WO2007032778A2 - Analyse des interactions entre liaisons au peptide mhc - Google Patents

Analyse des interactions entre liaisons au peptide mhc Download PDF

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Publication number
WO2007032778A2
WO2007032778A2 PCT/US2006/010809 US2006010809W WO2007032778A2 WO 2007032778 A2 WO2007032778 A2 WO 2007032778A2 US 2006010809 W US2006010809 W US 2006010809W WO 2007032778 A2 WO2007032778 A2 WO 2007032778A2
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mhc
proteins
binding
peptide
drb1
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PCT/US2006/010809
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WO2007032778A3 (fr
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Umesh S. Muchhal
John R. Desjarlais
Gregory L. Moore
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Xencor, Inc.
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Priority claimed from US11/226,928 external-priority patent/US20060084116A1/en
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Publication of WO2007032778A2 publication Critical patent/WO2007032778A2/fr
Publication of WO2007032778A3 publication Critical patent/WO2007032778A3/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
    • G01N33/541—Double or second antibody, i.e. precipitating antibody
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • Protein arrays also known as bioarrays
  • bioarrays used to study the binding between MHC proteins and peptides are described herein.
  • [ooo2] lmmunogenicity is a complex series of responses to a substance that is perceived as foreign and may include production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity responses, and anaphylaxis.
  • Properly modulating the immunogenicity of proteins may greatly improve the safety and efficacy of protein vaccines and protein therapeutics.
  • methods to predict the immunogenicity of novel engineered proteins will be critical for the development and clinical use of designed protein therapeutics.
  • the goal is typically to promote, in a large fraction of patients, a robust T cell or B cell-based immune response to a pathogen, cancer, toxin, or the like.
  • unwanted immunogenicity can reduce drug efficacy and lead to dangerous side effects. Immunogenicity has been clinically observed for most protein therapeutics, including drugs with entirely human sequence content.
  • APCs antigen presenting cells
  • T cells express MHC proteins that bind MHC agretopes, or peptides.
  • T cells express T-cell receptors (TCRs) that recognize T-cell epitopes in the context of peptide-MHC proteins
  • B cells express MHC molecules and B-cell receptors (BCRs) that recognize B-cell epitopes.
  • uptake by APCs is promoted by binding to any of a number of receptors on the surface of APCs.
  • particulate protein antigens may be more immunogenic than soluble protein antigens.
  • Immunogenicity may be dramatically reduced by blocking any of these recognition events. Similarly, immunogenicity may be enhanced by promoting these recognition events.
  • Several factors can contribute to protein immunogenicity, including but not limited to the protein sequence, the route and frequency of administration, and the patient population. Accordingly, modifying these and other factors may serve to modulate protein immunogenicity. Interaction of proteins and their processed peptides with the surface expressed MHC molecules is generally the first determinant in their ability to induce a immune response, and an analysis of this interaction could be used as a predictive and diagnostic tool to assess the "immunogenicity" of a protein.
  • Methods, apparatuses, and compounds for screening or detecting binding of candidate peptides to an MHC protein is provided.
  • a first component including at least one candidate peptides and a second component including at least one MHC protein are contacted.
  • One of the components is immobilized on a solid support. The presence, absence, or quantity of binding of the peptide and said MHC protein is then determined.
  • the peptides are immobilized on the support to form an array.
  • the array is then exposed to one or more MHC proteins.
  • the array of peptides is exposed to a library of MHC proteins.
  • At least one MHC protein is immobilized on the support to form an array.
  • the array is then exposed to one or more peptides.
  • the array of MHC proteins is exposed to a library of peptides.
  • the MHC protein or peptide can be labeled, such as with a fluorophore or fluorescent protein.
  • an MHC protein can include a label and an MHC protein.
  • a peptide can include the amino acid sequence of the peptide and a fluorophore.
  • the MHC protein or peptide can also be exposed to a secondary label, such as an epitope tag.
  • an MHC protein can include an attachment linker and an MHC protein.
  • the MHC proteins are selected from certain groups of alleles, or alleles that have certain levels of population coverage of alleles.
  • the MHC DR1 is expressed at a level of population coverage of at least 60%, MHC DR3/4/5 at a level of population coverage of at least 40%, MHC DP at a level of population coverage of at least 40%, and MHC DQ at a level of population coverage of at least 20%.
  • Other groups of alleles and population coverages are disclosed herein.
  • [ooii] Alternatively, methods of screening for binding of a first set of candidate peptides to a plurality of MHC proteins by calculating binding scores are provided.
  • an amino acid sequences of the first set of candidate peptides are input into a computer.
  • a scoring matrixgenerated by contacting a first component comprising a second set of candidate peptides with a second component comprising a plurality of MHC proteins and determining the degree of binding of said second set of candidate peptides and said plurality of MHC proteins is then input into the computer.
  • the binding score is then calculated based on the amino acid sequences and the scoring matrix.
  • the binding score is calculated using a score for at least nine (9) MHC pockets.
  • the present methods, apparatuses, and related compositions provided herein have a variety of uses. These uses include specific subsets of MHC proteins that are particularly representative of specific subsets of the human population are also provided.
  • the present methods, apparatuses, and related compositions provided herein also may be used to identify the agretopes in proteins that are responsible for immunogenicity based on MHC binding propensities.
  • the invention also teaches methods for the efficient production of large number of MHC-II constructs required for the aforementioned analysis.
  • FIG. 1 shows a schematic an embodiment of the MHC proteins of the present invention.
  • FIG. 2 shows a schematic of several embodiments of MHC proteins of the present invention.
  • Figure 3 is a picture of SDS gels of recombinant MHC ⁇ -subunit and ⁇ -submit expressed in insect cells.
  • HighFive® cells (2 x 106 in 2 ml) were transfected with 5 ug each of a & ⁇ subunit expression constructs (driven by constitutive promoter plE1).
  • Figure 4 is a picture of SDS gels of recombinant MHC ⁇ -subunit and /?-submit expressed in mammalian cells.
  • 293TTM cells (2 x 105 in 20 ml) were transfected with 20 ug each of a & ⁇ subunit expression constructs (driven by constitutive promoter pCMV).
  • Figure 5 shows a schematic of a scale-up of MHC expression and a picture of a SDS gel of a DR4 MHC with two different transfection reagents.
  • FIG. 6 shows a schematic of the purification of recombinant MHC proteins.
  • the MHC proteins may be purified using a modular purification protocol that yields >50% pure and concentrated preparation. This is stable and directly usable in binding assays.
  • This coomassie blue stained SDS-PAGE gel shows the purified MHCs of DR class showing the two bands representing a and ⁇ subunits for each.
  • Figure 7 shows a picture of an SDS gel of expression of multiple DRs using the modular constructs in insect cells. The yields are very comparable to DR4 example for ⁇ 80% of the DRs tested. The supernatants from cells transfected with listed DR constructs were analyzed by western blotting using a mixture of anti-his and anti-flag antibodies (two bands representing the tagged a and ⁇ subunits).
  • Figure 8 shows a diagraph of an MHC-peptide binding assay.
  • Figure 9 shows a graph of a peptide-binding assay showing the specific and competitive binding of biotinylated HA peptide to recombinant DR4 and DR1.
  • Various concentrations (25 to 400 nM) of two different batches of DR4 and DR1 were incubated with 400 nM of biotin-HA peptide with or without 10 fold molar excess of unlabelled HA peptide (C) in a 50 ul reaction volume.
  • the MHC bound bHA peptide was quantitated using Eu- streptavidin time resolved fluorescence assay.
  • Figure 10 is a schematic of a method for testing a therapeutic protein on an MHC bioarray of the present invention.
  • Figure 11 shows a schematic of a method for testing an array of peptides.
  • Figure 12 shows DR3/4/5 allele prevalences obtained from the National Marrow Donor Program.
  • Figure 13 shows DP allele prevalences obtained from the National Marrow Donor Program.
  • Figure 14 shows DQ allele prevalences obtained from the National Marrow Donor Program.
  • Figure 15 shows DR1 U.S. Hispanic allele prevalences obtained from the National Marrow Donor Program.
  • Figure 16 shows the MHC-peptide binding scoring matrix determined for DRB1*0401.
  • Figure 17 shows the MHC-peptide binding scoring matrix determined for DRB1*0701.
  • Figure 18 shows a scatterplot of calculated binding scores versus reported IC50 values for DRB1*0401 reported data.
  • Figure 19 shows a scatterplot of calculated binding scores versus reported IC50 values for DRB1*0701 reported data.
  • Figure 20 shows a receiver-operator curve for calculated binding scores versus reported IC50 values for DRB1*0401 reported data.
  • Figure 21 shows a receiver-operator curve for calculated binding scores versus reported IC50 values for DRB1*0701 reported data.
  • Methods, apparatuses, and compounds for screening or detecting binding of candidate peptides to an MHC protein is provided.
  • a first component including at least one candidate peptides and a second component including at least one MHC protein are contacted.
  • One of the components is immobilized on a solid support. The presence, absence, or quantity of binding of the peptide and said MHC protein is then determined.
  • the methods provide for the rapid and facile creation of
  • MHC protein bioarrays that may be used in a wide variety of methods and techniques.
  • the MHC proteins can be immobilized on the surface.
  • These MHC bioarrays may then be used in a wide variety of ways, including diagnosis (e.g. detecting the presence of specific peptides or agretopes), and screening (e.g. looking for target analytes that bind to specific proteins or detecting immunogenicity).
  • the present methods allow the rapid and facile creation of peptide bioarrays that may be used in a wide variety of methods and techniques.
  • the MHC protein targets that bind the peptide may be "captured" on the bioarray.
  • the present methods allow for competition between a peptide bound in an MHC molecule and a second, free peptide. Either the bound peptide or free peptide can be labeled.
  • the set of MHC proteins assembled in array format is particularly representative of a population of subjects who may be treated with the therapeutic protein of interest.
  • a set of MHC proteins would be those that are found most frequently within the general population (or as a good proxy, those found within the general US population) can be used.
  • the intended patient population for a particular therapeutic protein may possess certain MHC alleles more frequently than others.
  • populations can include a specific disease population (e.g. it is well established that the class Il MHC allele DRB1*1501 is frequently possessed by patients with multiple sclerosis) or a particularly ethnicity that is predisposed to a disease for genetic or geographical reasons.
  • MHC allele frequencies can either be determined directly by genotyping the patients, or by using existing data regarding the prevalence of MHC alleles within that population. The MHC alleles with the highest frequencies would then be produced and displayed in a readable array. Peptides representative of the protein sequence would then be analyzed for interaction with the arrayed MHC proteins in order to determine the presence of potential MHC agretopes within the protein. In a preferred embodiment, MHC alleles that have higher than 5% frequency within a target population will be arrayed. In alternative embodiments, the array size itself will determine the number of alleles - i.e. if the array holds 96 elements, the 96 highest frequency alleles from the target population could be arrayed.
  • the choice of arrayed MHC proteins would be influenced by the knowledge that a peptide from the protein does indeed interact with one or more of the MHC proteins. That MHC protein and related MHC proteins expected to have similar peptide binding preferences would then be assembled in array format for evaluating the offending peptide and variants thereof (e.g. variants designed to remove the ability to interact with MHC molecules).
  • MHC proteins selected for arrayed format will be a combination of high frequency alleles in a specific target population and high frequency alleles in the general population or a combination of high frequency alleles in a specific target population and alleles expected to interact with peptides within the therapeutic protein.
  • MHC proteins generally come in two separate classes designated class I and class II.
  • the molecules are generally designated by antigenic subtype.
  • Human MHC class I molecules also referred to as human leukocyte antigens (HLA) are designated HLA-A, -B, and -C.
  • Human MHC class Il molecules are designated HLA-DR, -DQ, and -DP.
  • MHC class I molecules are found on almost every nucleated cell of the body. MHC class I molecules are heterodimers that have a single transmembrane polypeptide chain (the (7-chain) and a ⁇ 2 microglobulin. The a chain has two polymorphic domains, a ⁇ a 2 , which binds peptides derived from cytosolic proteins. Because MHC class I molecules present peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often called the cytosolic or endogenous pathway.
  • MHC class I molecules are loaded with peptides generated in the cytosol. As viruses infect a cell by entering its cytoplasm, this cytosolic, MHC class l-dependent pathway of antigen presentation is the primary way for a virus-infected cell to signal T cells. MHC class I molecules generally interact exclusively with CD8 + ("cytotoxic") T cells (CTLs). The fate of the virus-infected cell is almost always apoptosis initiated by the CTL, effectively reducing the risk of infecting neighboring cells.
  • CTLs CD8 +
  • MHC Class Il molecules are found only on a few specialized cell types, particularly antigen-presenting cells (APCs) such as macrophages, B cells, and T cells. Like MHC class I molecules, class Il molecules are also heterodimers, but in this case consist of two homologous peptides, an a and ⁇ chain. The peptides presented by class Il molecules are derived from extracellular proteins. MHC class Il molecules bind peptides in a groove between the a and ⁇ chains. Because the peptide-binding groove of MHC class Il molecules is open at both ends, the peptides presented by MHC class Il molecules are generally between 15-24 amino acid residues long. Class Il molecules interact exclusively with CD4 + (“helper") T cells (THS). The helper T cells then help to trigger an appropriate immune response.
  • helper T cells helper T cells
  • MHC protein means the portion of an MHC class I or class Il lacking the transmembrane portion of membrane bound MHC class I and class Il proteins. MHC proteins are further capable of functioning as a capture binding ligand immobilized on a solid surface. Likewise, the MHC proteins may function as a target molecule when in solution. MHC class I constructs include a binding pocket that is closed on both ends. The MHC class I constructs are capable of binding a peptide 8-9 amino acids in length. Similarly, MHC class Il constructs include a binding pocket that is open on both ends, and capable of binding peptides between, for example, 14 and 25 amino acids long.
  • MHC class I and Il molecules both bind peptides in their respective binding pockets.
  • a peptide derived from a processed antigen is referred to as an "agretope.”
  • Peptides corresponding to agretopes can be screened according to the methods disclosed herein.
  • candidate peptide or grammatical equivalents herein is meant a peptide that is added to the bioarray for testing its binding to the MHC proteins.
  • candidate peptides are proteins as defined above.
  • the candidate peptides are naturally occurring proteins or fragments of naturally occurring proteins.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
  • libraries of procaryotic and eukaryotic proteins may be made for screening in the systems described herein.
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • Candidate peptides include a peptide being tested for activity, e.g. binding to an MHC protein.
  • peptide herein is meant at least two covalently attached amino acids.
  • MHC class I peptides are 8 or 9 amino acids in length, but can vary to between 7 and 10 amino acids in length.
  • MHC class Il peptides can vary from 15 to 24 amino acids in length. Optionally, they can vary from 10 amino acids to 30 amino acids or more in length.
  • the peptide may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids.
  • homo- phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as proline and hydroxyproline.
  • the side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configu ration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
  • Peptide inhibitors of enzymes find particular use.
  • the candidate peptides are naturally occurring proteins or fragments of naturally occurring proteins.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
  • libraries of procaryotic and eucaryotic proteins may be made for screening in the systems described herein.
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • the candidate peptides can comprise randomized peptides, either fully randomized or they are biased in their randomization.
  • randomized or grammatical equivalents herein is meant that each peptide consists of at least a portion of essentially random amino acids, respectively.
  • the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc.
  • the peptide length can be biased towards peptides that interact with known classes of molecules, such as MHC proteins.
  • libraries can be generated that have homology to known MHC binding peptides.
  • library herein is meant a plurality of molecules.
  • the library provides a sufficiently structurally diverse population of peptides to effect a probabilistically sufficient range of cellular responses to provide one or more cells exhibiting a desired response. Accordingly, an interaction library must be large enough so that at least one of its members will have a structure that gives it affinity for some molecule, protein, or other factor whose activity is necessary for completion of the signaling pathway.
  • the peptides may be linked to a fusion partner, alternatively with primary labels.
  • the peptides can also be selected based on agretopes. Methods of identifying, adding or remove class I or class Il MHC agretopes have been described. For example, vaccines may be made that are more effective at inducing an immune response by inserting agretopes with increased affinity for MHC class I or class Il molecules (see for example, WO 9833523; Sarobe, P., et al. J. Clin. Invest, 102:1239-1248 (1998); Thimme, R., et al. J.
  • MHC agretopes for the purpose of decreasing protein immunogenicity has also been disclosed (for example WO 98/52976, WO 02/079232, WO 00/34317, and WO 02/069232, all incorporated herein by reference in its entirety).
  • Addition or removal of MHC agretopes is a tractable approach for immunogenicity modulation because the factors affecting binding are reasonably well defined, the diversity of binding sites is limited, and MHC molecules and their binding specificities are static throughout an individual's lifetime.
  • immunogenicity may significantly affect the safety and efficacy of protein therapeutics and protein vaccines, methods to evaluate the immunogenicity of designed proteins intended for use as drugs or vaccines would be useful.
  • Class I MHC-bindin ⁇ agretopes loose] Peptides can be used as either the capture binging ligand or target molecule.
  • Class I MHC proteins for example, primarily bind fragments of intracellular proteins that are derived from infecting viruses, intracellular parasites, or internal proteins of the cell; proteins that are overexpressed in cancer cells are of special interest.
  • the resulting peptide-MHC proteins are transported to the surface of the APC, where they may interact with T cells via TCRs. This is the first step in the activation of a cellular program that may lead to cytolysis of the APC, secretion of lymphokines by the T cell, or signaling to natural killer cells.
  • MHC class I molecules show preferential restriction to CD8+ cells, (for example, Fundamental Immunology, 4th edition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 8, pp 263-285), incorporated herein by reference in its entirety.
  • Class I MHC ligands are generally octa- or nonapeptides (also known as 8-mers or 9-mers); they bind a groove in the class I MHC structure framed by two ⁇ -helices and a ⁇ -pleated sheet. Specific pockets in the binding groove recognize subsets of residues in the peptide, called anchor residues; these interactions confer some sequence selectivity. Class I MHC molecules also interact with atoms in the peptide backbone. The orientation of the peptides is determined by conserved side chains of the MHC I protein that interact with the N- and C-terminal residues in the peptide.
  • Any of a number of methods may be used to identify potential class I MHC agretopes, including but not limited to the computational and experimental methods described below. Rules for identifying MHC I binding sites have been described in Altuvia, Y., et al (1997) Human Immunology, 58:1-11 ; Meister, GE, et al (1995) Vaccine: 6:581-591 ; Parker, K.C., et al., (1994) J. Immunology, 152:163; Gulukota, K., et al., (1997) J. MoI.
  • MHC binding peptide such as SYPEITHI and MHCPEP may also be used to identify potential MHC I binding sites (Rammensee, H- G., et al., (1999) Immunogenetics, 50:213-219; Brusic, V., et al., (1998) Nucleic Acids Research, 26:368-371 ), all incorporated herein by reference in its entirety.
  • MHC binding motifs include allele-specific polynomial algorithms described by Fikes, J., et al., WO 01/41788, neural net (Gulukota, K, supra), polynomial (Gulukota, K., supra) and rank ordering algorithms (Parker, K.C., supra), all incorporated herein by reference in its entirety.
  • Class Il MHC molecules which are related to class I MHC molecules, primarily present extracellular antigens. Relatively stable peptide-MHC proteins may be recognized by TCRs; this recognition event is required for the initiation of most antibody-based (humoral) immune responses. MHC class Il molecules show preferential restriction to CD4+ cells (Fundamental Immunology, 4th edition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 8, pp 263-285, incorporated herein by reference in its entirety).
  • peptide-binding groove may be subdivided into "pockets", commonly named P1 through P9, where each pocket comprises the set of MHC residues that interacts with a specific residue in the peptide. Between two and four of these positions typically act as anchor residues.
  • the non-anchoring amino acids play a secondary, but still significant role (Rammensee, H., et al., (1999) Immunogenetics, 50:213-219, incorporated herein by reference in its entirety).
  • a number of polymorphic residues face into the peptide-binding groove of the MHC molecule.
  • the identity of the residues lining each of the peptide-binding pockets of each MHC molecule determines its peptide binding specificity.
  • the sequence of a peptide determines its affinity for each MHC allele.
  • structure-based methods are used. For example, methods may be used in which a given peptide is computationally placed in the peptide-binding groove of a given MHC molecule and the interaction energy is determined (for example, see WO 98/59244 and WO 02/069232). Such methods may be referred to as "threading" methods.
  • purely experimental methods may be used. Examples of physical methods include high affinity binding assays (Hammer, J., et al. (1993) Proc. Natl. Acad. Sci. USA, 91:4456-4460; Sarobe, P. et al. (1998) J. Clin.
  • potential MHC Il binding sites are identified by matching a database of published motifs, such as SYFPEITHI (Rammensee, H., et al., (1999) Immunogenetics, 50:213-219; or MHCPEP (Brusic, B., et al., supra), both incorporated herein by reference in its entirety. Sequence-based rules for identifying MHC Il binding sites, including but not limited to matrix method calculations, have been described in Sturniolo, T, et al. Nat. Biotechnol., 17:555-561 (1999); Hammer, J. et al., Behring. Inst. Mitt., 94: 124-132 (1994); Hammer, J.
  • the matrix method is used to calculate MHC- binding propensity scores for each peptide of interest binding to each allele of interest.
  • the matrix comprises binding scores for specific amino acids interacting with the peptide binding pockets in different human class Il MHC molecule. It is possible to consider all of the residues in each 9-mer window; it is also possible to consider scores for only a subset of these residues, or to consider also the identities of the peptide residues before and after the 9-residue frame of interest.
  • the scores in the matrix may be obtained from experimental peptide binding studies, and, optionally, matrix scores may be extrapolated from experimentally characterized alleles to additional alleles with identical or similar residues lining that pocket.
  • Vatrices that are produced by extrapolation are referred to as "virtual matrices".
  • viral matrices See Sturniolo, T., Bono, E., Ding, J., Raddrizzani, L., Tuereci, O., Sahin, U., Braxenthaler, M., Gallazzi, F., Protti, M.P., Sinigaglia, F., and Hammer, J. (1999) "Generation of tissue-specific and promiscuous HLA ligand databases using DNA micro arrays and virtual HLA class Il matrices" Nat. Biotech., 17, 555-61 (1999), all incorporated herein by reference in its entirety.) Populations
  • the set of MHC proteins assembled in array format is particularly representative of a population of subjects who may be treated with the therapeutic protein of interest.
  • the set of MHC proteins would be those that are found most frequently within the general population (or as a good proxy, those found within the general US population).
  • the intended patient population for a particular therapeutic protein may possess certain MHC alleles more frequently than others.
  • populations can include a specific disease population (e.g., it is well established that the class Il MHC allele DRB1*1501 is frequently possessed by patients with multiple sclerosis) or a particular ethnicity that is predisposed to a disease for genetic or geographical reasons.
  • MHC allele frequencies can either be determined directly by genotyping the patients, or by using existing data regarding the prevalence of MHC alleles within that population. The MHC alleles with the highest frequencies would then be produced and displayed in a readable array. Peptides representative of the protein sequence would then be analyzed for interaction with the arrayed MHC proteins in order to determine the presence of potential MHC agretopes within the protein. In a preferred embodiment, MHC alleles that have higher than 5% frequency within a target population will be arrayed. In alternative embodiments, the array size itself will determine the number of alleles - i.e., if the array holds 96 elements, the 96 highest frequency alleles from the target population could be arrayed.
  • bioarrays of the present invention may be designed for specific populations of individuals. Populations may be based upon race, geographic area, sex, disease, etc. Examples of populations also include individuals with the following indications: arthritis, psoriatic arthritis, ankylosing spondylitis, spondyloarthritis, spondyloarthropathies, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, reactive arthritis (Reiter Syndrome) scleroderma, Sjogren's syndrome, keratoconjunctivitis, keratoconjunctivitis sicca, TNF-receptor associated periodic syndrome (TRAPS), periodic fever, periprosthetic osteolysis, apthous stomatitis, pyoderma gangrenosum, uveitis, reticulohistiocytosis, inflammatory bowel diseases, sepsis and septic shock, Cr
  • NMDP National Marrow Donor Program
  • NMDP National Marrow Donor Program
  • bioarrays of the present invention may be designed to contain MHC heterodimers comprising highly prevalent MHC alleles.
  • Class Il MHC alleles that are present in at least 10 % of the US population include but are not limited to: DPA1*0103, DPA1*0201 , DPB1*0201 , DPB1*0401 , DPB1*0402, DQA1*0101 , DQA1*0102, DQA1*0201 , DQA1*0501 , DQB1*0201 , DQB1*0202, DQB1*0301 , DQB1*0302, DQB1*0501 , DQB1*0602, DRA*0101 , DRB1*0701 , DRB1*1501 , DRB1*0301 , DRB1*0101 , DRB1*1101 , DRB1*1301 , DRB3 * 0101 , DRB3 * 020202, DRA*0101
  • bioarrays of the present invention may be designed to contain MHC heterodimers comprising moderately prevalent MHC alleles.
  • Class Il MHC alleles that are present in 1% to 10% of the US population include but are not limited to: DPA1*0104, DPA1*0302, DPA1*0301 , DPB1*0101 , DPB1*0202, DPB1*0301 , DPB1* 0501 , DPB1*0601 , DPB1 * 0901 , DPB1*1001 , DPB1*1101 , DPB1*1301 , DPB1*1401 , DPB1*1501 , DPB1*1701 , DPB1*1901 , DPB1*2001 , DQA1*0103, DQA1*0104, DQA1*0301 , DQA1*0302, DQA1 * 0401 , DQB1*0303, DQB1 * 0402, DQB
  • Bioarrays of the present invention may also be designed to contain MHC heterodimers comprising less prevalent alleles.
  • Information about MHC alleles in humans and other species can be obtained, for example, from the IMGT/HLA sequence database (ebi.ac.uk/imgt/hla/).
  • bioarrays of the present invention may be designed to provide a specific population coverage of specific populations of individuals. Populations may be based upon race, geographic area, sex, disease, etc.
  • population coverage is defined as the portion of the given population for whom both alleles of a given locus are included in a given bioarray. Possible loci of interest may include, but are not limited to, DP, DQ, DR1 , and DR3/4/5.
  • the population coverage provided by a given bioarray may be calculated by the following equation, in which, the term "allele prevalence” is defined as the portion of the alleles of the given population that are the allele of interest:
  • the choice of arrayed MHC proteins would be influenced by the knowledge that a peptide from the protein does indeed interact with one or more of the MHC proteins. That MHC protein and related MHC proteins expected to have similar peptide binding preferences would then be assembled in array format for evaluating the offending peptide and variants thereof (e.g., variants designed to remove the ability to interact with MHC molecules).
  • MHC-II molecules The polymorphic nature of the individual subunits that comprise the MHC-II molecules allows for possibility of >5000 MHC molecules to be present in human population. This large number may be reduced for practical purposes based on specific population genetics data. It is therefore possible to prioritize a list of 50-400 MHC-II molecules (including all three classes, DR, DQ and DP) that if experimentally analyzed for their peptide binding propensities would serve as a very representative set for any given population. For the analysis of the immunogenicity profile of any given protein the binding analysis of sequentially overlapping peptides of about 9-15 amino acids which represent the entire sequence, with all of the selected MHC molecules is done using surface anchored proteins or peptides in array format.
  • MHC proteins selected for arrayed format will be a combination of high frequency alleles in a specific target population and high frequency alleles in the general population or a combination of high frequency alleles in a specific target population and alleles expected to interact with peptides within the therapeutic protein.
  • one or both of the components of the assay e.g. the
  • Fusion partner herein is meant a sequence that is associated with the component, that confers a common function or ability. Fusion partners can be heterologous (i.e. not native to the host cell), or synthetic (not native to any cell).
  • Suitable fusion partners include, but are not limited to: a) presentation structures, as defined below, which provide the peptides in a conformationally restricted or stable form; b) targeting sequences, defined below, which allow the localization of the component into a subcellular or extracellular compartment; c) rescue sequences as defined below, which allow the purification or isolation of either component; d) stability sequences, which confer stability or protection from degradation to the peptide, for example resistance to proteolytic degradation; e) dimerization sequences, to allow for peptide dimerization; or f) any combination of a), b), c), d), and e), as well as linker sequences as needed.
  • the fusion partner is a presentation structure.
  • presentation structure or grammatical equivalents herein is meant a sequence, which, when fused to assay components, causes the attached proteins and peptides to assume a conformationally restricted form. Proteins interact with each other largely through conformationally constrained domains. Although small peptides with freely rotating amino and carboxyl termini can have potent functions as is known in the art, the conversion of such peptide structures into pharmacologic agents is difficult due to the inability to predict side- chain positions for peptidomimetic synthesis.
  • presentation structures are preferably used with the MHC proteins or peptides.
  • synthetic presentation structures i.e. artificial polypeptides, are capable of presenting a randomized peptide as a conformationally-restricted domain.
  • presentation structures comprise a first portion joined to the N-terminal end of the randomized peptide, and a second portion joined to the C-terminal end of the peptide; that is, the peptide is inserted into the presentation structure, although variations may be made, as outlined below.
  • the presentation structures are selected or designed to have minimal biologically activity when expressed in the target cell or synthesized de novo.
  • presentation structures maximize accessibility to the peptide by presenting it on an exterior loop.
  • suitable presentation structures include, but are not limited to, minibody structures, loops on beta-sheet turns and coiled-coil stem structures in which residues not critical to structure are randomized, zinc-finger domains, cysteine-linked (disulfide) structures, transglutaminase linked structures, cyclic peptides, B-loop structures, helical barrels or bundles, leucine zipper motifs, etc.
  • the presentation structure is a coiled-coil structure, allowing the presentation of the randomized peptide on an exterior loop. See, for example, Myszka et al., Biochem. 33:2362-2373 (1994), hereby incorporated by reference.
  • the presentation structure is a minibody structure.
  • a "minibody” is essentially composed of a minimal antibody complementarity region.
  • the minibody presentation structure generally provides two randomizing regions that in the folded protein are presented along a single face of the tertiary structure. See for example Bianchi et al., J. MoI. Biol. 236(2):649-59 (1994), and references cited therein, all of which are incorporated by reference).
  • the presentation structure is a sequence that contains generally two cysteine residues, such that a disulfide bond may be formed, resulting in a conformational ⁇ constrained sequence.
  • the fusion partner is a rescue sequence (similar to a "secondary label" as described herein).
  • peptide rescue sequences include purification sequences such as the His6 tag for use with Ni affinity columns and epitope tags .
  • Suitable epitope tags include myc (for use with the commercially available 9E10 antibody), the BSP biotinylation target sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST.
  • the fusion partner is a stability sequence to confer stability to the assay component or the nucleic acid encoding it.
  • peptides may be stabilized by the incorporation of glycines after the initiation methionine (MG or MGGO), for protection of the peptide to ubiquitination as per Varshavsky's N-End Rule, thus conferring long half-life in the cytoplasm.
  • two prolines at the C-terminus impart peptides that are largely resistant to carboxypeptidase action.
  • the presence of two glycines prior to the pralines impart both flexibility and prevent structure initiating events in the di-proline to be propagated into the candidate peptide structure.
  • some stability sequences are as follows: MG(X)nGGPP (SEQ ID NO: 1), where X is any amino acid and n is an integer of at least four.
  • the fusion partner is a dimerization sequence.
  • a dimerization sequence allows the non-covalent association of one random peptide to another random peptide, with sufficient affinity to remain associated under normal physiological conditions. This effectively allows small libraries of random peptides (for example, 104) to become large libraries if two peptides per cell are generated which then dimerize, to form an effective library of 108 (104 X 104). It also allows the formation of longer random peptides, if needed, or more structurally complex random peptide molecules.
  • the dimers may be homo- or heterodimers.
  • Dimerization sequences may be a single sequence that self-aggregates, or two sequences, each of which is generated in a different retroviral construct. That is, nucleic acids encoding both a first random peptide with dimerization sequence 1 , and a second random peptide with dimerization sequence 2, such that upon introduction into a cell and expression of the nucleic acid, dimerization sequence 1 associates with dimerization sequence 2 to form a new random peptide structure.
  • dimerization sequences will encompass a wide variety of sequences. Any number of protein-protein interaction sites are known. In addition, dimerization sequences may also be elucidated using standard methods such as the yeast two hybrid system, traditional biochemical affinity binding studies, or even using the present methods.
  • the fusion partners may be placed anywhere (i.e. N-terminal, C-terminal, internal) in the structure as the biology and activity permits.
  • the fusion partner includes a linker or tethering sequence.
  • Linker sequences between the fusion partner and the other components of the constructs may be desirable to allow the MHC proteins or peptides to interact with their target unhindered.
  • useful linkers include glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 2) and (GGGS)n (SEQ ID NO: 3), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art.
  • Glycine-serine polymers are some since both of these amino acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components.
  • serine is hydrophilic and therefore able to solubilize what could be a globular glycine chain.
  • similar chains have been shown to be effective in joining subunits of recombinant proteins such as single chain antibodies.
  • the fusion partners may be modified, randomized, and/or matured to alter the presentation orientation of the randomized expression product.
  • determinants at the base of the loop may be modified to slightly modify the internal loop peptide tertiary structure, which maintaining the randomized amino acid sequence.
  • labels may be either direct or indirect detection labels, sometimes referred to herein as “primary” and “secondary” labels.
  • detection label or “detectable label” herein is meant a moiety that allows detection. Accordingly, detection labels may be primary labels (i.e. directly detectable) or secondary labels (indirectly detectable; this is analogous to a “sandwich” type assay).
  • labels fall into four classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal labels; c) colored or luminescent dyes or moieties; and d) binding partners. Labels can also include enzymes (horseradish peroxidase, etc.) and magnetic particles.
  • the detection label is a primary label.
  • a primary label is one that may be directly detected, such as a fluorophore.
  • Preferred labels include chromophores or phosphors but are preferably fluorescent dyes or moieties. Fluorophores may be either "small molecule" fluores, or proteinaceous fluores. Suitable dyes for use in the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, quantum dots (also referred to as "nanocrystals": see U.
  • the test molecule is labeled with a primary label.
  • a primary label is added chemically using functional groups on the label and the test molecule.
  • the functional group can then be subsequently labeled with a primary label. Suitable functional groups include, but are not limited to, amino groups, carboxy groups, maleimide groups, oxo groups and thiol groups, with amino groups and thiol groups being particularly preferred.
  • primary labels containing amino groups may be attached to secondary labels comprising amino groups, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross linkers, pages 155-200, incorporated by reference).
  • linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross linkers, pages 155-200, incorporated by reference).
  • linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross linkers, pages 155-200, incorporated by reference).
  • linkers as are known in the art
  • homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross linkers, pages 155-200, incorporated by reference).
  • the test molecule may be fused to a label protein such as GFP, using well
  • a secondary label is used.
  • a secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, can act on an additional product to generate a primary label (e.g. enzymes), or may allow the separation of the compound comprising the secondary label from unlabeled materials, etc.
  • Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, enzymes such as horseradish peroxidase, alkaline phosphatases, lucifierases, cell surface markers, etc.
  • the secondary label is a binding partner pair.
  • the label may be a hapten or antigen, which will bind its binding partner.
  • suitable binding partner pairs include, but are not limited to: antigens and antibodies (including fragments thereof (FAbs, etc.)); proteins and small molecules (including biotin/streptavidin); enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners. Nucleic acid - nucleic acid binding proteins pairs are also useful. In general, the smaller of the pair is attached to the NTP for incorporation into the primer.
  • binding partner pairs include, but are not limited to, biotin (or imino-biotin) and streptavidin, digeoxinin and Abs, and Prolinx reagents.
  • the binding partner may be attached to a solid support to allow separation of components containing the label and those that do not.
  • the binding partner pair comprises a primary detection label (for example, attached to the test molecule) and an antibody that will specifically bind to the primary detection label.
  • a primary detection label for example, attached to the test molecule
  • an antibody that will specifically bind to the primary detection label.
  • specifically bind herein is meant that the partners bind with specificity sufficient to differentiate between the pair and other components or contaminants of the system. The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding.
  • the dissociation constants of the pair will be less than about 10 4 -10 6 M "1 , with less than about 10 5 -10 9 M "1 , being preferred and less than about 10 7 -10 9 M '1 being particularly preferred.
  • the secondary label is a chemically modifiable moiety.
  • labels comprising reactive functional groups are incorporated into the test molecule.
  • the functional group can then be subsequently labeled (e.g. either before or after the assay) with a primary label.
  • Suitable functional groups include, but are not limited to, amino groups, carboxy groups, maleimide groups, oxo groups and thiol groups, with amino groups and thiol groups being particularly preferred.
  • primary labels containing amino groups may be attached to secondary labels comprising amino groups, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross linkers, pages 155-200, incorporated by reference).
  • the techniques outlined herein result in the addition of a detectable label to the test molecule, which binds to at least one of the candidate proteins (e.g., MHC proteins on a bioarray).
  • a detectable label e.g., MHC proteins on a bioarray.
  • Fluorescent labels are preferred, and standard fluorescent detection techniques can then be used.
  • detection can proceed with unlabeled test molecules when a "solution binding ligands” or “soluble binding ligands” or “signaling ligands” or “signal carriers” or “label probes” or “label binding ligands” are used.
  • the soluble binding ligand carries the label and will bind to the test molecule.
  • proteinaceous test molecules they may be fused to heterologous epitope tags, which can then bind labeled antibodies to effect detection.
  • epitope tags are known as outlined above.
  • MHC proteins are added to bioarrays comprising arrays of capture probes, under conditions that allow the formation of binding complexes between the capture sequences of the MHC proteins to the capture probes of the bioarray. This forms the protein arrays of the invention.
  • label means any detectable label.
  • suitable labels include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 1, 131 I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, /?-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • radioisotopes or radionuclides e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125
  • the label is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance.
  • Various methods for labelling proteins are known in the art and may be used in performing the present invention.
  • the covalent attachment of the fluorescent label may be either direct or via a linker.
  • the linker is a relatively short coupling moiety, that is used to attach the molecules.
  • a coupling moiety may be synthesized directly onto an MHC protein or peptide for example, and contains at least one functional group to facilitate attachment of the fluorescent label.
  • the coupling moiety may have at least two functional groups, which are used to attach a functionalized MHC protein or peptide to a functionalized fluorescent label, for example.
  • the linker is a polymer.
  • covalent attachment is accomplished either directly, or through the use of coupling moieties from the agent or label to the polymer.
  • the covalent attachment is direct, that is, no linker is used.
  • the MHC protein or peptide preferably contains a functional group such as a carboxylic acid which is used for direct attachment to the functionalized fluorescent label.
  • amino modified or hydrazine modified fluorescent labels will be used for coupling via carbodiimide chemistry, for example using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as is known in the art (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; see also the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both of which are hereby incorporated by reference).
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
  • the carbodiimide is first attached to the fluorescent label, such as is commercially available.
  • a fluorescent label is attached, either directly or via a linker, to the MHC proteins or peptides and thus serves as a first labeling moiety.
  • the first labeling moiety comprises a first partner of a binding pair, which may or may not be fluorescent, and a second labeling moiety, comprising the second partner of a binding pair, and at least one fluorescent label, as defined above.
  • a secondary label may be used.
  • the secondary label includes a primary label covalently attached to a molecule capable of binding to the MHC protein - peptide complex. Examples include MHC specific antibodies.
  • the bioarrays comprise a substrate.
  • substrate or “solid support” or other grammatical equivalents herein is meant any material appropriate for the attachment of capture probes and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates is very large.
  • Possible solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, ceramics, and a variety of other polymers.
  • the solid supports allow optical detection and do not themselves appreciably fluoresce.
  • the solid support may be coated with any number of materials, including polymers, such as dextrans, acrylamides, gelatins, agarose, etc.
  • Exemplary solid supports include silicon, glass, polystyrene and other plastics and acrylics.
  • the solid support is flat (planar), although as will be appreciated by those in the art, other configurations of solid supports may be used as well, including the placement of the probes on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • the size of the array can depend on the composition and end use of the array. Arrays containing from about 2 different capture probes to many thousands may be made. Generally, the array will comprise from two to as many as 100,000 or more, depending on the size of the pads, as well as the end use of the array. Preferred ranges are from about 2 to about 10,000, with from about 5 to about 1000 being preferred, and from about 10 to about 100 being particularly preferred.
  • the compositions of the invention may not be in array format; that is, for some embodiments, compositions comprising a single capture probe may be made as well.
  • multiple substrates may be used, either of different or identical compositions. Thus for example, large arrays may comprise a plurality of smaller substrates.
  • the bioarray substrates optionally comprise an array of capture probes.
  • capture probes proteins (e.g. antibodies) or chemicals (attached either directly or indirectly to the substrate as is more fully outlined below) that are used to bind the MHC proteins or peptides.
  • the capture probes may be attached either directly to the substrate, or indirectly, through the use of polymers or through the use of microspheres.
  • a library of different candidate peptides are used.
  • the library should provide a sufficiently structurally diverse population of randomized agents to effect a probabilistically sufficient range of diversity to allow binding to a particular target.
  • the candidate peptides are added to the array under conditions suitable for binding to the MHC proteins.
  • binding is under physiological or close to physiological conditions.
  • Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 4O 0 C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is preferably removed or washed away.
  • components of the invention are linked together with attachment linkers.
  • an MHC protein or peptide can be attached to the solid support using an attachment linker, or an MHC protein can be attached to a label with an attachment linker, etc.
  • attachment will generally be done as is known in the art, and will depend on the composition of the two materials to be attached.
  • attachment linkers are utilized through the use of functional groups on each component that can then be used for attachment.
  • Preferred functional groups for attachment are amino groups, carboxy groups, oxo groups, hydroxyl groups and thiol groups. These functional groups can then be attached, either directly or indirectly through the use of a linker.
  • Linkers are well known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155- 200, incorporated herein by reference).
  • Preferred attachment linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), with short alkyl groups, esters, amide, amine, epoxy groups and ethylene glycol and derivatives being preferred, with propyl, acetylene, and C 2 alkene being especially preferred, with the corresponding functionalities.
  • the attachment linkers facilitate covalent attachment.
  • covalently attached herein is meant that two moieties are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. In some cases, for example when thiol groups are used to attach components to a gold surface, the thiol-gold attachment is considered covalent under these conditions.
  • non covalent attachment can be done, for example through the absorption of MHC proteins to the solid supports of the invention.
  • attach proteins using recombinant methods.
  • the use of fluorescent proteins as the label for MHC proteins can be done by ligating the encoding nucleic acids together for expression of fusion proteins.
  • the MHC proteins or peptides are synthesized first, and then covalently attached to the solid supports. As will be appreciated by those in the art, this will be done depending on the composition of the MHC proteins or peptides and the solid supports.
  • the functionalization of solid support surfaces such as certain polymers with chemically reactive groups such as thiols, amines, carboxyls, etc. is generally known in the art.
  • the MHC proteins or peptides are attached using functional groups on the MHC protein or peptide.
  • MHC proteins or peptides containing carbohydrates may be attached to an amino-functionalized support; the aldehyde of the carbohydrate is made using standard techniques, and then the aldehyde is reacted with an amino group on the surface.
  • a sulfhydryl linker may be used.
  • sulfhydryl reactive linkers known in the art such as SPDP, maleimides, a- haloacetyls, and pyridyl disulfides (see for example the 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference) which can be used to attach cysteine containing proteinaceous agents to the support.
  • an amino group on the MHC protein or peptide may be used for attachment to an amino group on the surface.
  • a large number of stable bifunctional groups are well known in the art, including homobifunctional and heterobifunctional linkers (see Pierce Catalog and Handbook, pages 155-200).
  • carboxy groups (either from the surface or from the MHC protein or peptide) may be derivatized using well known linkers (see the Pierce catalog).
  • linkers see the Pierce catalog.
  • carbodiimides activate carboxy groups for attack by good nucleophiles such as amines (see Torchilin et al., Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275-308 (1991), expressly incorporated herein).
  • amine- amine crosslinking thiol-thiol crosslinking, amine-thiol crosslinking, amine-carboxylic acid crosslinking, and carbohydrate crosslinking to amines and thiols; see Molecular Probes Catalog, 1996, Sixth Edition, chapter 5, hereby incorporated by reference.
  • proteinaceous MHC proteins or peptides may also be attached using other techniques known in the art, for example for the attachment of antibodies to polymers; see Slinkin et al., Bioconi. Chem. 2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj.
  • MHC proteins or peptides may be attached in a variety of ways, including those listed above. What is important is that manner of attachment does not significantly alter the functionality of the MHC protein or peptide; that is, the MHC protein or peptide should be attached in such a flexible manner as to allow its interaction with its corresponding peptide or MHC protein.
  • library of MHC proteins or peptides is meant generally at least about 10 2 different compounds, with at least about 10 3 different compounds being preferred, and at least about 10 4 , 10 5 or 10 6 different compounds being particularly preferred.
  • each solid support contain a multiplicity of MHC proteins or peptides. That is, each solid support will contain at least about 10 MHC proteins or peptides, with at least about 100 being preferred, and at least about 1000 being especially preferred.
  • each solid support may contain one type of MHC protein or peptide, or more than one. That is, in a preferred embodiment, any single solid support contains a single type of candidate peptide. This may be preferred for a variety of reasons, including synthetic considerations, ease of characterization of downstream "hits", and fluorescent detection limits.
  • each solid support may contain more than one type of MHC protein or peptide.
  • it will generally be desirable to "amplify" the fluorescent signal i.e. have more than one fluorescent label per target to facilitate detection.
  • solid supports that each contain a single MHC protein or peptide. That is, there are a number of solid supports each containing a particular MHC protein or peptide. Thus, at least about 100 solid supports per MHC protein or peptide are used, with at least about 1000 being preferred and at least about 10,000 to 100,000 being especially preferred.
  • the library of candidate peptides are contained upon a plurality of solid supports.
  • array and “bioarray” herein are synonymous, and mean a plurality of capture binding ligands on a solid support.
  • the size of the array will depend on the composition and end use of the array.
  • first example of a bioarray is an array of MHC proteins.
  • a second example of a bioarray is an array of peptides.
  • the biomolecules in the array may be attached to a solid support, free in solution, deposited on a solid support, etc.
  • Array surfaces can comprise any number of different substrates, including silicon, glass, electrodes, plastics, etc.
  • the non-immobilized MHC protein includes at least a first fluorescent label.
  • Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, and Texas Red.
  • Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.
  • all the labeled MHC proteins or peptides contain the same fluorescent label.
  • the labeled MHC protein or peptide population is divided into at least two subpopulations, each comprising a different fluorescent label. This may be particularly preferred to reduce false positives; that is, only solid supports comprising both labels (i.e. solid supports with a single MHC protein or peptide type that bind targets with both labels) will constitute "real" interactions.
  • the target molecules are also bound to solid supports.
  • the target molecules are attached to the solid supports using preferably flexible linkers, to allow for interaction with solid support-bound agents.
  • a preferred system utilizes fluorescent solid supports; that is, the solid support to which the target molecules is attached can be fluorescent, thus serving as the first or second labeling moiety. See for example the Molecular Probes catalog, supra, chapter 6, hereby incorporated by reference.
  • the solid supports containing the MHC proteins or peptides are added to the target molecules under reaction conditions that favor agent-target interactions. Generally, this will be physiological conditions. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40 0 C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away.
  • the arrays can have a number of formats in which either the the MHC protein or peptides are immobilized on a solid surface to form an array.
  • a single MHC protein is immobilized at multiple locations on a solid surface to form an array.
  • the single MHC protein can correspond, for example, to a single MHC allele.
  • the MHC allele is immobilized to the solid surface as described supra.
  • the array can then be exposed to one or more peptides.
  • the peptides can be spotted onto the surface at each location in the array.
  • a pool of different peptides can be applied to the array of MHC proteins.
  • the peptides can be added to the array, and those that bind can be detected.
  • a plurality of MHC proteins are immobilized at various locations on the solid surface to form an array.
  • the MHC proteins can, for example, correspond to a plurality of known alleles for a specific type of MHC.
  • the MHC proteins can correspond to multiple different MHC subtypes of MHC molecules, such as a combination of class I and class Il molecules, or subtypes thereof.
  • the array can then be exposed to one or more peptides, such as a pool of peptides.
  • an array of peptides may be provided.
  • a single peptide can be provided at multiple locations on the solid surface to form an array.
  • the single peptide can correspond to a specific peptide, including a specific agretope presented at the surface of specific MHC molecules.
  • the peptide can be designed to bind in the binding groove of a subset of MHC alleles in a specific class or antigen subtype (e.g. HLA-A, B, C, DR, DQ, DP).
  • the peptide is immobilized to the solid surface as described supra.
  • the array can then be exposed to one or more MHC protein in solution form.
  • MHC protein in solution form.
  • a single MHC allele can be provided.
  • the MHC molecules can by within a specific HLA antigen subtype (e.g. HLA-A, B, C, DR, DQ, or DP) can be provided.
  • a pool of different peptides can be applied to the array of MHC proteins.
  • the peptides can be added to the array, and those that bind can be detected.
  • a variety of other reagents may be included in the assays. These include reagents like salts, neutral proteins, e.g.
  • albumin may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions.
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used.
  • the mixture of components may be added in any order that provides for the requisite binding.
  • the MHC protein may be identified. Since the location and sequence of each capture probe is known, the identification of a "hit" at a particular location will identify the particular MHC protein with the corresponding capture sequence. This capture sequence may be used to identify the coding region of the candidate protein. This may be done in a wide variety of ways, as will be appreciated by those in the art, including using PCR technologies. For example, using primers specific to the capture sequence, the nucleic acid encoding the candidate protein may be amplified and sequenced.
  • the methods and compositions of the invention comprise a robotic system.
  • Many systems are generally directed to the use of 96 (or more) well microtiter plates, but as will be appreciated by those in the art, any number of different plates or configurations may be used.
  • any or all of the steps outlined herein may be automated; thus, for example, the systems may be completely or partially automated.
  • Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications.
  • This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration.
  • These manipulations are cross-contamination- free liquid, particle, cell, and organism transfers.
  • This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.
  • microplates, tubes or any solid phase matrices include non-polar surfaces, highly polar surfaces, modified dextran coating to promote covalent binding, antibody coating, affinity media to bind fusion proteins or peptides, surface-fixed proteins such as recombinant protein A or G, nucleotide resins or coatings, and other affinity matrix are useful in this invention.
  • platforms for multi-well plates, multi-tubes, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, electroporator, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • thermocycler and thermoregulating systems are used for stabilizing the temperature of the heat exchangers such as controlled blocks or platforms to provide accurate temperature control of incubating samples from 4°C to 100 0 C.
  • the instrumentation will include a detector, which may be a wide variety of different detectors, depending on the labels and assay.
  • useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluroescence resonance energy transfer (FRET), SPR systems, luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation.
  • Flow cytometry or capillary electrophoresis formats may be used for individual capture of magnetic and other beads, particles, cells, and organisms.
  • the flexible hardware and software allow instrument adaptability for multiple applications.
  • the software program modules allow creation, modification, and running of methods.
  • the system diagnostic modules allow instrument alignment, correct connections, and motor operations.
  • the customized tools, labware, and liquid, particle, cell and organism transfer patterns allow different applications to be performed.
  • the database allows method and parameter storage. Robotic and computer interfaces allow communication between instruments.
  • the robotic workstation includes one or more heating or cooling components. Depending on the reactions and reagents, either cooling or heating may be required, which may be done using any number of known heating and cooling systems, including Peltier systems.
  • the robotic apparatus includes a central processing unit that communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • a central processing unit that communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • input/output devices e.g., keyboard, mouse, monitor, printer, etc.
  • one of the components of the invention is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.).
  • the component bound may be an envelope virus particle expressing the candidate protein or the target molecule, etc.
  • the insoluble support may be made of any composition to which the assay component may be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads.
  • microtiter plates and arrays are especially convenient because a large number of assays may be carried out simultaneously, using small amounts of reagents and samples.
  • bead-based assays may be used, particularly with use with fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • One preferred method of binding include the use of antibodies, more preferably antibodies which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support.
  • Other preferred methods includes direct binding to "sticky" or ionic supports, chemical crosslinking, the use of labeled components (e.g. the assay component is biotinylated and the surface comprises strepavidin, etc. ), the synthesis of the target on the surface, etc.
  • excess unbound material is removed by washing.
  • the sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
  • BSA bovine serum albumin
  • the target molecule is bound to the support, and an envelope virus particle expressing a candidate protein is added to the assay.
  • the envelope virus particle expressing a candidate protein is bound to the support and the target molecule is added.
  • Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells.
  • Determination of the binding of the target and the candidate protein may be done using a wide variety of assays, including, but not limited to labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, the detection of labels, functional assays (phosphorylation assays, etc.) and the like.
  • the determination of the binding of the candidate protein to the target molecule may be done in a number of ways.
  • one of the components preferably the soluble one, is labeled, and binding determined directly by detection of the label. For example, this may be done by attaching the envelope virus particle expressing a candidate protein to a solid support, adding a labeled target molecule (for example a target molecule comprising a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
  • a labeled target molecule for example a target molecule comprising a fluorescent label
  • This system may also be run in reverse, with the target (or a library of targets) being bound to the support and envelope viruses expressing candidate proteins, preferably comprising a primary or secondary label, added.
  • envelope virus particles expressing a candidate protein comprising fusions with GFP or a variant may be particularly useful.
  • Various blocking and washing steps may be utilized as is known in the art.
  • the bioarray may also be done using bead based systems.
  • assays may be run using beads or other solid supports such that libraries of sequences are made. The addition of envelope virus libraries then allows for the detection of candidate proteins that bind to specific sequences.
  • the binding of the candidate protein is determined through the use of competitive binding assays.
  • the competitor is a binding moiety known to bind to the target molecule such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the target and the binding moiety, with the binding moiety displacing the target.
  • Positive controls and negative controls may be used in the assays.
  • Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound. Similarly, ELISA techniques are generally preferred. In some embodiments, only one of the components is labeled. In an alternate embodiment, more than one component may be labeled with different labels.
  • a variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, co-factors such as cAMP, ATP, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
  • bioarrays of the present invention may be designed for specific populations of individuals. Populations may be based upon race, geographic area, sex, disease, etc. Examples of populations also include individuals with the following indications: arthritis, psoriatic arthritis, ankylosing spondylitis, spondyloarthritis, spondyloarthropathies, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, reactive arthritis (Reiter Syndrome) scleroderma, Sjogren's syndrome, keratoconjunctivitis, keratoconjunctivitis sicca, TNF-receptor associated periodic syndrome (TRAPS), periodic fever, periprosthetic osteolysis, apthous stomatitis, pyoderma gangrenosum, uveitis, reticulohistiocytosis, inflammatory bowel diseases, sepsis and septic shock
  • the extracellular peptide binding domains are expressed with C-terminal leucine zippers to facilitate dimerization. (See Figures 1 and 2 and included amino acid sequences).
  • the constructs may include C-terminal purification tags, for example, 6xhis, flag, c-myc, etc. Additional sequences may be attached or modifications made to provide anchoring to a solid surface may be used. These sequences or modifications preferably added at the C-terminus.
  • sequences and modifications include but are not limited to biotinylation of the constructs, fusions with the protein (e.g., Fc, albumin, etc.), linker sequences of from 3 to 50 amino acids (preferably combinations of GIy and Ser) or modification with small molecules, all to enhance anchoring the construct to a solid surface.
  • protein e.g., Fc, albumin, etc.
  • linker sequences of from 3 to 50 amino acids (preferably combinations of GIy and Ser) or modification with small molecules, all to enhance anchoring the construct to a solid surface.
  • the expression constructs encoding both a and /?-subunits are co-transfected in production cell lines in transient manner.
  • the preferred expression cells include HighFive, Sf9 or Drosophila S2 insect cells, or for mammalian expression, the 293T cells are preferred. Stable cell lines may also be established for production of selected MHCs. For purification, the affinity chromatography using either the specific tag or anti-MHC antibodies are preferred methods.
  • Example 2 Expression in insect cells.
  • 5 ug each of the plasmid DNAs for a and ⁇ subunit expression constructs were used for transfecting 2 x 106 HiFive cells plated in 150mm tissue culture dishes.
  • the plasmid DNAs were mixed with 50 ul of a liposome based transfection reagent, CellFectin (Invitrogen) in 2 ml of a serum-free medium, ESF-921 (ExpressionSystems LLC) and incubated for 20 min at RT for DNA-liposome complexes to form.
  • the mix was added slowly to insect cells already plated in 150mm dishes followed by a 4hr incubation at RT with very gentle rocking (5 rpm).
  • Example 3 Expression in mammalian cells.
  • Example 4 Expression of recombinant MHC molecules of each class ( ⁇ LA-DR. HLA-DP & HLA-DCO
  • the recombinant MHC molecules were produced in insect cells using a non-lytic system involving transient transfection of cells with both a and ⁇ subunit expression constructs.
  • the expression constructs for each of the subunits contained the respective extracellular domain attached at the c-terminus to the fos/jun leucine zipper dimerization domains and a his/flag tag sequence via a flexible linker (VDGGGGG) (SEQ ID NO: 4) as described in Figure 2.
  • VDGGGGG flexible linker
  • the a subunits contain a c-terminal fos LZ and a His tag, where as the ⁇ subunits contain c-terminal jun LZ and the flag tag.
  • Co-transfection with plasmid DNAs encoding these two modular subunits yields heterodimeric MHC molecules in the medium supernatant driven by system appropriate signal sequence.
  • the expression-reading frame was joined at the N-terminus with Honeybee Melittin signal sequence to facilitate efficient secretion of the correctly folded heterodimer MHCs in the media supernatant.
  • the extracellular domains of the MHC molecules were amplified from corresponding cDNA clones obtained from ATCC and fused to synthetic DNAs containing the leucine zipper and the tag sequence using standard PCR based protocols. This modular insert was cloned downstream of the signal sequence in the expression vector. Scale-up of MHC expression was performed as shown in Figure 5. Using this approach, the MHC proteins may be expressed in various expression systems as sampled in Table 1.
  • the recombinant MHC molecules expressed in cells may be purified using Ni-NTA affinity chromatography as described for the DR4 (DRA1*0101 & DRB1*0401 ) test molecule, Figure 6.
  • the supernatant was concentrated 2OX using a tangential flow ultrafiltration cassette (Millipore, Pelicon) with a MWCO of 10000 d.
  • the concentrated supernatant was buffer exchanged with binding buffer (50 mM Tris-HCI, pH 8.0 & 500 mM NaCI) using the same ultrafiltration device.
  • binding buffer 50 mM Tris-HCI, pH 8.0 & 500 mM NaCI
  • the slurry was poured into a chromatography column and washed with 50 mM Tris-HCI, pH 8.0 , 500 mM NaCI and 30 mM imidazole followed by one more wash with 50 mM Tris-HCI, pH 8.0 & 1.0 M NaCI .
  • the bound protein was eluted with 50 mM Tris-HCI, pH 8.0, 100 mM NaCI and 250 mM imidazole in 10% glycerol.
  • the eluted protein was buffer exchanged into PBS with 10% glycerol. This yields a partially purified (-50%) and concentrated preparation is stable and may be used in directly in all binding assays and surface captures.
  • Example 6 MHC-peptide binding assays.
  • the recombinant MHC molecules produced as described above may be used to test the binding of various peptides either in solution or as captured or arrayed on a solid surface.
  • the peptide binding may be determined in a direct binding experiment where the peptide is labeled (e.g., with radioactivity, fluorescence, biotin etc.) or in a competition format where the reference peptide is labeled but the test peptide(s) are unlabeled.
  • Ahx aminohexanoic acid spacer
  • Peptide binding affinities may be determined by incubation of the labeled (biotin, fluorescence, radiolabeled) peptide over the surface and then evaluating the amount of bound peptide using appropriate detection methodologies. A dose response experiment using multiple concentrations of the peptide in sequence may be done to evaluate the specific ED50 values for each peptide-MHC combination. The dissociation rates may also be calculated by monitoring the label after loading as a function of time. Fluorescently labeled (for example, FAM, fluorescein, Alexa, Cy dyes) peptides would be the preferred format for this analysis.
  • a competition based method may also be used to analyze the relative binding affinities of unlabelled peptides using a single labeled reference peptide pre-loaded onto MHC molecules as long as the reference peptide binds to all of them.
  • An SPR based approach may also be used to study the binding interactions using an SPOT-Matrix method.
  • MHC protein arrays having about 2 to about 1000 different MHCs may be prepared by surface capture on glass, plastic, nitrocellulose, hydrogels or other derivatized surfaces using either direct binding or binding via specific interactions. Specific interactions include but are not limited to antibodies against a common tag, streptavidin for a biotinylated MHC or protein A or albumin or Fc for a fusion MHC). Control proteins and multiples of same MHCs may be used as internal controls.
  • every pad on the array has the same capture molecule, and each MHC protein has the same capture sequence.
  • the array is used more as a general affinity capture surface, in a manner similar to phage display panning.
  • the MHC proteins are bound to the array (which can also be a continuous surface, rather than spatially separate addresses) and test molecules added. Washing and competitive assays may be done to test for protein-protein interactions and affinity.
  • the present invention may be used to study MHC-peptide binding interaction in an array format where either MHC or the peptide would be used as the arrayed partner.
  • the MHC bioarray may comprise more than one MHC molecule, more preferably 2- 10000, and even more preferably from 2-100.
  • Either the MHC proteins or the peptides may be attached to a solid surface in an ordered format.
  • the attached molecules may be selected based on: 1 ) a specific population prevalence; 2) a specific disease state association/disease susceptibility; 3) a specific structural subclass(s) of MHCs; or 4) other criteria.
  • the MHCs may be natural MHC isolated from cells, or recombinant produced with a natural ectodomain sequence or recombinant with a modified ectodomain sequence.
  • the peptide bioarrays may comprise of a selection of 2-100000, more preferably a range of 2-1000.
  • the peptides are attached to a solid surface in an ordered format.
  • the peptides may be selected, for example, from the following groups: 1) a peptide scan of one or more protein sequences; 2) randomly selected from genome sequencing; 3) peptides containing sequence similar to those occurring in natural proteins with one or more modifications; 4) completely synthetically created sequences; 5) other criteria.
  • the peptides may be 6-30 amino acids long, with preferred range being 8-16.
  • the peptides may have spacer amino acids, may be attached to surface via biotin or directly coupled to surface during synthesis or attached using other chemistries.
  • the bioarray may be reacted with labeled/unlabeled specific peptides or population of peptides to characterize the binding interaction.
  • the bioarray may be reacted with labeled/unlabeled specific MHCs or population of MHCs to characterize the binding interaction.
  • the interactions may be used to identify immunogenic epitopes on the proteins, de-immunize protein sequences, select populations with specific MHCs that could be used for clinical trials or therapeutic use, improve the potency of a vaccine, etc.
  • a therapeutic protein may be analyzed using an MHC bioarray of the present invention.
  • the target molecule may be expressed as multiple overlapping peptides, preferably from 8 to 16 amino acids in length. These peptides may be run over an MHC bioarray as described herein to study the MHC-peptide binding.
  • the MHC bioarray may be optimized to a target population for the therapeutic, such as people with type Il diabetes in the US population; Alzheimer's patients; MS patients, arthritic patients; etc.
  • the present invention may be used with an array of peptides.
  • the peptides may be bound to the surface in an oriented manner and the binding of individual MHCs may be evaluated.
  • binding is evaluated using either an SPR based method or detection using an MHC specific labeled antibody.
  • a peptide array may be use for the analysis of a smaller number of MHC molecules against a larger set of peptides.
  • libraries of different MHC proteins may be used. However, as will be appreciated by those in the art, different members of the library may be reproduced or duplicated, resulting in some libraries members being identical.
  • Example 11 Predicting Immune Response to Vaccine Candidate
  • the present invention may be used to predict the immune response to a vaccine candidate.
  • the proteins of the vaccine candidate may be expressed as multiple overlapping peptides, preferably from 8 to 16 amino acids in length.
  • the peptides may then be run over an MHC bioarray.
  • the bioarray will include a combination of MHC proteins to represent over 99% of the target population. Analysis of the binding may then be used to predict the effectiveness of the candidate vaccine.
  • Bioarrays may be designed to provide a specified level of population coverage for a given population at a given loci.
  • a greedy algorithm was used to design bioarray allele sets for given levels of population coverage.
  • the designed sets provide minimal allele sets for the specified level of population coverage for a given population at a given loci.
  • Allele prevalences in the general U.S. population for the DR1 locus were extracted from Schreuder et al. (2005) Hum. Immunol. Feb;66(2): 170-210, incorporated entirely by reference. Allele prevalences in the general U.S. population for the DP, DQ, and DR3/4/5 loci were obtained from the NMDB (see Figures 12-14).
  • Table 2 lists some allele sets that provide 50% population coverage over the general U.S. population for the DR1 locus.
  • Table 3 lists some allele sets that provide 60% population coverage over the general U.S. population for the DR1 locus.
  • Table 4 lists some allele sets that provide 70% population coverage over the general U.S. population for the DR1 locus.
  • Table 5 lists some allele sets that provide 80% population coverage over the general U.S. population for the DR1 locus.
  • Table 6 lists some allele sets that provide 90% population coverage over the general U.S. population for the DR1 locus.
  • Table 7 lists some allele sets that provide 95% population coverage over the general U.S. population for the DR1 locus.
  • Table 8 lists some allele sets that provide 60% population coverage over the general U.S. population for the DP locus.
  • Table 9 lists some allele sets that provide 90% population coverage over the general U.S. population for the DP locus.
  • Table 10 lists some allele sets that provide 35% population coverage over the general U.S. population for the DQ locus.
  • Table 10 Bioarray allele sets that provide 35% population coverage over the general U.S. population for the DQ locus.
  • Table 11 lists some allele sets that provide 60% population coverage over the general U.S. population for the DQ locus.
  • Table 12 lists some allele sets that provide 50% population coverage over the general U.S. population for the DR3/4/5 locus.
  • Table 13 lists some allele sets that provide 75% population coverage over the general U.S. population for the DR3/4/5 locus.
  • Example 13 Designing MHC Bioarrays with Specified Population Coverage for a Specific Ethnic or Racial Group
  • Bioarrays may be designed to provide a specified level of population coverage for a given population at a given loci.
  • a greedy algorithm was used to design bioarray allele sets for given levels of population coverage for the U.S. Hispanic population. Allele prevalences in the U.S. Hispanic population for the DR1 locus were obtained from the NMDB (see Figure 15).
  • Table 14 lists some allele sets that provide 90% population coverage over the U.S. Hispanic population for the DR1 locus.
  • the X 9 library and the 171 sublibraries were employed in competition assays with Biotin- AhX-AhX-PKYVKQNTLKLAT-CO-NH 2 for binding to a bioarray of DRB1 * 0401 and DRB 1*0701 molecules prepared as described above.
  • IC50 values indicate the relative preference for an amino acid when placed in a particular pocket in a particular MHC molecule. For instance, comparing the X 9 IC50 with the Ac-X 4 -F-X 4 -NH 2 IC50 yields the relative preference of phenylalanine in pocket 5.
  • peptide binding scoring matrices for DRB1*0401 and DRB1*0701 were developed using the following equation.
  • GDTRPRFL ⁇ QVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVG ⁇ YRAVTELGRPDAEYWNSQKDLLEQ KRAAVDTYCRHNYGWESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKT GWSTGLIQNGDWTFQTLVMLETVPRSG ⁇ VYTCQVEHPSLTSPLTVEWRARSESAQSK

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Abstract

La présente invention concerne des procédés, appareils et composés de recherche systématique ou de détection de la liaison de candidats peptides à une protéine MHC. En l'occurrence, on prend un premier composant comprenant au moins des candidats peptides et un deuxième composant comprenant au moins une protéine MHC, et on les met en contact. L'un des composants est immobilisé sur un support solide. Il ne reste plus qu'à déterminer la présence, l'absence, ou la quantité de liaison et la protéine MHC considérée.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2003082197A2 (fr) * 2002-03-22 2003-10-09 Gpc Biotech Ag Composes immunosuppresseurs, methodes et utilisations associees
US20040230380A1 (en) * 2002-01-04 2004-11-18 Xencor Novel proteins with altered immunogenicity
WO2005073248A1 (fr) * 2004-01-30 2005-08-11 King's College London Peptides pour le traitement de maladies auto-immunes

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Publication number Priority date Publication date Assignee Title
US20040230380A1 (en) * 2002-01-04 2004-11-18 Xencor Novel proteins with altered immunogenicity
WO2003082197A2 (fr) * 2002-03-22 2003-10-09 Gpc Biotech Ag Composes immunosuppresseurs, methodes et utilisations associees
WO2005073248A1 (fr) * 2004-01-30 2005-08-11 King's College London Peptides pour le traitement de maladies auto-immunes

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Title
GODKIN ANDREW ET AL: "Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2)" INTERNATIONAL IMMUNOLOGY, vol. 9, no. 6, 1997, pages 905-911, XP002419884 ISSN: 0953-8178 *
HAMMER J ET AL: "Precise prediction of majour histocompatibility complex class II-peptide interaction based on peptide side chain scanning" JOURNAL OF EXPERIMENTAL MEDICINE, TOKYO, JP, vol. 180, December 1994 (1994-12), pages 2353-2358, XP002322265 ISSN: 0022-1007 cited in the application *
MARSHALL KEITH W ET AL: "Prediction of peptide affinity to HLA DRB1*0401" JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 154, no. 11, 1995, pages 5927-5933, XP002375653 ISSN: 0022-1767 cited in the application *

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