparison were then performed as described herein. As illustrated by the spectra of fraction 15 for each (A and B), the ion maps were consistent with one another; subjecting an ion (514.0) from each B*1508 fraction to NanoES-MS/MS further demonstrated reliability of the protocols employed.
  • FIG. 15 is a tabulation of the pooled Edman sequencing motifs for peptides extracted from B*1501 purified by two different MAbs.
  • FIG. 16 is a tabulation and corresponding pictorial representation of pooled Edman sequencing motifs for peptides extracted from B*1508, B*1512, B*1503, B*1518, and B*1510. Edman degradation was carried out as described herein, and the data were analyzed as described hereinbefore with respect to FIG. 15. Ribbon diagrams of the class I antigen binding groove to the right of each motif show the structural residues of the ⁇ ./ ⁇ 2 antigen binding groove among each of the alleles which differ with respect to B*1501 according to Table 1; they are indicated in black and numbered from the N terminus of the mature class I ⁇ -chain.
  • FIG. 17 is a graphical showing RP-HPLC separation of ligands from BBM.1- purified B*1501.
  • the UV trace obtained during separation as described herein of approximately 150 ⁇ g of peptides extracted from BBM.l-purified B*1501 reveals the bulk of absorbance to occur along the gradient (black line) between 10-30% buffer B (indicated at the right).
  • the fractions collected are numbered; although all fractions were examined, only those subsequently selected for analysis and data presentation in FIGS. 18 and 19 are marked by dots. It is noted that the chosen fractions were distributed evenly across the entire region of interest and included a wide variety that were of high as well as low UV absorbance and/or resolution.
  • FIG. 18 is four graphs showing the percentages of RP-HPLC ligand fractions from BBM.l-purified B*1501 demonstrating particular amino acid occupancies by Edman degradation. Sequence complexity among the peptides was summarized by averaging the frequency of occurrence for amino acid residues at P2 through up to P12 for the fractions marked by dots in FIG. 17. Amino acids are grouped along the x-axis according to their physicochemical natures (charged, polar, or hydrophobic), as specifically designated in the chart for P10 and indicated within all four charts by differential shading. Residues observed at P2 and P9 in former B*1501 motif descriptions are indicated in bold italics. Since it was not derivatized, cysteine was undetectable and is therefore excluded.
  • FIG. 19 is four tabular examples of Edman sequence complexity among RP-HPLC ligand fractions from BBM.l-purified B*1501. While each of the fractions marked by dots in FIG. 15 was subjected to Edman sequencing, the results obtained from fractions 10, 15, 28, and 31 are shown here to specifically illustrate some of the diverse trends observed among constituents of the ligand mixture. Dominant and strong assignments were made as described hereinbefore for FIG. 13; weak assignments were made for amino acids demonstrating a 1.5 to less than 2.0-fold picomolar increase over previous cycles. No assignments are shown for PI due to lower confidence since comparison with former cycles was impossible. The positional assignments in fraction 15, which are identified in bold italics, appear to correspond to a hexamer, IAVGYV, derived from HLA class I ⁇ -chain 23 - 28 (Prilliman et al. 1998).
  • FIG. 20 is five graphs summarizing teh length diversity among the 449 characterized HLA-B15 ligands.
  • Graphed data from the ligands listed in Tables A-E summarizes length diversity among the peptides respectively characterized from B*1501, B*1503, B*1508, B*1510, and B*1512.
  • FIG. 21 is two graphs showing N- and C-regional diversity observed through alignments of B*1501 ligands. Frequency of occurrence for amino acids at the first (A) and final (B) four positions among the ligands characterized from B*1501 (Table A). The graphs were generated from separate N- and C-terminal data alignments.
  • FIG. 22 is two graphs showing N- and C-regional diversity observed through alignments of B*1503 ligands. Frequency of occurrence for amino acids at the first (A) and final (B) four positions among the ligands characterized from B*1503 (Table B). The graphs were generated from separate N- and C-terminal data alignments.
  • FIG. 23 is two graphs showing N- and C-regional diversity observed through alignments of B*1508 ligands. Frequency of occurrence for amino acids at the first (A) and final (B) four positions among the ligands characterized from B*1508 (Table C). The graphs were generated from separate N- and C-terminal data alignments.
  • FIG. 24 is two graphs showing N- and C-regional diversity observed through alignments of B*1510 ligands. Frequency of occurrence for amino acids at the first (A) and final (B) four positions among the ligands characterized from B*1510 (Table D). The graphs were generated from separate N- and C-terminal data alignments.
  • FIG. 25 is two graphs showing N- and C-regional diversity observed through alignments of B*1512 ligands. Frequency of occurrence for amino acids at the first (A) and final (B) four positions among the ligands characterized from B*1512 (Table E). The graphs were generated from separate N- and C-terminal data alignments. The information shown here for B*1512 ligands is skewed in relation to that obtained for ligands from the other four allotypes since (i) a comparatively fewer B*1512 peptides were sequenced, and (ii) over half of the B*1512 peptides characterized were specifically investigated as ion map differences with B*1501.
  • FIG. 26 is a graphical and tabular representation showing ligand overlaps identified by NanoES-MS mapping and characterized by NanoES-MS/MS during comparative analysis of B*1508, B*1501, B*1503, and B*1510 extracts.
  • Ribbon diagrams of the class I antigen binding groove indicate residue substitutions (black, numbered from the first residue of the mature ⁇ -chain) between each B15 molecule with respect to B*1501.
  • the peptides are categorized into three different groups from top to bottom as follows: ligands that overlap B*1508 and B*1501; ligands that overlap B*1508, B*1501, and B*1503; and ligands that overlap B*1501 and B*1503.
  • FIG. 27 is a pictorial representation of the proposed N-proximal and C- terminal anchoring of a nonamer overlapping B*1508, B*1501, and B*1503.
  • the shared C-terminal anchoring preferences for Tyr in the NQZHGSAEY ligand among B*1508, B*1501, and B*1503 as defined by their respective motifs (FIGS. 15 and 16) are shaded black, while the varied N-proximal anchoring preferences likewise reflected in the motifs are shaded gray.
  • Ligand residues are numbered sequentially from the N terminus.
  • FIG. 28 is a pictorial schematic summarization the summary of structure- function relationships among the HLA-B15 allotypes from which ligands were characterized.
  • HLA-B15 allotypes for which a motif is known are indicated in bold, while allotypes with currently undefined motifs are indicated in italics. Arrows are representative of single mutagenic events (which are specifically defined for each arrow); bold arrows emphasize evolutionary pathways between structures for which motifs have been described either here or elsewhere.
  • P2 and P9 designations are listed. Tentative designations have been made for the italicized allotypes by extrapolation from known motifs in light of the polymorphisms present.
  • P2/P9 specificities for B*1519 are based upon B*1512.
  • P2/P9 specificities for B*1539 are based upon what has been observed between B*4402 and B*4403 (Fleischhauer et al. 1994).
  • P2/P9 specificities for B*1529 are based upon B*1508.
  • P2/P9 specificities for B*1523 are based upon what has been observed between B*1502 and B*1513 (Table 7), as well as observations between B*0801 and B*0802 (Arnett et al. 1998).
  • P2/P9 specificities for B*1547 are based upon the relationship previously discussed between B*1503 and B*4801 (Martinez-Naves et al. 1997).
  • P2/P9 specificities for B*1537 are based upon B*1402 (DiBrino et al. 1994). The three sections (A, B, and C) into which the molecules have been divided are discussed in the text. For reference, a ribbon diagram of the class I antigen binding groove indicating residue substitutions (black, numbered from the first residue of the mature ⁇ -chain) between the molecules with respect to B*1501 is provided.
  • FIG. 29 is a pictorial representation of the PCR strategy for the production of soluble HLA from gDNA according to the methods of the present invention.
  • FIG. 30 is a gel image of the primary PCR of 3A394.
  • FIG. 31 is a gel image of the secondary PCR of 3A394.
  • FIG. 32 is a gel image showing 3A394 and pcDNA3.1 digested with EcoR I and Xba I.
  • FIG. 33 is a gel image showing the restriction digests of 3A394 clones.
  • FIG. 34 is a graph showing the comparative binding of three monoclonal antibodies to four soluble HLA molecules.
  • FIG. 35 is a pictorial representation of the MHC.
  • FIG. 36 is a pictorial representation of an HLA molecule.
  • FIG. 37 is a pictorial representation of the HLA binding groove that binds antigenic peptides.
  • FIG. 38 is a pictorial representation of the antigen processing and assembly of the MHC class I/peptide complex.
  • FIG. 39 is a pictorial representation of HLA peptide loading and movement of the HLA molecule to the cell surface.
  • FIG. 40 is a pictorial representation of a tetramer biotinylated sHLA complex of the present invention.
  • FIG. 41 is a pictorial representation of a bacterial expression vector encoding a biotinylation substrate peptide sequence.
  • FIG. 42 is a pictorial representation of the biotinylation of sHLA.
  • FIG.43 is a pictorial representation of the conjugate used to confirm biotinylation of the sHLA molecule
  • FIG. 44 is a graph showing production of sHLA-B* 0702 by T 2 transfectants after peptide pulsing.
  • FIG. 45 is a graph showing the elution curve of the Elisa assay used to confirm sHLA production.
  • FIG.46 is a graph showing that an increase in time results in greater biotinylation.
  • FIG. 47 is a graph showing separation of biotinylated class I from free biotin.
  • Tables 1-24 and Tables A-E are set forth below on Pages 156-192.
  • PCR and sequencing primers for creating and verifying sHLA constructs. Designated restriction enzyme cut sites are underlined on the 5' and 3' PCR primers, and the regions of the 3' PCR primers that inserted a stop codonn at position 300 are shown in bold italics. Sequencing primers were Cy5-labelled; the regions that they either sequenced through or hybridized with are indicated in parentheses.
  • HLA-B15 ligands identically matching database source proteins, by category. The ligands are categorized according to source protein functions. Residues for the specific ligands are numbered from the initiating Met of their respective source proteins. Ligands from HLA-B15 molecules not studied here (B*1502, B*1509, and B*4601) are referenced herein.
  • B*1501 ligands N- and C-regional occupancies observed at >10% among 126 ligands. The positional occupancy percentages for residues among the ligands from Table A greater than 10% are specifically shown; dashes indicate occupany rates below 10% for the given side chains. N- regional positions and N sum values are highlighted in black, while C-regional positions and C sum values are highlighted in gray.
  • B*1503 ligands N- and C-regional occupancies observed at >10% among 74 ligands. The positional occupancy percentages for residues among the ligands from Table B greater than 10% are specifically shown; dashes indicate occupany rates below 10% for the given side chains. N- regional positions and N sum values are highlighted in black, while C-regional positions and C sum values are highlighted in gray.
  • B*1508 ligands N- and C-regional occupancies observed at >10% among 96 ligands. The positional occupancy percentages for residues among the ligands from Table C greater than 10% are specifically shown; dashes indicate occupany rates below 10% for the given side chains. N- regional positions and N sum values are highlighted in black, while C-regional positions and C sum values are highlighted in gray.
  • B*1510 ligands N- and C-regional occupancies observed at >10% among 123 ligands. The positional occupancy percentages for residues among the ligands from Table D greater than 10% are specifically shown; dashes indicate occupany rates below 10% for the given side chains. N- regional positions and N sum values are highlighted in black, while C-regional positions and C sum values are highlighted in gray.
  • B*1512 ligands N- and C-regional occupancies observed at >10% among 30 ligands. The positional occupancy percentages for residues among the ligands from Table E greater than 10% are specifically shown; dashes indicate occupany rates below 10% for the given side chains. N- regional positions and N sum values are highlighted in black, while C-regional positions and C sum values are highlighted in gray.
  • B*1501 epitopes selected from the EBV gp85 structural antigen.
  • the EBV gp85 protein (accession number 1334905, Arrand et al. 1981) was manually scanned for epitopes with consideration to B*1501.
  • epitope candidates matching the length and sequence constraints of the B*1501 pooled motif (FIG. 15) are listed.
  • epitope candidates matching the B*1501 motif-prescribed P2 and C- terminal occupancies but demonstrating relaxed length constraints (7 to 11 residues, according to the B*1501 panel in FIG. 20) are listed.
  • epitope candidates matching the B*1501 motif-prescribed nonameric length but demonstrating P2 flexibility (according to FIG. 21A) are listed.
  • Table 24 HLA types and sHLA molecules preduced.
  • HLA class I and class II ligands characterizing naturally processed HLA class I and class II ligands is a key element behind the basic understanding of how polymorphism impacts ligand presentation.
  • technical and scientific challenges including both extreme sample heterogeneity and limited sample sizes complicate such examinations.
  • Thousands of distinct peptides are present within a ligand extract prepared from a single type of class I molecule, and the immunoprecipitation/extraction protocols typically employed to recover peptide ligands yield sparse quantities on the order of ⁇ 20 ⁇ g (Hunt et al. 1992; Henderson et al. 1993). These factors often require specialized biochemical expertise not necessarily available in either the common laboratory or core facility.
  • Class I major histocompatibility complex (MHC) molecules bind and display peptide antigens upon the cell surface.
  • the peptides they present are derived from either normal endogenous proteins ("self") or foreign proteins ("nonself"), such as products of malignant transformation or intracellular pathogens such as viruses.
  • class I molecules convey information regarding the internal fitness of a cell to immune effector cells including but not limited to CD8 + cytotoxicT lymphocytes (CTLs), which are activated upon interaction with "nonself” peptides and which lyse or kill the cell presenting such "nonself” peptides.
  • CTLs cytotoxicT lymphocytes
  • Class II MHC molecules designated HLA class II in humans, also bind and display peptide antigens upon the cell surface.
  • 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. Therefore, 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 have deleterious and even lethal effects on the host.
  • 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, which help eliminate such pathogens both by helping B cells make antibodies against microbes as well as toxins produced by such microbes and by activating macrophages to destroy ingested microbes.
  • immune effector cells including but not limited to CD4 + helper T cells, which help eliminate such pathogens both by 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 HLA molecules exhibit extensive polymorphism, which is 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 among the population. Such extensive HLA diversity in 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, HLA proteins are needed to study transplantation, autoimmunity, and for developing vaccines.
  • 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 different MHC molecules, the testing of numerous individual MHC molecules is a prerequisite for understanding differences in disease susceptibility between individuals. Therefore, purified MHC molecules representative of the hundreds of different HLA types existing throughout the world's population are highly desirable for unraveling disease susceptibilities and resistances and for designing therapeutics.
  • HLA protein available were 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 envisions a method of producing MHC molecules which are secreted from mammalian cells in a bioreactor unit. Substantial quantities of individual MHC molecules are obtained by modifying class I or class II molecules so they are secreted. 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. In this way the cells producing secreted MHC remain alive and therefore continue to produce MHC. Problems of purity are overcome because the only MHC molecule secreted from the cell is the one that has specifically been constructed to be secreted.
  • transfection of vectors encoding such secreted MHC molecules into cells which may express endogenous, surface bound MHC provides a method of obtaining a highly concentrated form of the transfected MHC molecule as it is secreted from the cells. Greater purity can be assured by transfecting the secreted MHC molecule into MHC deficient cell lines.
  • 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.
  • the culturing of MHC secreting cells in bioreactors allows for a continuous production of individual MHC proteins in a concentrated form.
  • the initial HLA molecules selected for examination and production were from the HLA-B15 family, a hypothetical schematic of which is presented in FIG. 7.
  • the HLA-B15 family represents a broad and diverse group of molecules comprised of nearly 50 evolutionarily related allotypes differing almost sequentially by 1-15 peptide binding groove residues, and they are observed throughout numerous ethnic populations (Hildebrand et al. 1994); serological and DNA-based typing thus far confirm distribution of B15 alleles among Caucasians, Amerindians (North and South), Mexicans, Blacks (African and American), Indians, Egyptians, Pakistanis, Chinese, Japanese, Koreans, and Thais.
  • the majority of HLA B-locus polymorphisms known to exist are represented among the members of this allelic family.
  • HLA-B*1501 appears to be the "ancestral allele.”
  • B*1501 (Pohla et al. 1989; Choo et al. 1993; Hildebrand et al. 1994; Lin et al. 1996)
  • B*1503 Domena et al. 1993
  • B*1508 Hildebrand et al. 1994
  • B*1510 Domena et al. 1993; Rodriguez et al. 1993; Rodriguez et al. 1996)
  • B*1512 Hildebrand et al. 1994
  • B*1518 (formerly B*7901, Choo et al. 1991; Lin et al. 1996; Rodriguez et al. 1996) (Table 1 and FIG. 8).
  • B*1508 differs from B*1501 by a single mutagenic event in the. ⁇ x helix, while B*1512 differs by a single mutagenic event in the ⁇ 2 helix; the remaining three alleles demonstrate a progressive series of polymorphisms throughout their binding grooves imposed by sequential mutagenic events during their divergent evolution from B*1501.
  • PCR products were introduced into mammalian expression vectors.
  • Initial constructs (truncated B*1501, B*1503, and B*1508) were prepared with the PSR ⁇ -neo vector (Lin et al. 1990), which has formerly been used to express non-truncated HLA molecules (Barber et al. 1997; Martinez-Naves et al. 1997), while other constructs (truncated B*1501, B*1503, B*1508, B* 1510, B*1512, and B*1518) were additionally prepared with either pcDNA3 or pcDNA3.1(-) (Invitrogen).
  • DNA from each of the construct clones was prepared using Qiagen Midi kits for transfection of the class I-negative B-LCL 721.221.
  • Cells growing in log phase in RPMI-1640 + 2 mM L-glutamine + phenol red + 20% FCS were pelleted and electroporation was performed as described (Gumperzetal. 1995) prior to beginning selection with 1.5 mg/mL G418.
  • putative transfectant wells were screened for sHLA production using a sandwich ELISA (Prilliman et al. 1997).
  • Transfectant wells positive for sHLA production were then subcloned by limiting dilution to establish cell lines optimally secreting greater than 1 ⁇ g/mL of class I molecules in static culture over 48 h. Satisfactorily subcloned transfectants were then expanded, frozen in RPMI-1640 + 20% FCS + 10% DMSO, and stored at -135°C.
  • hollow-fiber bioreactors have been applied in place of in vivo hybridoma culture and MAb harvest from ascites (Evans et al. 1996) in order to continuously produce large quantities of pure immunoglobulins, they were utilized to produce and harvest the sHLA of the present invention.
  • the Unisyn Technologies CP-3000 the standard flow path and primary components of which are diagrammatically simplified in FIG. 9, was selected for hollow-fiber bioreactor culture of successfully established transfectants.
  • basal media is pumped into the fully-assembled system from a 200 L barrel; the media flows from the 4 L reservoir tank into the hollow-fiber networks of the four bioreactors, which provide 2.7 m 2 of surface area per cartridge, and then exits as waste.
  • ECS media and sHLA harvest are tandemly pumped into and out of the 270 mL cartridges, respectively, with each bioreactor receiving/yielding equal media/harvest volumes as regulated by in-line solenoids.
  • the CP-3000 was set up according to the manufacturer's protocol. After the system was completely prepared, at least 1 x 10 9 viable cells of a transfectant were grown in roller bottles of RPMI-1640 + 2 mM L-glutamine + phenol red + 10% FCS. The cells were pelleted and inoculated into the ECS of the bioreactor cartridges. ECS feed and harvest bottles were then attached to their corresponding lines, and the basal and recirculation rates were initially set to 100 and 1000 mL/h, respectively; the ECS was usually not activated until 24- 36 h following inoculation. The system was then monitored at least twice daily over 4-6 weeks, with adjustments made as necessary.
  • BBM.l and W6/32 were used to affinity purify B*1501, the first molecule prepared.
  • W6/32 alone was employed to isolate the remaining molecules from bioreactor harvests. This MAb has been frequently used by others in purifying HLA (Falk et al. 1991; Barber et al. 1997).
  • isolated peptides were purified of free amino acids and salts prior to fractionation. This was done on a 2.1 x 100 mm C18 column (Vydac) with a steep RP-HPLC gradient using a DYNAMAX HPLC system (Rainin). The gradient was generated by increasing to 100% buffer B (0.06% TFA in 100% acetonitrile) in 1 min, holding for 10 min, and returning to buffer A (0.1% TFA in HPLC-grade water) in 1 min. The column was loaded with peptides reconstituted in the minimum volume of buffer A required for solubilization. During the run, the region corresponding to absorbance at 214 nm was
  • the purified ligands were next fractionated by RP-HPLC.
  • approximately 150 ⁇ g of peptides as calculated from the ELISA-based total mass of sHLA bound to the affinity column and an estimated 50% handling loss, were loaded in 10% acetic acid onto a 1.0 x 150 mm C18 column (Michrom Bioresources, Inc.) and separated using an initial gradient of 2-10% buffer B (0.085% TFA in 95% acetonitrile) in 0.02 min followed by a linear gradient of 10-60% buffer B in 60 min at 40 ⁇ L/min on a 2.1 x 150 mm C18 column (Michrom Bioresources, Inc.); buffer A was 0.1% TFA in 2% acetonitrile.
  • NanoES The specific ionization interface, NanoES, chosen here as an ES source functions on the principles described by developers Wilm and Mann (Wilm and Mann 1996). To establish and validate the procedure, comprehensive peptide mapping and sequencing were first performed among fractions 6 through 19 (FIG. 11), which represented a region of relatively rich ligand concentration (data not shown), for B*1501, B*1503, B*1508, and B*1510; once this was accomplished, a more focused, and therefore less extensive, comparison was subsequently made between B*1501 and B*1512.
  • the capillary was then positioned directly in front of the API Ii (PE SCIEX) triple quadrupole mass spectrometer's orifice, and 20- 30 scans were collected as separate data files for the mass range 325-1400 m/z while operating the instrument at positive polarity. This procedure was performed sequentially to obtain constituent mass data for samples drawn from each RP-HPLC fraction.
  • API Ii PE SCIEX
  • Spectral "ion maps” were generated from the TICs acquired for each fraction.
  • the maps obtained from corresponding fractions of peptides eluted from different HLA-B15 molecules were aligned (FIG. 13A), and ions of interest for NanoES-MS/MS were located.
  • the ion maps were typically compared following baseline subtraction and smoothing. Putative ligand matches or, in the case of B*1512, mismatches among the ions were identified through a combination of data centroiding and direct visual assessment.
  • NanoES-MS/MS was performed by loading into a NanoES capillary tip, as described above, the desired volume of a fraction for which data was to be acquired.
  • the volume loaded depended upon the relative sample flow rate achieved after opening the capillary tip and how long data acquisition was intended to proceed. Typically 3-4 ⁇ L were loaded at a time to collect MS/MS data for 20-25 mid- to low-intensity ions from a given fraction.
  • the source head was positioned and the capillary opened as before. Separate data files were collected for each ion subjected to collisional dissociation.
  • NanoES-MS/MS data from ions of potentially overlapping peptides was aligned to confirm or refute the presence of shared ligands among different HLA-B15 molecules, as shown for one ion confirmed as an overlapping peptide across B*1501, B*1503, and B*1508 in FIG. 13, (B and C). Reproducibility of the protocol in its entirety is demonstrated in FIG. 14.
  • N and C values for the four ligand positions at either terminus were determined by summing occurrence frequencies (using an arbitrarily-defined baseline of 10%); N Sum was subsequently calculated from the four N values, and C sum was calculated from the four C values.
  • Peptides from HLA-B15 molecules were subjected to pooled Edman sequencing as well as more extensive examinations, including fractional Edman sequencing and mass spectrometric characterization of individual ligands. This was done to: (i) confirm the production/purification methods employed; and (ii) evaluate the relative nature and complexity of the peptides contained in extracts of naturally presented ligands.
  • B-locus allotypes that present peptides with Pro at P2 demonstrate a shallower B-pocket within their binding grooves than does B*1501, which exhibits a Ser at ⁇ -chain position 67 rather than a more constricting residue such as Phe (Barber et al. 1997).
  • B*1512 motif appeared nearly identical to that obtained from B*1501; by extension, considering that B*1519 differs from B*1512 in ⁇ 3 , which does not contribute to the peptide binding groove, it is predicted that B*1519 would bear the same motif as B*1501 and B*1512.
  • B*1503 diverges somewhat from the other three molecules presented above in showing a distinct preference for ligands with a neutral, polar Gin or positively-charged Lys as the P2 anchor; the aliphatic Met was evident here as well, though to a lesser degree than noted for the hydrophilic Gin and Lys residues (Prilliman et al. 1999). Like B*1501, B*1508, and B*1512 however, aromatic residues Tyr and Phe defined a hydrophobic P9 anchor. The only other class I molecules with motifs whose definitions thus far indicate a Lys at P2 are B*3902 (Falk et al. 1995) and B 801 (Martinez-Naves et al.
  • B*1503 structurally bear B-pockets identical to B*1503 except for a single L ⁇ T or L ⁇ E substitution, respectively, at the ⁇ 2 helical residue 163 (Chelvanayagam 1996).
  • the B-pocket of B*1503 is indistinguishable from that of B*4802 (Chelvanayagam 1996), whose motif remains undetermined but is likely to follow suit with those of B*1503 and these other molecules at the second ligand position.
  • An assortment of polar, charged, and hydrophobic residues is evident at P3 of the B*1503 motif.
  • the B*1510 motif demonstrated a strict preference for ligands bearing a basic, hydrophilic His as a P2 anchor.
  • a hydrophobic P9 anchor was described by residues including Leu and Phe.
  • the B*1510 motif strongly resembled that previously defined for B*1509, which exhibits nearly identical anchor preferences with His at P2 and Leu, Phe, and Met at P9 (Barber et al. 1997).
  • B*1510 and B*1509 differ structurally only by a substitution of N ⁇ D in ⁇ 2 at the ⁇ -sheet floor position 114, which takes part in forming several specificity pockets within the peptide binding groove (FIG. 3).
  • B*1518 would have for its motif a P2 anchor of His (as seen for B*1510 and B*1509) and a P9 of Tyr and Phe (as seen with B*1501, B*1503, B*1508, and B*1512).
  • B*1518 differs from B*1510 solely at position 116; two other HLA-B molecules that differ exclusively at this position are B*3501 and B*3503: they differ by a S ⁇ F substitution here, which would sterically mimic the substitution between B*1518 and B*1510 and confer B*1510-like P9 preferences (Steinle et al. 1995; Kubo et al. 1998).
  • More than 400 individual ligands extracted from the five distinct HLA-B15 allotypes were characterized according to the methodology of the present invention.
  • the ligands characterized here were from ion map masses found in multiple B15 allotypes as demonstrated in FIG. 13A. Selected ions were then dissociated by NanoES-MS/MS, and the resulting fragment information was compared and interpreted, as described hereinabove, to determine if the ions represented sequence-identical or merely mass-identical ligand matches.
  • the single peptide sequences ranged from 7 to 12 amino acids in length and demonstrated (i) greater heterogeneity at their N-terminal/ proximal regions than their C termini, and (ii) varying degrees of observed ligand overlap, both of which will be examined In the subsequent sections of this chapter.
  • the endogenous peptides eluted from B*1501, B*1503, B*1508, B*1510, and B*1512 varied in length from 7 to 12 amino acids as shown (FIG. 20).
  • An overall length breakdown of the peptides listed in Tables A-E demonstrates that approximately 6% are heptamers, 21% are octamers, 50% are nonamers, 19% are decamers, 3% are undecamers, and 1% are dodecamers.
  • Further emerging from the length characterization of individual ligands is the observation that peptides bound by each of the B15 molecules spanned ranges of 5 to 6 amino acids in length. For example, peptides eluted from B*1501, B*1510, and B*1512 were 7-11 amino acids in length, while those from B*1503 and B*1508 were 7-12 amino acids in length.
  • HLA ligands examples of ligands from this study with homology to stretches of known proteins are shown in Table 4.
  • the peptides yielding 100% identical BLAST database hits were grouped into seven categories, which were defined here according to the common natures of their potential source proteins: HLA ligands, replication/transcription/translation ligands, biosynthetic/degradative modification ligands, signalling/modulatory ligands, transporter/chaperone ligands, structural/cytokinesis ligands, and unknown function ligands.
  • the eIF3-p66 61 . 69 nonamer SQFGGGSQY (Falk et al. 1995; Barber et al. 1996) was found here within B*1501, B*1503, B*1508, and B*1512 extracts.
  • the decamer YMIDPSGVSY which is homologous to proteasome subunit C8 150 - 159 , was also previously described as a ligand for B*1502 (Barber et al. 1997), B*1508 (Barber et al. 1997), and B*4601 (Barber et al. 1996); it was found here presented by B*1501, B*1508, and B*1512.
  • B*1502, B*1513, and B*1508 have a Pro at P2, while the lack of a strong Pro at P2 in both B*1501 and B*1503 corresponds to polymorphism at heavy chain positions 63 and 67.
  • B*1501 appears to lose the propensity for Pro at P2 due to polymorphism at position 63
  • B*1508 appears to lose a Gin at P2 resulting from polymorphism at 67.
  • comparisons within the B15 family highlight how substitutions at positions 63 and 67 of the class I heavy chain ⁇ x helix appear to confer differential interaction with P2 of the peptide ligand.
  • Allotypes B*1509, B*1510, and B*1518 recognize a positively charged His at P2 and have the same residues at 24 and 45 as B*1503, but the differences at positions 63 and 67, which separate B*1503 from the other three molecules, again modulate the contour of P2 such that different positively charged P2 residues fit respectively into the B*1503 and B*1509/B*1510/B*1518 B-pocket categories.
  • Pro and Ala likewise appear with frequencies comparable to or exceeding those of the W6/32-purified pooled motif residues for B*1501 (FIG. 21A), and B*1508 ligands illustrate P2 inclinations for a rich array of side chains in addition to the motif-prescribed residues Pro and Ala which include Gly, Val, Met, Leu/He, Ser, Thr, and Gln/Lys. Similar variety is observed within the limited B*1512 ligand data set (FIG. 25A). In contrast, the B-pocket composition for B*1510 indicates His as the sole dominant/strong P2 occupant (Table 6), and among individual ligands characterized from B*1510 His is noted at a markedly higher degree than are alternative residues (FIG.
  • B*1501, B*1503, B*1508, and B*1512 a dominant C terminus was especially prominent among the ligands characterized from them, while B*1510 exhibited a P2 anchor nearly as strong as its primary C-terminal residue preference (FIG. 24, A and B).
  • the aromatic residues Phe and, even more prominently, Tyr occupied the C-terminal positions of most peptides bound by the first four B15 molecules, which appeared to agree with P9 of their respective motifs (FIGS. 15 and 16).
  • HLA-B15 molecules nine have F-pocket functionality in the same category (B*1501, B*1502, B*1503, B*1508, B*1512, B*1516, B*1517, B*1518, and B*4601), with preferences for Tyr, Phe, and/or Met, despite the fact that they exhibit amino acid substitutions at nine different positions throughout the ⁇ x helix and ⁇ 2 sheets.
  • Val (C 1 ) and Pro (C 2 ) were especially prominent C-proximal residues observed among the B*1510 ligands; the overriding presence of Pro, which distinguished this region of B*1510-derived peptides from those of the other allotypes, can likely be attributed to steric influences imposed by the Tyr at ⁇ 2 position 116 in B*1510, which additionally interacts with the C- and E-pockets of the peptide binding groove (FIG. 3). Further distinguishing several B*1510 ligands from but rare occurrences among B*1501, B*1503, B*1508, and B*1512, Pro frequently appeared as well in various C-proximal sequence combinations with Ala or Val (Table 8).
  • the amino acid residues characterized from each of the five HLA-B15 allotypes with occupancy rates of at least 10% for the first four (N- terminal/proximal) and last four (C-terminal/proximal) positions among ligands, respectively, are condensed in Tables 9-13. Presenting the data already discussed in this manner effectively emphasizes C-terminal dominance and N- proximal flexibility. By comparison, the data illustrates the limitations of pooled Edman motifs in being able to adequately reflect a consensus of the individual peptides contained within a given ligand extract.
  • each allotype is comprised of two amino acid specificities as shown by more than 80% of characterized peptides in all cases (Tables 9-13).
  • Tables 9-13 comparing observed N- and C-regional occupancies among the characterized ligands underscores the flexibility of N-proximal versus the dominance of C-terminal preferences among the B*1501, B*1503, B*1508, B*1510, and B*1512 binding grooves.
  • B*1501 and B*1503 were identified, and four ligands were found to overlap across B*1508, B*1503, and B*1501.
  • FIG. 26 depicts, in the context of the class I peptide binding cleft, the locations of polymorphisms that individuate B*1508, B*1501, B*1503, and B*1510 and highlights the anchoring residues for the peptide overlaps according to the N-proximal and C- terminal specificities of their respective presenting molecule's motif (FIGS. 15 and 16).
  • a further example provided here of how C-proximal auxiliary anchors might positively impact endogenous ligand binding is that eight of the peptides overlapping both the B*1508 and B*1501 antigen binding grooves bear Thr at C 1 , C 2 , or C "3 , and in four cases the peptides that bind B*1508/B*1501 or B*1508/B*1501/B*1503 are heptamers with Thr occupying P7, their C-terminal positions (FIG. 26).
  • Thr provides a C-terminal anchor for this ligand not evident in the pooled motifs of the three allotypes.
  • the B*1501/B*1503 overlap AQFASGAGZ may instead be additively stabilized through the N-proximal anchors indicated at P2 and P3 as well as at the N-terminal position, since Ala demonstrated significant PI occupancy among both B*1501 and B*1503 ligands, as previously shown (FIGS. 21 and 22, A).
  • Tapasin is not a requirement for ligand loading via the typical endogenous processing pathway (Lewis et al. 1998; Peh et al. 1998), and aside from its proposed role in serving as a bridge between a class I dimer and the peptide transporter until release of mature trimers upon peptide binding, the exact role of tapasin during class I assembly is unknown (Pamer and Cresswell 1998). Interactions between nascent class I molecules and TAP1/TAP2 have, however, been shown to be influenced either directly or indirectly by ⁇ 3 and positions 116 and 156 of ⁇ 2 (Suh et al. 1999; Kulig et al. 1998; Neisig et al. 1996).
  • B*1510 is capable of accommodating ligands with the properties favored by the B*1501, B*1503, and B*1508 binding grooves.
  • Data both from individual ligands (Table D) and fractional Edman sequencing (Table 3) indicate that Tyr can occupy the C-terminal position, and specific examples in Table 4, including the spleen mitotic checkpoint BUB3 53 . 60 octamer YQHTGAVL and the splicing factor U2AF large chain 179 . 187 nonamer TQAPGNPVL, attest to B-pocket flexibility.
  • B*1501 and B*1512 demonstrated the highest overlap frequency between the allotypes at 70% among ions subjected to NanoES-MS/MS.
  • B*1503 and B*1508 respectively exhibited 14% and 9% overlap frequencies, while as shown earlier B*1510 completely failed to reveal overlaps with B*1501.
  • the trend distinctly illustrates that the polymorphisms which distinguish the B*1503, B*1508, B*1510, and B*1512 peptide binding grooves from B*1501 are not functionally equivalent in terms of their impacts upon class I ligand association. However, it is also evident that they do not create concrete barriers to ligand binding.
  • Comparative analyses of closely related soluble MHC class I molecules produced by the recombinant methods described herein, provide a means for assessing the functional impact of individual ⁇ -chain polymorphisms.
  • the primary impetus for characterizing peptides extracted from class I molecules is to more precisely understand the influence of structural polymorphism upon the presentation of endogenous ligands. This is important since a fundamental realization of how naturally processed peptides bind to both individual and multiple class I allotypes can then be translated into protein and/or peptide- based therapies intended to elicit protective CTL responses. Therefore, an accurate interpretation of sequence data from such class I-bound peptides, either individual or pooled, should in turn further the selection of optimal viral and tumor-associated ligands to expedite the development of successful therapeutic applications.
  • a step in developing therapies intended to elicit protective CTLs requires the selection of pathogen- and tumor-specific peptide ligands for presentation by MHC class I and class II molecules. Binding/reconstitution assays provide information that is biased due to their technical inconsistency and/or in vitro nature, while Edman sequencing of extracted class I peptide pools generates "motifs" that indicate that the optimal peptides are nonameric ligands bearing conserved P2 and P9 anchors; motifs have frequently been used to provide the search parameters for selecting potentially immunogenic epitopes that might be successfully presented by particular allotypes (Pamer et al. 1991; DiBrino et al. 1994; Kast et al. 1994; Davenport et al.
  • ligands were purified from different sHLA molecules produced in hollow-fiber bioreactors, mapped by RP-HPLC and NanoES-MS, and sequenced by NanoES-MS/MS, all according to the methodology of the present invention.
  • FIG. 28 summarizes the ⁇ -chain substitutions and motif-derived P2 and P9 anchors for the HLA-B15 allotypes examined here, as well as additional allotypes indicated earlier in FIG. 5 that appear to serve as evolutionary intermediates between or extensions from B*1503, B*1510, B*1512, and B*1518.
  • B*4601 is included since it differs from B*1501 by a single mutagenic event and overlaps with it were among peptides characterized in this study (Table 4).
  • the allotypes B*1501, B*1503, B*1508, B*1512, and B*4601 have been shown to bind variously overlapping ligands; based upon the structurally-predicted motif anchors of the remaining molecules in this section including B*1519, B*1529, B*1539, and B*1547, it is suspected that ion mapping and characterization would reveal further overlaps according to the model shown in FIG. 27.
  • the allotypes B*1509 and B*1510 have demonstrated overlapping ligands and will probably share some with B*1537.
  • the comparative peptide mapping strategy succeeded in identifying endogenously processed ligands which bind variously across the allotypes B*1501, B*1503, B*1508, and B*1512, but not B*1510. Overlapping peptide ligands appeared to favorably bind the first four B15 molecules since these allotypes share identical C- terminal anchoring pockets, whereas B*1510 diverges in this region. Endogenous peptide loading into the HLA-B15 allotypes therefore requires that a conserved C terminus be firmly anchored in the appropriate specificity pocket while N-proximal residues act more flexibly in terms of both location and sequence specificity to anchor the ligand into this binding groove region.
  • This alternative method of the present invention begins by obtaining genomic 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 some cases 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. In other cases 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 insure fidelity of the PCR.
  • Faithful truncated (i.e. sHLA) clones of the desired MHC molecule are then transfected into a mammalian cell line.
  • sHLA truncated clones of the desired MHC molecule
  • 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 expression or express endogenous class I. It is important to note that cells expressing endogenous class I may spontaneously release MHC into solution upon natural cell death.
  • 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 which will be attached to the protein may be added by the PCR reaction or may be encoded by the vector into which the gDNA fragment is cloned, and such His tail will further aid in purification of the class I molecules away from endogenous class I molecules.
  • Tags beside a histidine tail have also been demonstrated to work and are logical to those skilled in the art of tagging proteins for downstream purification.
  • 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 - this is observed when trying to find a match 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. Alternatively, in the method of the present invention, only a saliva sample, a hair root, an old freezer sample, or less than a milliliter (0.2 ml) of blood would be required to isolate the gDNA.
  • 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 or in mammalian cells or from cDNA in several species of cells using the methods of the present invention described herein.
  • Current experiments to obtain an MHC allele for protein expression typically start from mRNA, which requires a fresh sample of mammalian cells that express the MHC molecule of interest. Working from gDNA does not require gene expression or a fresh biological sample. It is also important to note that RNA is inherently unstable and is not easily obtained as is gDNA.
  • RNA messenger RNA
  • experiments using the methodology of the present invention show that . >5 milliliters of blood that is less than 3 days old is required to obtain sufficient RNA for MHC cDNA synthesis.
  • gDNA the breath of MHC molecules that can be readily produced is expanded. This is a key factor in a system as polymorphic as the MHC system; hundreds of MHC molecules exist, and not all MHC molecules are readily available from MRNA.
  • MHC molecules unique to isolated populations or of MHC molecules unique to ethnic minorities This is especially true of MHC molecules unique to isolated populations or of MHC molecules unique to ethnic minorities.
  • Starting class I or class II protein expression from the point of genomic DNA simplifies the isolation of the gene of interest and insures a more equitable means of producing MHC molecules for study; otherwise, one would be left to determine whose MHC molecules are chosen and not chosen for study, as well as to determine which ethnic population from which fresh samples cannot be obtained should not have their MHC molecules included in a diagnostic assay.
  • 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 the present invention does not require viable cells, and therefore the degradation which plagues RNA is not a problem.
  • any number of gDNA and cDNA MHC molecules can be produced.
  • the first product is the soluble class I MHC protein, which may be purified and utilized in various experimental strategies, including but not limited to epitope testing.
  • Epitope testing is a method for determining how well discovered or putative peptide epitopes bind individual, specific class I or class II MHC proteins.
  • Epitope testing with secreted individual MHC molecules has several advantages over the prior art, which utilized MHC from cells expressing multiple membrane- bound MHCs. While the prior art method could distinguish if a cell or cell lysate would recognize an epitope, such method was unable to directly distinguish in which specific MHC molecule the peptide epitope was bound.
  • MHC molecules Lengthy purification processes might be used to try and obtain a single MHC molecule, but doing so limits the quantity and usefulness of the protein obtained.
  • the novelty of the current approach is that individual MHC specificities can be utilized in sufficient quantity through the use of recombinant, soluble MHC proteins. Because MHC molecules participate in numerous immune responses, studies of vaccines, transplantation, immune tolerance, and autoimmunity can all benefit from individual MHC molecules provided in sufficient quantity.
  • MHC molecules A second important product obtained from mammalian cells secreting individual MHC molecules is the peptide cargo carried by MHC molecules.
  • Class I and class II MHC molecules are really a trimolecular complex consisting of an alpha chain, a beta chain, and the alpha/beta chain's peptide cargo to be reviewed by immune effector cells. Since it is the peptide cargo, and not the MHC alpha and beta chains, which marks a cell as infected, tumorigenic, or diseased, there is a great need to characterize the peptides bound by particular MHC molecules.
  • characterization of such peptides will greatly aid in determining how the peptides presented by a person with MHC-associated diabetes differ from the peptides presented by the MHC molecules associated with resistance to diabetes.
  • having a sufficient supply of an individual MHC molecule, and therefore that MHC molecules bound peptides provides a means for studying such diseases. Because the method of the present invention provides quantities of MHC protein previously unobtainable, unparalleled studies of MHC molecules and their important peptide cargo can now be facilitated.
  • Primers have been designed for HLA-A, -B and -C loci in order to produce a truncated amplicon of the human class I MHC using a two-stage PCR strategy.
  • the first stage PCR uses a primer set that amplify from the 5' Untranslated region to Intron 4. This amplicon is used as a template for the second PCR which results in a truncated version of the MHC Class I gene by utilizing a 3' primer that sits down in exon 4 at codon 298 (including the leader peptide), the 5' primer remains the same as the 1 st PCR.
  • An overview of this PCR strategy can be seen in FIG. 29.
  • the primers for each locus are listed in Table 17. Different HLA-B locus alleles require primers with different restriction cut sites depending on the nucleotide sequence of the allele. Hence there are two 5' and two 3' truncating primers for the HLA-B locus.
  • Amplification primers (in ng/ul): a. A locus: 5' sense PP5UTA (300); 3'antisense PPI4A (300) b. B locus (Not B*39's): sense PP5UTB (300); antisense PPI4B (300) c. B locus (B*39's): sense 5UTB39 (300); antisense PPI4B (300) d. C Locus: sense 5PKCE (300); antisense PPI4C (300).
  • H 2 O Dionized ultra filtered water (DIUF) Fisher Scientific, W2-4,41. PCR nt mix (lOmM each deoxyribonucleoside triphosphate [dNTP]), Boehringer Manheim, #1814, 362. Pfu DNA Polymerase, Promega, M7741. P f u D A Polymerase lOx reaction Buffer with MgSO 4 , 200m ⁇ ? Tris-HCL,pH 8.8,100mM KCl, lOOmM (NH 4 ) 2 SO 4 , 20 M MgSO 4 , lmg/ml nuclease free BSA,1% TritonrX- 100. Template 1:100 dilution of the primary PCR product.
  • Amplification primers in ng/ul: a. A-locus: 5' sense PP5UTA (300), 3' antisense PP3PEI (300) b. B-locus: sense PP5UTB (300), antisense PP3PEI (300) c. B-locus: sense PP5UT (300), antisense PP3PEIH (300) d. B-locus B39's: sense 5UTB39 (300), antisense PP3PEIH (300) e. C-locus: sense 5PKCE (300), antisense PP3PEI (300) f. C-locus Cw*7's: sense 5PKCE (300), antisense 3PEIHC7 (300)
  • T4 DNA ligase buffer 500mM Tris-HCL, lOOmM MgCL 2 , lOOmM DTT, lOmM ATP, 250ug/ml BSA, pH 7.5.
  • NEB buffer 2 500mM NaCl, 100 mM Tris-HCl, lOOmM MgCI 2 , lOmM DDT, pH 7.9. lOOx BSA, New England Biolabs.
  • Z-Competent E. coli Transformation Buffer Set Zymo Research, T3002. E. coli strain JM109. LB Plates with lOOug/ml ampicillin. LB media with lOOug/ml ampicillin
  • PagePlus 40% concentrate Amresco, E562, 500ml. Urea, Amersham Pharmacia Biotech, 17-0889-01, 500g. N'N'N'N'-tetramethylethyleneiamine (TEMED), APB. Ammonium persulphate (10% solution), APB. Boric acid,APB. EDTA-disodium salt, APB. Tris, APB. Bind-Saline, APB. Isopropanol, Sigma. Glacial Acetic Acid, Fisher Biotech. DIUF water, Fisher Scientific. Ethanol 200- proof.
  • TEMED N'N'N'N'-tetramethylethyleneiamine
  • Biorad Gene Pulser with capacitance extender Bio-Rad Laboratories. Gene Pulser Cuvette, Bio-Rad Laboratories. Cytomix: 120mM KCl, 0.15mM CaCI 2 , lOmMK.HPOyKH ⁇ , pH 7.6, 25mM Hepes,pH 7.6, 2mM EGTA, pH 7.6, 5mM MgCI 2 , pH 7.6 with KOH. RPMI 1640+ 20% Foetal Calf Serum + Pen/strep. Haemacytometer. Light Microscope. CO 2 37° Incubator. Cells in log phase. Methods used for the production of soluble human HLA Class I and II proteins in mammalian cells from gDNA. 1. Primary PCR
  • the cut sites incorporated into the PCR primers for each individual PCR will determine the enzymes used.
  • the expression vector pcDNA3.1(-) will be cut in a similar manner.
  • Electroporations are performed as described in "The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKbl, a putative HLA receptor. Gumperz, J.E., V. Litwin, J.H. Phillips, L.L. Lanier and P. Parham. J. Exp. Med. 181 : 1133-1144, 1995," which is herein expressly incorporated by reference in its entirety. 15. Screening for production of Soluble HLA
  • 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. It is directed against monomorphic determinants on HLA-A, -B and -C heavy chains, which recognizes only mature complexed class I molecules and recognizes a conformational epitope on the intact MHC molecule containing both beta2-microglobulin (b2m) and the heavy chain (HC).
  • mAb monoclonal antibodies
  • W6/32 binds a compact epitope on the class I 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. Immu ⁇ ogenetics 1999 Apr;49(4):312-20.).
  • 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 beta2-microglobulin (Shields MJ, Ribaudo RK.
  • W6/32 Mapping of the monoclonal antibody W6/32: sensitivity to the amino terminus of beta2- icroglobulin. Tissue Antigens 1998 May;51(5):567-70). W6/32 is available biotinylated (Serotec MCA81B) offering additional variations in ELISA procedures.
  • Anti-human b2m (HRP) (DAKO P0174) recognizes denatured as well as complexed b2m. Although in principle anti-b2m reagents could be used for the purpose of identification of HLA molecules, they are less suitable when association of heavy chain and b2m is weak. The patterns of class I molecules precipitated with W6/32 and anti-b2m are usually indistinguishable [Vasilov, 1983 #10].
  • Rabbit anti-b2-microglobulin dissociates b2-microglobulin from heavy chain as a consequence of binding (Rogers, M.J., Appella, E., Pierotti, M. A., Invernizzi, G., and Policyani, G. (1979) Proc Natl. Acad. Sci. U.S.A. 76, 1415- 1419). It also has been reported that rabbit anti-human b2-microglobulin dissociates b2-microglobulin from HLA heavy chains upon binding (Nakamuro, K., Tanigaki, N., and Pressman, D. (1977) Immunology 32, 139-146.). This anti-human b2m antibody is also available unconjugated (DAKO A0072).
  • 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.
  • the W6/32 - anti-b2m 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-b2m) 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 4 factors:
  • 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). Although this is well above the capacity of a microtiter plate, the binding will occur more rapidly. Higher concentrations will speed the binding of antigen to the polystyrene but the capacity of the plastic is only about 100 ng/well (300 ng/cm 2 ), so the extra protein will not bind. If using W6/32 of unknown composition or concentration, first titrate the amount of standard antibody solution needed to coat the plate versus a fixed, high concentration of labeled antigen. Plot the values and select the lowest level that will yield a strong signal. Do not include sodium azide in any solutions when horseradish peroxidase is used for detection.
  • microtiter plate Immediately coat the microtiter plate with 100 ⁇ l per well using a multichannel pipet. Standard polystyrene will bind antibodies or antigens when the proteins are simply incubated with the plastic. The bonds that hold the proteins are non-covalent, but the exact types of interactions are not known. Shake the plate to ensure that the antigen solution is evenly distributed over the bottom of each well. Seal the plate with plate sealers (sealplate adhesive sealing film, nonsterile, 100 per unit; Phenix (1-800 767-0665); LMT-Seal-EX) or sealing tape to Nunc-ImmunoTM Modules (# 236366). Incubate at 4 ° C overnight. Avoid detergents and extraneous proteins.
  • prewetting 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. Prewetting is absolutely necessary on the more difficult liquids such as 3% BSA. Do not prewet, if your intention is to mix your pipetted sample thoroughly with an already present solution. However, prewet only for volumes greater than 10 ⁇ l. In the case of pipettes for volumes less than 10 ⁇ l the residual liquid film is as a rule taken into account when designing and adjusting the instrument. The tips must be changed between each individual sample. With volumes ⁇ 10 ⁇ l special attention must also be paid to drawing in the liquid slowly, otherwise the sample will be significantly warmed up by the frictional heat generated. Then slowly withdraw the tip from the liquid, if necessary wiping off any drops clinging to the outside. To dispense the set volume hold the tip at a slight angle, press it down uniformly as far as the first stop.
  • 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. After liquid has been discharged with the metering stroke, a short pause is made to enable the liquid running down the inside of the tip to collect at its lower end. Then press it down swiftly to the second stop, in order to blow out the tip with the extended stroke with which the residual liquid can be blown out. In cases that are not problematic (e.g. aqueous solutions) this brings about a rapid and virtually complete discharge of the set volume. In more difficult cases, a slower discharge and a longer pause before actuating the extended stroke can help.
  • HLA absolute amount of antigen
  • 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). Use a tube of the standard stock solution to prepare successive dilutions according to the scheme below. While standard curves are necessary to accurately measure the amount of antigen in test samples, they are unnecessary for qualitative "yes/no" answers.
  • 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 H 2 SO 4 , producing an orange-brown product and read at 492 nm.
  • OPD fresh from tablets (Sigma, P6787; 2 mg/tablet). The solid tablets are convenient to use when small quantities of the substrate are required.
  • 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 your assay is high, this may be due to insufficient blocking. Finally analyze your readings. Prepare a standard curve constructed from the data produced by serial dilutions of the standard antigen. To determine the absolute amount of antigen, compare these values with those obtained from the standard curve.
  • 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. Set appropriate parameters to record the loading conditions on the recorder.
  • 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.
  • Cleaning- in-place is a procedure, which removes contaminants such as lipids, precipitates or denatured proteins that may remain in the column after regeneration. Such contaminations are especially likely when working with crude materials.
  • the procedure helps to maintain the capacity, flow properties and general performance. Mock elute the column using buffers with alternating pH. Start running over 10 gel volumes of 0.2 N acetic acid followed by 10 gel volumes of 50 mM diethylamine, pH 11.3 at a speed of 10 ml/min. Repeat three times and always equilibrate with 10 gel volumes PBS between buffer changes.
  • the AKTATM prime system has to be cleaned carefully. Start with the cleaning of line 5, where the sample was hooked up. Rinse the system pump and include the fraction collector line. First clean the inlet tubings, by manually running the system pump and flushing with 0.2 N acetic acid at 30 ml/min followed by 0.1 N NaOH. Always equilibrate with PBS. Don't forget to add a line between the injection valve and the UV detector as a bridge, as replacement of the column. Finally, rinse with 20% ethanol. If the column was sanitized because of bacterial contamination, rinse with 70% ethanol.
  • the methodology for this assay is essentially the same as the standard Elisa described above, except that instead of coating the plate with w6/32, the soluble HLA molecules are coated directly to it.
  • the three different antibodies are then utilized in three different ways to detect the bound HLA.
  • W6/32 is biotin labeled, therefore Vectastain kit and OPD are used for detection, anti b 2 M is conjugated to horse radish peroxidase and so can be directly detected using OPD and with hclO a secondary anti-mouse IgG horse radish peroxidase conjugated antibody and OPD are used.
  • Table 19 shows the optical density readings and concentration of this extraction.
  • FIG 30 2% agarose gel showing the primary PCR product of 3A394 (5 th lane from the right) at approximately 2kb in size.
  • the PCR product was gel purified and this along with unpurified PCR was used in the secondary PCR at a 1 : 100 dilution.
  • FIG. 32 is a gel showing 3A394 and pcDNA3.1 digested with EcoR I and Xba I. Once the ligations, at three different insert to vector ratios, and transformation were completed colonies were picked, grown overnight and then the plasmid DNA was extracted. A restriction digest was performed to screen for insert as shown in FIG. 33 is a restriction digests of 3A394 clones. A vector to insert ratio of 1:6 is the most efficient.
  • the concentration of the vector was established by optical density (Table 20 ) and then diluted to 500ng/ul.
  • the clone 3A394PC1 was then grown up and the plasmid containing A*1102 Truncated (A*1102T) was prepared for electroporation by performing a large scale plasmid extraction. The concentration of this was determined using optical density which can be seen in Table 21.
  • the cells were then placed under G418 selection to screen for positive transfectants, those exhibiting G418 resistance were screened for soluble HLA production using the Elisa, Table 23. Only two replicates were tested for this sample and all were done using undiluted supernatant.
  • FIG. 34 is a graph showing the comparative binding of three monoclonal antibodies to four different soluble HLA molecules.
  • A*1102T is sample 3A394TPClwell 1 from genomic DNA the other three are produced from cDNA. Three different amounts of soluble HLA were coated to the plate for each allele.
  • results of the comparative binding assay demonstrate several properties of the soluble HLA produced from genomic DNA. Coating a plate with more protein will not necessarily yield a higher signal to protein ratio. Soluble HLA from genomic DNA gives results comparable to that of cDNA constructs. The fact that all three antibodies bind this confirms the correct epitopes of the recombinant molecules are present.
  • the Elisa data allows us to test test how functional these molecule are. By using w6/32 and anti b 2 M to establish production levels we also provide information as to how much of the protein is in a trimeric form.
  • the comparative Elisa data helps back this up as the ratio of w6/32:hcl0 needs to be greater than 1.0 in order for there to be more conformational molecule than denatured, this is shown to be the case.
  • An exemplary useful product which can be obtained from the mammalian cell line expressing such a genomic DNA construct is a cDNA clone encoding the desired class I or class II molecule.
  • the cDNA clone encoding the desired class I or class II molecule is formed from the mRNA molecule encoding the desired class I molecule isolated from such mammalian cell line.
  • the cDNA clone may be utilized for functional testing, as described in more detail herein below.
  • gDNA clones can be used as a mechanism to obtain cDNA clones of the desired class I or class II HLA molecule.
  • the cDNA clones may be transfected into a cell which is unable to splice introns and process the mRNA molecule and therefore would not express the MHC molecule encoded by the genomic DNA, such as insect cells or bacterial cells.
  • these cell lines will also be deficient in peptide processing and loading, and therefore the soluble MHC molecules expressed from such cells will not contain peptides bound therein (referred to as free heavy chain HLA).
  • free heavy chain HLA peptides bound therein
  • Such soluble, free heavy chain HLA can effectively be tested for epitope binding as well. That is, MHC made in cells which do not naturally load peptide can be experimentally loaded with the peptide of choice.
  • the heavy chain, light chain, peptide trimer can be reassembled in vitro using a high affinity peptide to facilitate assembly.
  • a cell deficient in peptide processing can be pulsed with peptide such that the trimolecular MHC complex forms.
  • DNA encoding a peptide also encoding an appropriate targeting signal
  • MHC molecules could also be co-transfected into the cell with the MHC so that the MHC molecule which emerges from the cell is loaded only with the desired peptide. In this way MHC molecules could be loaded with a single low affinity peptide so that replacement with test peptides in a binding assay are more controlled.
  • an advantage of secreting individual MHC molecules from a cell that naturally loads peptide is that the MHC molecule of interest is naturally loaded with thousands of different peptides.
  • a synthetic peptide can therefore be compared to thousands of naturally loaded peptides.
  • the peptide-MHC complex can be multimerized to form soluble peptide-MHC dimers or tetramers, or other multiple soluble peptide-MHC - mers, such as fivemers, sixmers, etc. which serve as ligands for CTLs.
  • the tetramers can be mixed with CTLs in vitro or with CTLs from the blood of human subjects to identify antigenic peptides responsible for immune responses in humans.
  • Altman et al discloses a method of functional testing using tetramer technology; however, the method of Altman, however, only discloses one soluble MHC molecule which has been utilized in such a method, and Altman's method faces the same disadvantages and defects described above for the prior art, that is, the method envisions isolating individual mRNA/cDNA molecules from hundreds of different, typed cell lines, and then manipulating the cDNA molecules to produce the desired soluble MHC molecule.
  • the methods of the present invention envision combining the tetramer technology with amplification of genomic DNA, cloning the genomic DNA fragment, and transfection of the resulting construct into a mammalian cell line followed by isolation of cDNA from such transfected cell line and transfection into a cell line deficient in peptide processing and loading, thereby removing the need to isolate hundreds of different, typed cell lines for obtaining the different cDNAs.
  • MHC/peptide tetramers are widely utilized in the phenotypic analysis of T cells and in the study of T cell responses to pathological conditions such as viral infections and cancer.
  • Current methodology for tetramer production consists of expressing the MHC class I heavy chain in bacterial or insect cells and refolding the heavy chain in the presence of ⁇ -2-m ⁇ croglobulin and a specific peptide ligand in vitro.
  • the methodology of the present invention had two specific aims, although this should not be regarded as limiting: 1) to engineer a cDNA construct of a class I heavy chain containing a BirA substrate peptide (bsp) sequence at its 3' end (C-terminus) which would enable its subsequent biotinylation and 2) to develop a novel means of tetramer production using a mammalian expression system.
  • the mammalian system used was a B cell/T cell hybrid, the antigen- processing mutant cell line CEM x 721.174.T2 (T2).
  • T2 cells When pulsed with established HLA B*0702-presented peptides from HIV-infected CD4+ T cells, T2 cells transfected with the recombinant, truncated B*0702 HLA heavy chain secreted specific MHC/peptide complexes. Following enzymatic biotinylation, these complexes were combined with avidin to form B7 tetramers.
  • a mammalian expression system affords several advantages over a prokaryotic system, such as allowing normal glycosylation of the class I heavy chain and eliminating the need to refold the MHC/peptide complex in vitro following expression. The MHC class I molecules are therefore naturally folded in the cell rather than artificially folded outside the cell.
  • MHC major histocompatibility complex
  • ⁇ 2 m ⁇ 2 - microglobulin
  • HLA human leukocyte antigen
  • sHLA soluble human leukocyte antigen
  • bsp BirA substrate peptide or biotinylation substrate peptide
  • CTL cytotoxic T lymphocytes
  • PCR polymerase chain reaction
  • ELISA enzyme- linked immunosorbent assay
  • HRP horseradish peroxidase
  • PE phycoerythrin
  • PBS phosphate buffered saline
  • TAP Transporters associated with Antigen Processing
  • TCR T cell receptor
  • ER endoplasmic reticulum
  • kDa kiloDaltons.
  • MHC class I The structure and function of MHC class I. Unlike B lymphocytes which can interact with antigen in its intact form, T lymphocytes only recognize protein antigen broken down into peptide fragments and presented in association with specialized cell-surface molecules. These molecules are the gene products of the major histocompatibility complex (MHC) (FIGS. 1 and 35), a region on chromosome six known to encode proteins that are critical for immunologic specificity and transplantation histocompatitiblity. MHC molecules, also termed human leukocyte antigens (HLA), are cell-surface glycoproteins that function primarily in communicating to T cells the presence of intracellular or extracellular invaders.
  • HLA human leukocyte antigens
  • MHC/peptide complexes are recognized by the T cell receptor (TCR), an interaction enabling T cells to discriminate between MHC molecules bearing "foreign” antigen (viral/tumor/bacterial peptides) and MHC molecules displaying "self” peptides. An immune response can then be initiated against cells determined to harbor foreign antigen on the basis of the avidity of this binding interaction.
  • TCR T cell receptor
  • MHC molecules are of two types, designated class I and class II.
  • class II molecules are located on antigen presenting cells (APCs), such as macrophages or dendritic cells, and present peptides from exogenously synthesized protein, usually the breakdown products from phagocytosed bacteria or protozoa. Thus, class II molecules primarily function to warn the immune system of extracellular invaders.
  • the class II/peptide complex is recognized by and interacts with TCRs of CD4+ T cells (T helper or Th cells).
  • Class I molecules are located on platelets and all nucleated cells of the body and bind peptides derived from endogenously synthesized proteins.
  • class I molecules examples include both "self” proteins, such as those normally produced in healthy cells, and “foreign” proteins, such as the viral proteins in virus-infected cells or the abnormal proteins produced in tumor cells.
  • class I molecules primarily function to report to the immune system the health of the intracellular environment.
  • the TCRs of CD8+ cytotoxic T lymphocytes (CTLs) interact with the MHC class I/peptide complex.
  • class I molecules are heterodimers comprised of two noncovalently bound polypeptide chains, a larger "heavy” chain ( ⁇ ) and a smaller “light” chain ( ⁇ -2-microglobulin, or ⁇ 2 m).
  • the two outermost extracellular domains, a- ⁇ and ⁇ 2 together form the groove that binds antigenic peptide (FIG. 37).
  • interaction with the TCR occurs at this region of the protein.
  • the ⁇ 3 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.
  • the C-terminal intracellular and transmembrane domains of the heavy chain are not amplified by PCR and thus deleted, resulting in a functional MHC/peptide complex that is secreted from the cell and still capable of interaction with the TCR.
  • the heavy chain of the class I heterodimer is cotranslationally inserted into the lumen of the ER (FIG. 38).
  • the extracellular (intralumenal) domains of newly synthesized ⁇ chains are glycosylated and become immediately associated with the ER chaperone protein calnexin.
  • Calnexin is a membrane-bound molecule that functions to temporarily keep the heavy chain in a partially folded state.
  • the subsequent noncovalent interaction of free ⁇ 2 m with the heavy chain causes the release of calnexin, and the heavy chain/ ⁇ 2 m complex becomes sequentially associated with the chaperones calreticulin and tapasin.
  • These chaperones position the class I heterodimer in a manner that enables it to be loaded with the processed peptides once they are transported into the ER by the TAP (transporter associated with antigen processing) complex, located within the ER membrane.
  • proteosome functions to prepare the endogenously synthesized proteins for presentation by MHC class I molecules.
  • proteins viral or otherwise
  • the TAP complex actively transports the peptides into the ER where the MHC heterodimer awaits.
  • peptides are further trimmed to lengths of 8-11 amino acids.
  • a peptide will be loaded into the binding cleft of the class I heavy chain to form the MHC heavy chain/light chain/peptide heterotrimeric complex, which is subsequently routed to the cell surface via the Golgi apparatus (FIG. 39).
  • T2 TAP- deficient and therefore has no cell-surface class I expression
  • T2 cells the system of mammalian expression utilized in the experiments herein, are capable of cell surface MHC class I expression when pulsed with exogenous peptide of sufficient affinity for the particular class I allele.
  • the interaction of the MHC/peptide complex with the TCR is the central event in the initiation of most antigen-specific immune responses.
  • the TCR recognizes the antigen complex by forming intermolecular contacts with both the class I molecule and the antigenic peptide.
  • the outcome of this interaction i.e. whether or not an immune response is generated) is dependent upon the density and duration of the TCR-MHC/peptide binding.
  • an immunogenic MHC/peptide complex will bind the TCR with greater avidity than a non- immunogenic MHC antigen complex.
  • a monomeric MHC/peptide complex even if immunogenic, dissociates rapidly from a TCR, indicating that multiple TCRs must interact with multiple MHC/peptide complexes in order to activate T cells.
  • Altman et al. disclosed a suggested methodology to analyze antigen-specific T cell populations by multimerizing the MHC/peptide complex into tetramers.
  • a tetramer consists of four biotinylated MHC/peptide complexes non-covalently bound to an avidin molecule (FIG. 40).
  • tetramers Compared to a monomeric MHC/peptide complex, tetramers have slower rates of dissociation from CTLs since they are able to bind more than one TCR on that particular CTL. This unique characteristic makes tetramers very useful as immunological stains.
  • MHC/peptide complexes synthesized in mammalian cells specifically cells of the antigen-processing mutant cell line T2 so that every MHC/peptide complex secreted would contain the same antigenic peptide. It is desirable to reflect as much as possible the actual in vivo interaction between human cells expressing cell-surface MHC/peptide complexes and cytotoxic T lymphocytes.
  • oligonucleotide primers designated A, B, and C, were purchased from commercially available sources (i.e. operon):
  • Primer A a 5' primer:
  • Primer B a 3' primer:
  • Primer C a 3' primer:
  • Primers A and B were used in PCR #1 and the template cDNA was a recombinant sHLA-B*0702 truncated gene with a 6-histidine tail in pcDNA3.1(- ), a mammalian expression vector.
  • PCR #1 was designed to incorporate 5'Xho I (CTC GAG) and 3' Hind III (AAG CTT) cut sites and to amplify the truncated B*0702 heavy chain lacking a 3' stop codon.
  • PCR reactions for this project were performed with a proofreading taq polymerase (PFU Polymerase, Promega) and were subjected to 25 cycles of 95°C for 1 minute (strand separation), 59°C for 1 minute (primer annealing), and 72°C for 2 minutes (DNA synthesis), followed by a final extension time of 7 minutes at 72°C.
  • the product of PCR #1 was purified using a QIAquick PCR purification kit (QIAGEN) and was subsequently digested for 2 hr at 37°C with Xho I and Hind III.
  • a bacterial expression vector C-Terminal Biotin AviTag Vector, Avidity; catalog number pAC-6) (FIG.
  • the B*0702t-no stop/ AviTag vector DNA was isolated and prepared using a DNA Miniprep kit (Promega) and served as the template cDNA for PCR #2. Primers A and C were used in PCR #2, which was designed to maintain a 5'Xho I cut site, incorporate a 3' EcoR I cut site (GAA TTC) distal to the bsp, and amplify the B*0702t gene with the bsp on its 3' end.
  • the PCR product was purified with the QIAquick purification kit and, along with the mammalian expression vector pcDNA3.1(-), was digested with Xho I and EcoR I restriction enzymes.
  • Digest products were gel purified (Freeze N Squeeze), ligated together, and transformed into competent JMl ⁇ 9 E. coli cells, which were then incubated overnight at 37°C on LB/ampicillin agar. Colony PCR was performed to check selected colonies for insert of the B*0702t-bsp gene into pcDNA(-). DNA from clones with insert was prepared (Miniprep kit) and sequenced using cycle sequencing. Sequences were analyzed and a good clone was identified. The plasmid DNA of the good clone was isolated and prepared for transfection using a DNA Midiprep kit (QIAGEN).
  • T2 Cells of the human cell line T2 were cultured in RPMI 1640 media + 20% fetal calf serum, 1% penicillin/streptomycin, and 0.25% phenol red. A total of 1.7 X 10 7 T2 cells were transfected with 30 ⁇ g of B*0702t-bsp/pcDNA3.1(-) DNA by electroporation using the Gene Pulser (BioRad) at 0.25 V and 960 ⁇ FD. Transfected cells were selected in a medium containing 40% RPMI 1640, 40% conditioned media, 20% fetal calf serum, 2% penicillin/streptomycin, 0.2% phenol red, and 1.5 mg/mL G418 neomycin (Cellgro).
  • Surviving cells were pulsed with the synthetic HIV GAG peptide NH 2 -S-P-R-T-L-N-A-W-V-COOH at 20 ug/mL and then incubated for 24 hours at 37°C. Transfectants were then screened by ELISA using W6/32 (8 ⁇ g/mL), which is directed against the entire HLA heavy chain/light chain ( ⁇ 2 m)/peptide complex, as the primary (capture) antibody and anti- ⁇ 2 m conjugated to HRP (diluted 1: 1000) as the secondary (conjugate) antibody (Dako).
  • High-producing cells were then expanded and cultured (continuously being repulsed with peptide), and their supernatant was collected and centrifuged to remove cell debris.
  • the specific MHC/peptide complexes were purified using an affinity chromatography system (AKTATM prime). In brief, the supernatant was passed over a 38 mL bed volume XK 26 column of cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech) coupled to W6/32. The protein was then eluted intact in basic buffer (0.1 M glycine-NaOH, pH 11.0).
  • the purified sHLA-bsp/peptide complex was enzymatically biotinylated on a single lysine residue within the bsp by incubation with BirA (Avidity; other names for this enzyme include biotin protein ligase, biotin ligase, biotin operon repressor protein, biotin holoenzyme synthetase, and biotin-[acetyl-CoA carboxylase] synthetase) (FIG. 42).
  • Incubation time was varied under constant conditions to determine the time required for maximum biotinylation efficiency. Four identical reactions were incubated for either 1, 4, 8, or 16 hours at 37°C.
  • Biotinylation mixture components were as follows: sHLA-bsp, 6 ⁇ M; MgOAc (Biomix B, Avidity), 1.9 mM; adenosine triphosphate (Biomix B, Avidity), 1.9 mM; BirA, 0.8 ⁇ M; TrisHCl, pH 8.0, 9.4 mM; biotin (Biomix B, Avidity), 10 ⁇ M; Pefabloc SC Plus (Roche), a protease inhibitor, 0.42 mM.
  • Biotinylation was confirmed using a modified ELISA, with W6/32 (8 ⁇ g/mL) as the capture antibody and ABC Vectastain (Vector Laboratories), a kit containing avidin and biotinylated HRP, as the conjugate (FIG. 43).
  • Biotinylated sHLA was separated from free biotin by applying the biotinylation reaction product to a Bio-Spin chromatography column (BioRad, Bio-Spin 30 Tris columns). Tetrameric sHLA/peptide complexes were produced by the stepwise addition (l/10 th volume, waiting 10 minutes between each addition) of the conjugate UltraAvidin-R-phycoerythrin (UltraAvidin-R-PE; Leinco Technologies) to the biotinylated class I complex.
  • UltraAvidin-R-phycoerythrin UltraAvidin-R-PE; Leinco Technologies
  • the final mixture contained a 1:4 molar ratio of UltraAvidin-R-PE: biotinylated class I, which is the same as a 1:1 molar ratio UltraAvidin-R-PE biotin binding sites: biotinylated class I.
  • the volumes of avidin and biotinylated class I were determined by calculating the molar concentrations of each (in moles/ ⁇ L) and then making certain that the number of moles of biotin binding sites in the avidin equaled the number of moles of biotinylated class I.
  • Tetramers were purified by gel filtration on a Superdex S-200 column (Pharmacia; Molecular Biology Resource Facility, University of Oklahoma Health Sciences Center).
  • the construct successfully created using the AviTag bsp vector contained a 7-residue sequence (W-K-L-P-A-G-G) between the truncated B7 heavy chain at its C-terminus and the bsp.
  • W-K-L-P-A-G-G 7-residue sequence
  • the peptide binding groove is at the N-terminus of the heavy chain, it was undetermined at the time of transfection whether this 7-residue linker would in any way affect MHC protein folding or peptide binding capability. Because class I molecules must be properly folded and loaded with peptide before being directed to the cell surface, only the specific MHC/peptide complexes should be in the supernatant.
  • the W6/32 monoclonal capture antibody of the ELISA positively identified the presence of MHC-bsp/peptide complexes secreted into the supernatant by producing transfectants (FIG. 44).
  • the 7 residue linker did not alter protein folding or peptide loading.
  • the bsp addition to the molecule perhaps affected column loading, as only ⁇ 50% recovery of the purified protein was achieved when pre-column and post-column protein amounts were compared. A large percentage of this "lost" protein was detected both in the wash, indicating nonspecific binding to the column during loading, and in the flow-thru, possibly indicating that the additional 22 amino acids (15 amino acids in the bsp and 7 amino acids linking the bsp to the class I, discussed above) on the C-terminus of the molecule interfered with loading.
  • FIG. 46 demonstrates that increasing the incubation time of the biotinylation reaction, with all other variables unchanged, allows for greater , biotinylation.
  • tetramers Upon purification of the tetramers via gel filtration (Superdex-200), tetramers (MW 498.6 kDa) were separated on the basis of size from any other possibly-present molecule contained in the reaction mixture [free avidin-PE conjugate (300 kDa— -avidin 60 kDa and Phycoerythrin 240 kDa); free biotinylated class I (49.65 kDa); unbiotinylated class I-bsp, since biotinylation efficiency was not likely 100% (49.4 kDa); free biotin, despite the purification step (243 Daltons); monomers (349.65 kDa); dimers (399.3 kDa); or trimers (448.94 kDa)].
  • tetramers as reagents to stain T cells specific to an epitope is widely known and utilized. Additionally, the use of tetramers in vaccine development and immune modulation is a reality in present biomedical research.
  • Several potential applications of tetramers in modulating the human immune system include tolerance induction in autoimmune diseases and adoptive transfer of antigen-specific T cells in the clinic. If the specific epitope causing the immune response in an autoimmune disease is known, a tetrameric complex specific to the patient's HLA type and containing the immunogenic epitope could be made and, in theory, given therapeutically to the patient in a dose large enough to induce tolerance.
  • Liposomes which are artificial antigen presenting cells that can be made and have incorporated in their membranes specific MHC/peptide complexes, are a cousin to the tetramer and would likely be used, rather than tetramers, for this type of immune modulation.
  • Adoptive transfer of T cells specific to an antigen of a disease-causing agent could be achieved by using tetramers (specific to the patient and the antigen) to sort live T cells, culturing these T cells in vitro to increase their numbers, and then transferring the cells back into the patient in hopes of enhancing the patient's immune response.
  • a cell line deficient in peptide processing but still efficient in peptide loading may be utilized for both epitope and functional testing, so that a putative epitope can be expressed or pulsed into a cell and loaded into the HLA molecule in the ER of such cell.
  • the cDNA construct isolated as described above may be ligated into a mammalian expression vector which also contains a DNA fragment encoding a peptide of interest attached to a fragment encoding a signal peptide so that the peptide of interest will be retained in the ER of the cell for loading, and such construct transfected into the mammalian cell line deficient in peptide processing but which retains the ability to load peptide in the HLA molecules, such as the T2 cell line.
  • the peptide of interest is produced together with the HLA molecule.
  • the soluble HLA molecule (with or without a His or biotinylation signal tail) can then be purified and utilized as a reagent that has been produced in mammalian cells (fully glycosylated, etc.) and is loaded with the single co-transfected peptide.
  • random oligomers could be made and cloned into such a mammalian expression vector, and the soluble HLA molecules could again be purified and used to characterize T cells or other immune effector cells.
  • the cells expressing the HLA molecule could be pulsed with a single synthetic peptide or multiple synthetic peptides and analyzed as described above to identify bound peptides.
  • Any of the HLA molecule-peptide complexes could be multimerized to form dimers, tetramers, etc. and tested for their ability to serve as ligands for CTLs and induce immune responses in humans.
  • the method of the present invention involves production of MHC class I and class II molecules beginning from gDNA and/or cDNA.
  • the gDNA clones encoding a given MHC molecule can be truncated to be secreted rather than bound at the cell surface.
  • This truncated version of the MHC molecule can be produced in mammalian or insect/bacterial cells such that milligram or greater quantities of an individual class I or class II molecule can be obtained.
  • the secreted MHC class I molecules can be naturally loaded with thousands of endogenous peptides in mammalian cells, while the secreted MHC class II molecules can be naturally loaded with thousands of endocytic peptides in mammalian cells.
  • the secreted MHC proteins can be produced in cells that do not load the MHC molecule with peptide ligand. Production of MHC proteins in cells which do not load the MHC molecule with peptide facilitates the loading of the MHC molecule via co-transfection with constructs encoding a given peptide(s).
  • the MHC peptide-loading deficient MHC transfectant can be pulsed with peptides or DNA encoding peptides. The resulting individual secreted MHC molecules are useful for studies of peptide loading (i.e. in vaccine development), for characterizing human immune responses to a given MHC molecule loaded with a particular peptide(s), and to the development of diagnostics where one needs sufficient MHC protein in order to directly assess reactivity to different MHC proteins.
  • Another important component of the secreted MHC molecules described here is that naturally loaded peptides can be eluted from the MHC molecules and characterized. Substantial quantities of peptide can be obtained from individual MHC molecules, and the peptides can be selectively characterized. Unique information results from having a sufficient supply of eluted peptide, and this information is essential to databases and predictive algorithms which are essential to the vaccine architect.
  • 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.
  • HLA5UT PCR (5'; inserts Sail site) GGGCG ⁇ ££ ⁇ CGGACTCAGAATCTCCCCAGACGCCGAG
  • SHLA3TM PCR (3'; inserts stop codon and H dIII site) CCGC ⁇ ClirCATCTCAGGGTGAG
  • 5PXI PCR (5'; inserts Xbal site) GGGCI-CIAGAGGACTCAGAATCTCCCCAGACGCCGAG
  • CD CO Ml 3 universal sequencing (m l8, end through ⁇ 3 ) TGTAAAACGACGGCCAGT (m l9, leader through 2 )
  • T7 promoter sequencing (T7 promoter forward priming site) TAATACGACTCACTATAGGG pcDNA3.1/BGH sequencing (BG ⁇ reverse priming site) TAGAAGGCACAGTCGAGG
  • NQFQALLQY polypyrimidine tract-binding protein (220- B*1512
  • FVSNHAY aldolase A (358-364) B*1501, 1508 I GPPGSVY ubiquitin-protein ligase (83-91) B*1501, B*1502, 1508,
  • YMIDPSGVSY proteasome subunit C8 (150-159) B*1501, B*1502, 1508,
  • WAPITTGY calcyclin binding protein (63-71) B*1501, 1508 GHSPPTSSL tyrosine-protein kinase JAK3 (491-499) B*1510 LPPPPPPPP Fas antigen ligand (54-62) B*1503 NHANGLTL serine/threonine protein phosphatase PP2A ( ⁇ B*1510 and ⁇ ) (229-236)
  • EHVASSPAL 13S Golgi transport complex 90 kD subunit B*1510
  • NMNDLVSEY tubulin ⁇ chain (414-422) B*1508 THTQPGVQL septin 2 homolog (70-78) B*1509, B*1510 SHA SAWL ⁇ -adaptin (249-257) B*1509, B*1510
  • GQYPTQPTY KIAA0058 5-13; like Mus muluscus proline- B*1503 rich protein
  • PLEKQLFYY (125-133) MLSAPLEKQLF (121-131) APLEKQLFY (124-132)
  • ALSINGDKF 159-167)
  • PLEKQLFY 125-132
  • NTRPHSYVF 140-148)
  • TMFEVSVAF (290-298) YVALSINGDKF (157-167) LTSAQSGDY (216-224)
  • DLRW AKSF (314-322) FQYTGAMTSKF (167-177) YSLVIVTTF (224-232)
  • HLTTEKQEY (366-374) AMTSKFLMGTY (172-182) VIVTTFVHY (227-235)
  • AVSNAVDGF (505-513)
  • SLVIVTTF 225-232
  • DTETLTTMF (284-292)
  • ALYE S TY (564-572) LVIVTTF (226-232) ATVKGMQSY (338-346)
  • HW AII Y (679-687) FVHYANFHNFY (232-242) ESGLFSPCY (471-479)
  • WLAIILYF (680-688) TMTAASY (255-261) S C LSLRF (476-484)
  • VHKIVMF (696-704) TMTAASYARY 255-264) IIPLINVTF (544-552) VHKIVMFF (697-705) ELDTETLTTMF 282-292) TTYLSSSLF (570-578)
  • VLAIILY ( 681-687)
  • VLAIILYF ( 681-688)
  • VLAIILYFIAF ( 681-691)
  • ⁇ X s ⁇ M ⁇ is t. i i I ⁇ ⁇ ! ⁇ i X ! ⁇ ⁇ . X M , - ⁇ N 'ti N ?- S-.IM > X Jd ⁇ ⁇ : • ⁇ - --
  • MHC class I genes of the channel catfish sequence analysis and expression. Immunogenetics 49: 303-311.
  • HLA class I-restricted human cytotoxic T-cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen. Proc. Natl. Acad. Sci. USA 88: 10445-10449.
  • HLA and HIV-1 heterozygote advantage and B*35-Cw*04 disadvantage. Science 283: 1748- 1752.
  • HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides. J. Exp. Med. 185: 367-371.
  • HLA-B14 peptide binding site can accommodate peptides with different combinations of anchor residues. J. Biol. Chem. 269: 32426-32434.
  • proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7. Proc. Natl. Acad. Sci. USA 91: 9213-9217.
  • HLA-Aw68 Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle. Nature 360: 364-366.
  • HLA- A2.1- associated peptides from a mutant cell line a second pathway of antigen presentation. Science 255: 1264-1266.
  • HLA-B15 a widespread and diverse family of HLA-B alleles. Tissue Antigens 43: 209-218.
  • Serum angiotensin-1 converting enzyme activity processes a human immunodeficiency virus 1 gpl60 peptide for presentation by major histocompatibility complex class I molecules. J. Exp. Med. 175: 1417-1422.
  • Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference. Immunogenetics 47: 256-263.
  • Cytotoxic T-cells specific for a single peptide on the M2 protein of respiratory syncytial virus are the sole mediators of resistance induced by immunization with M2 encoded by a recombinant vaccinia virus. J. Virol. 69: 1261-1264.
  • HLA-A*0201 presents TAP-dependent peptide epitopes to cytotoxic T lymphocytes in the absence of tapasin. Eur. J. Immunol. 28: 3214-3220.
  • Proteasomes can either generate or destroy MHC class I epitopes: evidence for nonproteasomal epitope generation in the cytosol. J. Immunol. 161 : 112-121.
  • HLA-B27 reveals nonamer self-peptides bound in an extended conformation. Nature 353: 321-325. Madden, D.R., J.C. Gorga, J.L. Strominger and D.C. Wiley (1992). The three- dimensional structure of HLA-B27 at 2.lA resolution suggests a general mechanism for tight peptide binding to MHC. Cell 70: 1035-1048.

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Abstract

L'invention concerne de manière générale au moins un procédé et un dispositif de production d'antigènes solubles du CMH, et plus particulièrement, mais pas exclusivement, un procédé et un dispositif de production de molécules solubles des classes I et II du système HLA. L'invention comprend aussi les molécules des classes I et II du système HLA ainsi produites et leur utilisation. Selon les procédés de l'invention, les molécules des classes I et II du système HLA peuvent être produites à partir de matière première d'ADNg (ADN génomique) ou d'ADNc.
EP01994366A 2000-12-18 2001-12-18 Procede et dispositif de production d'antigenes et utilisation de ceux-ci Withdrawn EP1353950A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US25641000P 2000-12-18 2000-12-18
US25640900P 2000-12-18 2000-12-18
US256409P 2000-12-18
US256410P 2000-12-18
US974366 2001-10-10
US09/974,366 US7541429B2 (en) 2000-10-10 2001-10-10 Comparative ligand mapping from MHC positive cells
PCT/US2001/049744 WO2002062846A2 (fr) 1999-12-17 2001-12-18 Procede et dispositif de production d'antigenes et utilisation de ceux-ci

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EP1353950A2 true EP1353950A2 (fr) 2003-10-22

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EP (1) EP1353950A2 (fr)
CA (1) CA2438376A1 (fr)
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6867283B2 (en) 2001-05-16 2005-03-15 Technion Research & Development Foundation Ltd. Peptides capable of binding to MHC molecules, cells presenting such peptides, and pharmaceutical compositions comprising such peptides and/or cells

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US6001365A (en) * 1992-02-19 1999-12-14 The Scripps Research Institute In vitro activation of cytotoxic T cells
EP0726777A4 (fr) * 1993-10-25 2002-04-24 Anergen Inc Expression procaryote de proteines du cmh
NZ333915A (en) * 1996-08-16 2000-11-24 Harvard College Soluble monovalent and multivalent MHC class II fusion proteins

Non-Patent Citations (1)

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Title
See references of WO02062846A2 *

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IL156473A0 (en) 2004-01-04
CA2438376A1 (fr) 2002-08-15
WO2002062846A2 (fr) 2002-08-15
WO2002062846A3 (fr) 2003-07-24

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