[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2007053644A2 - Cartographie comparative des ligands de cellules positives vis-a-vis du mhc de classe i - Google Patents

Cartographie comparative des ligands de cellules positives vis-a-vis du mhc de classe i Download PDF

Info

Publication number
WO2007053644A2
WO2007053644A2 PCT/US2006/042549 US2006042549W WO2007053644A2 WO 2007053644 A2 WO2007053644 A2 WO 2007053644A2 US 2006042549 W US2006042549 W US 2006042549W WO 2007053644 A2 WO2007053644 A2 WO 2007053644A2
Authority
WO
WIPO (PCT)
Prior art keywords
class
cell line
molecule
individual
molecules
Prior art date
Application number
PCT/US2006/042549
Other languages
English (en)
Other versions
WO2007053644A3 (fr
Inventor
William H. Hildebrand
Angela D. Wahl
Curtis P. Mcmurtrey
Original Assignee
The Board Of Regents Of The University Of Oklahoma
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Board Of Regents Of The University Of Oklahoma filed Critical The Board Of Regents Of The University Of Oklahoma
Publication of WO2007053644A2 publication Critical patent/WO2007053644A2/fr
Publication of WO2007053644A3 publication Critical patent/WO2007053644A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • the present invention relates generally to a methodology of epitope testing for the identification of peptides that bind to an individual soluble MHC Class I or Class Il molecule as well as to peptides identified by such methodology.
  • Class I major histocompatibility complex (MHC) molecules designated HLA class I in humans, bind and display peptide antigen ligands upon the cell surface.
  • the peptide antigen ligands presented by the class I MHC molecule are derived from either normal endogenous proteins ("self) or foreign proteins ("nonself") introduced into the cell. Nonself proteins may be products of malignant transformation or intracellular pathogens such as viruses.
  • class I MHC molecules convey information regarding the internal fitness of a cell to immune effector cells including but not limited to, CD8 + cytotoxic T lymphocytes (CTLs), which are activated upon interaction with "nonself peptides, thereby lysing or killing the cell presenting such "nonself peptides.
  • CTLs cytotoxic T lymphocytes
  • Class Il MHC molecules designated HLA class Il in humans, also bind and display peptide antigen ligands upon the cell surface. Unlike class I MHC molecules which are expressed on virtually ail nucleated cells, class Il MHC molecules are normally confined to specialized cells, such as B lymphocytes, macrophages, dendritic cells, and other antigen presenting cells which take up foreign antigens from the extracellular fluid via an endocytic pathway. The peptides they bind and present are derived from extracellular foreign antigens, such as products of bacteria that multiply outside of cells, wherein such products include protein toxins secreted by the bacteria that often times have deleterious and even lethal effects on the host (e.g. human).
  • class II molecules convey information regarding the fitness of the extracellular space in the vicinity of the cell displaying the class Il molecule to immune effector cells, including but not limited to, CD4 + helper T cells, thereby helping to eliminate such pathogens the examination of such pathogens is accomplished by both helping B cells make antibodies against microbes, as well as toxins produced by such microbes, and by activating macrophages to destroy ingested microbes.
  • immune effector cells including but not limited to, CD4 + helper T cells
  • Class I and class Il HLA molecules exhibit extensive polymorphism generated by systematic recombinatorial and point mutation events; as such, hundreds of different HLA types exist throughout the world's population, resulting in a large immunological diversity. Such extensive HLA diversity throughout the population results in tissue or organ transplant rejection between individuals as well as differing susceptibilities and/or resistances to infectious diseases. HLA molecules also contribute significantly to autoimmunity and cancer. Because HLA molecules mediate most, if not all, adaptive immune responses, large quantities of pure isolated HLA proteins are required in order to effectively study transplantation, autoimmunity disorders, and for vaccine development. [0006] There are several applications in which purified, individual class I and class Il MHC proteins are highly useful.
  • Such applications include using MHC-peptide multimers as immunodiagnostic reagents for disease resistance/autoimmunity; assessing the binding of potentially therapeutic peptides; elution of peptides from MHC molecules to identify vaccine candidates; screening transplant patients for preformed MHC specific antibodies; and removal of anti-HLA antibodies from a patient. Since every individual has differing MHC molecules, the testing of numerous individual MHC molecules is a prerequisite for understanding the differences in disease susceptibility between individuals. Therefore, purified MHC molecules representative of the hundreds of different HLA types existing throughout the world's population are highly desirable for unraveling disease susceptibilities and resistances, as well as for designing therapeutics such as vaccines. [0007] Class I HLA molecules alert the immune response to disorders within host cells.
  • Peptides which are derived from viral- and tumor-specific proteins within the cell, are loaded into the class I molecule's antigen binding groove in the endoplasmic reticulum of the cell and subsequently carried to the cell surface. Once the class I HLA molecule and its loaded peptide ligand are on the cell surface, the class I molecule and its peptide ligand are accessible to cytotoxic T lymphocytes (CTL). CTL survey the peptides presented by the class I molecule and destroy those cells harboring ligands derived from infectious or neoplastic agents within that cell.
  • CTL cytotoxic T lymphocytes
  • Discerning virus- and tumor-specific ligands for CTL recognition is an important component of vaccine design.
  • Ligands unique to tumorigenic or infected cells can be tested and incorporated into vaccines designed to evoke a protective CTL response.
  • Several methodologies are currently employed to identify potentially protective peptide ligands.
  • One approach uses T cell lines or clones to screen for biologically active ligands among chromatographic fractions of eluted peptides (Cox et al., Science, vol 264, 1994, pages 716- 719, which is expressly incorporated herein by reference in its entirety). This approach has been employed to identify peptide ligands specific to cancerous cells.
  • a second technique utilizes predictive algorithms to identify peptides capable of binding to a particular class I molecule based upon previously determined motif and/or individual ligand sequences (De Groot et al., Emerging Infectious Diseases, (7) 4, 2001 , which is expressly incorporated herein by reference in its entirety). Peptides having high predicted probability of binding from a pathogen of interest can then be synthesized and tested for T cell reactivity in various assays, such as but not limited to, precursor, tetramer and ELISpot assays. [0010] However, there has been no readily available source of individual HLA molecules. The quantities of HLA protein available have been small and typically consist of a mixture of different HLA molecules.
  • HLA molecules traditionally involves growth and lysis of cells expressing multiple HLA molecules. Ninety percent of the population is heterozygous at each of the HLA loci; codominant expression results in multiple HLA proteins expressed at each HLA locus.
  • To purify native class I or class Il molecules from mammalian cells requires time-consuming and cumbersome purification methods, and since each cell typically expresses multiple surface-bound HLA class I or class Il molecules, HLA purification results in a mixture of many different HLA class I or class Il molecules.
  • interpretation of results cannot directly distinguish between the different HLA molecules, and one cannot be certain that any particular HLA molecule is responsible for a given result.
  • FIG. 1 Overview of 2 stage PCR strategy to amplify a truncated version of the human class I MHC.
  • FIG. 2 Flow chart of the epitope discovery of C-terminal-tagged sHLA molecules.
  • Class I positive transfectants are infected with a pathogen of choice, and sHLA is preferentially purified utilizing the tag.
  • Subtractive comparison of MS ion maps yields ions present only in infected cell, which are then MS/MS sequenced to derive class I epitopes.
  • the present invention combines methodologies for assaying the binding of peptide epitopes to individual, soluble MHC molecules with methodologies for the production of individual, soluble MHC molecules and with a method of epitope discovery and comparative ligand mapping (including methods of distinguishing infected/tumor cells from uninfected/non-tumor cells).
  • the method of production of individual, soluble MHC molecules has previously been described in detail in parent application U.S. Publication No. 2003/0166057, filed December 18, 2001, entitled “METHOD AND APPARATUS FOR THE PRODUCTION OF SOLUBLE MHC ANTIGENS AND USES THEREOF," the contents of which are hereby expressly incorporated herein in their entirety by reference.
  • genomic DNA or cDNA encoding at least one class I molecule is obtained, and an allele encoding an individual class I molecule in the genomic DNA or cDNA is identified.
  • the allele encoding the individual class I molecule is PCR amplified in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule.
  • the PCR product is then cloned into an expression vector, thereby forming a construct that encodes the individual soluble class I molecule, and the construct is transfected into a cell line to provide a cell line containing a construct that encodes an individual soluble class I molecule.
  • the cell line must be able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules.
  • the cell line is then cultured under conditions which allow for expression of the individual soluble class I molecules from the construct, and these conditions also allow for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell.
  • the secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto are then isolated.
  • the construct that encodes the individual soluble class I molecule may further encode a tag, such as a HIS tail or a FLAG tail, which is attached to the individual soluble class I molecule and aids in isolating the individual soluble class I molecule.
  • a tag such as a HIS tail or a FLAG tail
  • the peptide of interest may be chosen based on several methods of epitope discovery known in the art. Alternatively, the peptide of interest may be identified by a method for identifying at least one endogenously loaded peptide ligand that distinguishes an infected cell from an uninfected cell.
  • Such method includes providing an uninfected cell line containing a construct that encodes an individual soluble class I molecule, wherein the uninfected cell line is able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules.
  • a portion of the uninfected cell line is infected with at least one of a microorganism (such as HIV, HBV or influenza), a gene from a microorganism or a tumor gene, thereby providing an infected cell line, and both the uninfected cell line and the infected cell line are cultured under conditions which allow for expression of individual soluble class I molecules from the construct.
  • a microorganism such as HIV, HBV or influenza
  • the culture conditions also allow for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell.
  • the secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto are isolated from the uninfected cell line and the infected cell line, and the endogenously loaded peptide ligands are separated from the individual soluble class I molecules from both the uninfected cell line and the infected cell line.
  • the endogenously loaded peptide ligands are then isolated from both the uninfected cell line and the infected cell line, and the two sets of endogenously loaded peptide ligands are compared to identify at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the infected cell line that is not presented by the individual soluble class I molecule on the uninfected cell line, or to identify at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule in a substantially greater amount on the infected cell line when compared to the uninfected cell line.
  • the comparison described herein above may also identify at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the uninfected cell line that is not presented by the individual soluble class I molecule on the infected cell line, or that is presented in a substantially greater amount on the uninfected cell line when compared to the infected cell line.
  • substantially greater amount refers to an amount that is detectably greater than another amount; for example, the term “presented in a substantially greater amount” as used herein refers to an at least 1-fold increase in a first amount of presentation when compared to a second amount of presentation.
  • the tables provided herein disclose "Fold Increase” amounts for the peptides identified by the methods of the present invention.
  • proteomics may eventually allow for sequencing all epitopes from a diseased cell so that comparative mapping, i.e., comparison of infected cells to healthy cells, would no longer be required. Microarrays and other proteomic data should provide insight as to the healthy cell.
  • a source protein from which the endogenously loaded peptide ligand is obtained can be identified. Such source protein may be encoded by at least one of the microorganism, the gene from a microorganism or the tumor gene with which the cell line was infected to form the infected cell line, or the source protein may be encoded by the uninfected cell line.
  • the present invention is also directed to isolated peptide ligands for an individual class I molecule isolated by the methods described herein.
  • the isolated peptide ligand has a length of from about 7 to about 13 amino acids and consists essentially of a sequence selected from the group consisting of SEQ ID NOS: 1- 315.
  • the isolated peptide ligand has a length of from about 7 to about 13 amino acids and consists essentially of a sequence selected from the group consisting of SEQ ID NOS: 99-301.
  • the isolated peptide ligand has a length of from about 7 to about 13 amino acids and consists essentially of a sequence selected from the group consisting of SEQ ID NOS: 302-315.
  • the isolated peptide ligand described herein above may be an endogenously loaded peptide ligand presented by an individual class I molecule in a substantially greater amount on an infected cell when compared to an uninfected cell.
  • the peptide ligands of the present invention may be isolated by a method that includes providing a cell line containing a construct that encodes an individual soluble class I molecule, wherein the cell line is able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules.
  • the cell line is cultured under conditions which allow for expression of the individual soluble class I molecules from the construct, and also allowing for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell.
  • the isolated peptide ligands of the present invention may be identified by a method that includes providing an uninfected cell line containing a construct that encodes an individual soluble class I molecule, wherein the cell line is able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules. A portion of the uninfected cell line is infected with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing an infected cell line.
  • the uninfected cell line and the infected cell line are cultured under conditions which allow for expression of the individual soluble class I molecules from the construct, and also allow for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell.
  • the secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto are isolated from both the uninfected cell line and the infected cell line; then, the endogenously loaded peptide ligands are separated from the individual soluble class I molecules from the uninfected cell, and the endogenously loaded peptide ligands are separated from the individual soluble class I molecules from the infected cell.
  • the endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line are then isolated and compared. Finally, at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule in a substantially greater amount on the infected cell line when compared to the uninfected cell line is identified.
  • the uninfected cell line containing the construct that encodes the individual soluble class I molecule may be produced by a method that includes obtaining genomic DNA or cDNA encoding at least one class I molecule and identifying an allele encoding an individual class I molecule in the genomic DNA or cDNA.
  • the allele encoding the individual class I molecule is PCR amplified in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule.
  • the PCR product is cloned into an expression vector to form a construct that encodes the individual soluble class I molecule, and the construct is tranfected into an uninfected cell line.
  • the construct may further encode a tag, such as but not limited to, a HIS tail or a FLAG tail, which is attached to the individual soluble class I molecule, and the tag aids in isolating the individual soluble class I molecule.
  • the tag may be encoded by a PCR primer utilized in the PCR step, or the tag may be encoded by the expression vector into which the PCR product is cloned.
  • the at least one endogenously loaded peptide ligand may be obtained from a protein encoded by at least one of the microorganism, the gene from the microorganism or the tumor gene with which the portion of the uninfected cell line is infected to form the infected cell line.
  • the at least one endogenously loaded peptide ligand may be obtained from a protein encoded by the uninfected cell line. Production of Individual, Soluble MHC Molecules
  • the methods of the present invention may, in one embodiment, utilize a method of producing MHC molecules (from genomic DNA or cDNA) that are secreted from mammalian cells in a bioreactor unit.
  • MHC molecules from genomic DNA or cDNA
  • Substantial quantities of individual MHC molecules are obtained by modifying class I or class Il MHC molecules so that they are capable of being secreted, isolated, and purified.
  • Secretion of soluble MHC molecules overcomes the disadvantages and defects of the prior art in relation to the quantity and purity of MHC molecules produced. Problems of quantity are overcome because the cells producing the MHC do not need to be detergent lysed or killed in order to obtain the MHC molecule. In this way the cells producing secreted MHC remain alive and therefore continue to produce MHC.
  • MHC secreting cell lines allow for high concentrations of individual MHC proteins to be obtained. Highly concentrated individual MHC proteins provide an advantage in that most downstream protein purification strategies perform better as the concentration of the protein to be purified increases. Thus, the culturing of MHC secreting cells in bioreactors allows for a continuous production of individual MHC proteins in a concentrated form.
  • the method of producing MHC molecules utilized in the present invention and described in detail in U.S. Serial No. 10/022,066 begins by obtaining genomic or complementary DNA which encodes the desired MHC class I or class Il molecule. Alleles at the locus which encode the desired MHC molecule are PCR amplified in a locus specific manner.
  • locus specific PCR products may include the entire coding region of the MHC molecule or a portion thereof.
  • a nested or hemi-nested PCR is applied to produce a truncated form of the class I or class Il gene so that it will be secreted rather than anchored to the cell surface.
  • FIG. 1 illustrates the PCR products resulting from such nested PCR reactions.
  • the PCR will directly truncate the MHC molecule.
  • Locus specific PCR products are cloned into a mammalian expression vector and screened with a variety of methods to identify a clone encoding the desired MHC molecule.
  • the cloned MHC molecules are DNA sequenced to ensure fidelity of the PCR.
  • Faithful truncated clones of the desired MHC molecule are then transfected into a mammalian cell line.
  • a mammalian cell line When such cell line is transfected with a vector encoding a recombinant class I molecule, such cell line may either lack endogenous class I MHC molecule expression or express endogenous class I MHC molecules.
  • One of ordinary skill of the art would note the importance, given the present invention, that cells expressing endogenous class I MHC molecules may spontaneously release MHC into solution upon natural cell death, infection, transformation, etc.
  • the transfected class I MHC molecule can be "tagged" such that it can be specifically purified away from spontaneously released endogenous class I molecules in cells that express class I molecules.
  • a DNA fragment encoding a HIS tail may be attached to the protein by the PCR reaction or may be encoded by the vector into which the PCR fragment is cloned, and such HIS tail, therefore, further aids in the purification of the class I MHC molecules away from endogenous class I molecules.
  • Tags beside a histidine tail have also been demonstrated to work, and one of ordinary skill in the art of tagging proteins for downstream purification would appreciate and know how to tag a MHC molecule in such a manner so as to increase the ease by which the MHC molecule may be purified.
  • genomic DNA fragments contain both exons and introns as well as other non-translated regions at the 5' and 3' termini of the gene.
  • gDNA genomic DNA
  • mRNA messenger RNA
  • Transfection of MHC molecules encoded by gDNA therefore facilitates reisolation of the gDNA, mRNA/cDNA, and protein.
  • MHC molecules in non-mammalian cell lines such as insect and bacterial cells require cDNA clones, as these lower cell types do not have the ability to splice introns out of RNA transcribed from a gDNA clone.
  • the mammalian gDNA transfectants of the present invention provide a valuable source of RNA which can be reverse transcribed to form MHC cDNA.
  • the cDNA can then be cloned, transferred into cells, and then translated into protein.
  • such gDNA transfectants therefore provide a ready source of mRNA, and therefore cDNA clones, which can then be transfected into non-mammalian cells for production of MHC.
  • the present invention which starts with MHC genomic DNA clones allows for the production of MHC in cells from various species.
  • a key advantage of starting from gDNA is that viable cells containing the MHC molecule of interest are not needed. Since all individuals in the population have a different MHC repertoire, one would need to search more than 500,000 individuals to find someone with the same MHC complement as a desired individual - such a practical example of this principle is observed when trying to find a donor to match a recipient for bone marrow transplantation. Thus, if it is desired to produce a particular MHC molecule for use in an experiment or diagnostic, a person or cell expressing the MHC allele of interest would first need to be identified.
  • the MHC molecule of interest could be obtained via a gDNA clone as described herein, and following transfection of such clone into mammalian cells, the desired protein could be produced directly in mammalian cells or from cDNA in several species of cells using the methods of the present invention described herein.
  • RNA is inherently unstable and is not as easily obtained as is gDNA. Therefore, if production of a particular MHC molecule starting from a cDNA clone is desired, a person or cell line that is expressing the allele of interest must traditionally first be identified in order to obtain RNA.
  • cDNA may be substituted for genomic DNA as the starting material
  • production of cDNA for each of the desired HLA class I types will require hundreds of different, HLA typed, viable cell lines, each expressing a different HLA class I type.
  • fresh samples are required from individuals with the various desired MHC types.
  • genomic DNA as the starting material allows for the production of clones for many HLA molecules from a single genomic DNA sequence, as the amplification process can be manipulated to mimic recombinatorial and gene conversion events.
  • Several mutagenesis strategies exist whereby a given class I gDNA clone could be modified at either the level of gDNA or at the cDNA resulting from this gDNA clone.
  • the process of producing MHC molecules utilized in the present invention does not require viable cells, and therefore the degradation which plagues RNA is not a problem.
  • Peptide epitopes unique to infected and cancerous cells can be directly identified by the methods of the present invention, which include producing sHLA molecules in cancerous and infected cells and then sequencing the epitopes unique to the cancerous or infected cells. Such epitopes can then be tested for their binding to various HLA molecules to see how many HLA molecules these epitopes might bind.
  • This direct method of epitope discovery is described in detail in US 09/974,366 and is briefly described herein below.
  • the method of epitope discovery included in the present invention (and described in detail in US Serial No.
  • 09/974,366 includes the following steps: (1) providing a cell line containing a construct that encodes an individual soluble class I or class Il MHC molecule (wherein the cell line is capable of naturally processing self or nonself proteins into peptide ligands capable of being loaded into the antigen binding grooves of the class I or class Il MHC molecules); (2) culturing the cell line under conditions which allow for expression of the individual soluble class I or class Il MHC molecule from the construct, with such conditions also allowing for the endogenous loading of a peptide ligand (from the self or non-self processed protein) into the antigen binding groove of each individual soluble class I or class Il MHC molecule prior to secretion of the soluble class I or class Il MHC molecules having the peptide ligands bound thereto; and (3) separating the peptide ligands from the individual soluble class I or class Il MHC molecules.
  • Class I and class Il MHC molecules are really a trimolecular complex consisting of an alpha chain, a beta chain, and the alpha/beta chain's peptide cargo (i.e. the peptide ligand) which is presented on the cell surface to immune effector cells. Since it is the peptide cargo, and not the MHC alpha and beta chains, which marks a cell as infected, tumorigenic, or diseased, there is a great need to identify and characterize the peptide ligands bound by particular MHC molecules.
  • the alpha/beta chain's peptide cargo i.e. the peptide ligand
  • characterization of such peptide ligands greatly aids in determining how the peptides presented by a person with MHC- associated diabetes differ from the peptides presented by the MHC molecules associated with resistance to diabetes.
  • having a sufficient supply of an individual MHC molecule, and therefore that MHC molecule's bound peptides provides a means for studying such diseases.
  • the method of the present invention provides quantities of MHC protein previously unobtainable, unparalleled studies of MHC molecules and their important peptide cargo can now be facilitated and utilized to distinguish infected/tumor cells from uninfected/non-tumor cells by unique epitopes presented by MHC molecules in the disease or non-disease state.
  • the method of the present invention includes the direct comparative analysis of peptide ligands eluted from class I HLA molecules (as described previously in U.S. Publication No. 2002/097672).
  • the teachings of U.S. Publication No. 2002/097672 demonstrates that the addition of a C-terminal epitope tag (such as a 6-HIS or FLAG tail) to transfected class I molecules has no effects on peptide binding specificity of the class I molecule and consequently has no deleterious effects on direct peptide ligand mapping and sequencing, and also does not disrupt endogenous peptide loading.
  • the method described in parent application U.S. Publication No. 2002/097672 further relates to a novel method for detecting those peptide epitopes which distinguish the infected/tumor cell from the uninfected/non-tumor cell.
  • the results obtained from the present inventive methodology cannot be predicted or ascertained indirectly; only with a direct epitope discovery method can the unique epitopes described therein be identified. Furthermore, only with this direct approach can it be ascertained that the source protein is degraded into potentially immunogenic peptide epitopes. Finally, this unique approach provides a glimpse of which proteins are uniquely up and down regulated in infected/tumor cells.
  • HLA-presented peptide epitopes which mark the infected/tumor cell are three-fold.
  • diagnostics designed to detect a disease state i.e., infection or cancer
  • epitopes unique to infected/tumor cells represent vaccine candidates.
  • the present invention describes and claims epitopes which arise on the surface of cells infected with HIV. Such epitopes could not be predicted without natural virus infection and direct epitope discovery.
  • the epitopes detected are derived from proteins unique to virus infected and tumor cells.
  • epitopes can be used for virus/tumor vaccine development and virus/tumor diagnostics.
  • the process indicates that particular proteins unique to virus infected cells are found in compartments of the host cell they would otherwise not be found in. Thus, uniquely upregulated or trafficked host proteins are identified for drug targeting to kill infected cells.
  • epitopes detected as unique to infected/tumor cells may serve as direct targets (i.e., through diagnostic, vaccine or therapeutic means), such epitopes may also be utilized to influence the environment around a diseased cell so that these treatments and therapies are effective, and thus allowing the immune responses to see the diseased cell.
  • the presently disclosed and claimed invention, as well as the parent application U.S. Publication No. 2002/097672, describe, in particular, peptide epitopes unique to HIV infected cells. Peptide epitopes unique to the HLA molecules of HIV infected cells were identified by direct comparison to HLA peptide epitopes from uninfected cells by the method illustrated in the flow chart of FIG. 2.
  • Such method has been shown to be capable of identifying: (1) HLA presented peptide epitopes, derived from intracellular host proteins, that are unique to infected cells but not found on uninfected cells, and (2) that the intracellular source-proteins of the peptides are uniquely expressed/processed in HIV infected cells such that peptide fragments of the proteins can be presented by HLA on infected cells but not on uninfected cells.
  • the method of epitope discovery and comparative ligand mapping also, therefore, describes the unique expression of proteins in infected cells or, alternatively, the unique trafficking and processing of normally expressed host proteins such that peptide fragments thereof are presented by HLA molecules on infected cells.
  • HLA presented peptide fragments of intracellular proteins represent powerful alternatives for diagnosing virus infected cells and for targeting infected cells for destruction (i.e., vaccine development).
  • a group of the host source-proteins for HLA presented peptide epitopes unique to HIV infected cells represent source-proteins that are uniquely expressed in cancerous cells.
  • a peptide fragment (SEQ ID NO: 12) of reticulocalbin is uniquely found on HIV infected cells.
  • a literature search indicates that the reticulocalbin gene is uniquely upregulated in cancer cells (breast cancer, liver cancer, colorectal cancer).
  • cancer cells breast cancer, liver cancer, colorectal cancer.
  • HLA presented peptide fragments of host genes and gene products that distinguish the tumor cell and virus infected cell from healthy cells have been directly identified.
  • the epitope discovery method is also capable of identifying host proteins that are uniquely expressed or uniquely processed on virus infected or tumor cells. HLA presented peptide fragments of such uniquely expressed or uniquely processed proteins can be used as vaccine epitopes and as diagnostic tools.
  • the methodology of targeting and detecting virus infected cells is not meant to target the virus-derived peptides. Rather, the methodology of the present invention indicates that the way to distinguish infected cells from healthy cells is through alterations in host encoded protein expression and processing. This is true for cancer as well as for virus infected cells.
  • the methodology according to the present invention results in data which indicates, without reservation, that proteins/peptides distinguish virus/tumor cells from healthy cells.
  • a cell line producing individual, soluble MHC molecules is constructed as described herein before and in US Publication No. 2003/0166057.
  • a portion of the transfected cell line is cocultured with a virus of interest, resulting in high-titre virus and providing infected cells.
  • influenza virus the infection is not productive in the bioreactor and does not result in the production of high titer virus. Because of this, fresh influenza virus was added to the coculture.
  • the viruses of interest are HIV, influenza and WNV.
  • a portion of the cell line producing individual, soluble MHC molecules may be transformed to produce a tumor cell line.
  • the non-infected cell line and the cell line infected with HIV are both cultured in hollow-fiber bioreactors as described herein above and in detail in US Publication No. 2003/0166057, and the soluble HLA-containing supernatant is then removed from the hollow-fiber bioreactors.
  • the uninfected and infected harvested supernatants were then treated in an identical manner post-removal from the cell-pharm.
  • MHC class l-peptide complexes were affinity purified from the infected and uninfected supernatants using W6/32 antibody. Following elution, peptides were isolated from the class I molecules and separated by reverse phase HPLC fractionation. Separate but identical (down to the same buffer preparations) peptide purifications were done for each peptide-batch from uninfected and infected cells.
  • Fractionated peptides were then mapped by mass spectrometry to generate fraction-based ion maps. Spectra from the same fraction in uninfected/infected cells were manually aligned and visually assessed for the presence of differences in the ions represented by the spectra. Ions corresponding to the following categories were selected for MS/MS sequencing: (1) upregulation in infected cells (at least 1.5 fold over the same ion in uninfected cells), (2) downregulation in infected cells (at least 1.5 fold over the same ion in the uninfected cells), (3) presence of the ion only in infected cells, or (4) absence of ion in infected cells that is present in uninfected cells.
  • peptides were assigned to one of the above categories, including checking the peptide fractions preceding and following the peptide fraction by MS/MS to ensure that the peptide of interest was not present in an earlier or later fraction as well as generation of synthetic peptides and subjection to MS/MS to check for an exact match.
  • one early quality control step involves examining the peptide's sequence to see if it fits the "predicted motif defined by sequences that were previously shown to be presented by the MHC molecule utilized.
  • Table I lists peptide ligands that have been identified as being presented by the B*0702 and A*0201 or B*1801 class I MHC molecule in cells infected with the HIV MN-1 virus but not in uninfected cells, and also lists one peptide ligand that has been identified as not being presented by the B*0702 class I MHC molecule in cells infected with the HIV MN-1 virus that is presented in uninfected cells.
  • One of ordinary skill in the art can appreciate the novelty and usefulness of the present methodology in directly identifying such peptide ligands and the importance such identification has for numerous therapeutic (vaccine development, drug targeting) and diagnostic tools.
  • Table I identifies the sequences of peptide ligands identified to date as being unique to HIV infected cells.
  • Class I sHLA B*0702, A*0201 or B*1801 was harvested from T cells infected and not infected with HIV.
  • Peptide ligands were eluted from B*0702, A*0201 or B*1801 and comparatively mapped on a mass spectrometer so that ions unique to infected cells were apparent.
  • Ions unique to infected cells (and one ligand unique to uninfected cells) were subjected to mass spectrometric fragmentation for peptide sequencing.
  • Restricting allele for Sequences marked with a (•) is HLA-B*0702.
  • Restrictin allele for Se uences marked with a D is HLA-A*0201 or HLA-B*1801.
  • a human T cell line was utilized for infection with HIV.
  • This cell line, Sup-T1 possesses its own class I; HLA-A and -B types are A*2402, A * 6801 , B*0801, and £*1801.
  • HLA-B*1801 is shed after HIV infection.
  • A*0201 prefers peptides with an L at position 2 (P2) and an L or V at P9. Most of the peptides that did not match the A*0201 motif had an E at P2 and a Y or F at P9.
  • the methodology used herein is to use sHLA to determine what is unique to unhealthy cells as compared to healthy cells. Using sHLA to survey the contents of a cell provides a look at what is unique to unhealthy cells in terms of proteins that are processed into peptides.
  • the data summarized in TABLE I shows that the epitope discovery technique described herein is capable of identifying sHLA bound epitopes and their corresponding source proteins which are unique to infected/unhealthy cells.
  • peptide ligands presented in individual class I MHC molecules in an uninfected cell that are not presented by individual class I MHC molecules in an uninfected cell can also be identified.
  • the peptide "GSHSMRY” (SEQ ID NO:98), for example, was identified by the method of the present invention as being an individual class I MHC molecule which is presented in an uninfected cell but not in an infected cell.
  • the source protein for this peptide is MHC Class I Heavy Chain, which could be derived from multiple alleles, i.e., HLA-B*0702 or HLA-G, etc.
  • the utility of this data is at least threefold.
  • Third, realization of the source protein can lead to therapies and diagnostics which target the source protein.
  • an epitope unique to unhealthy cells also indicates that the source protein is unique in the unhealthy cell.
  • Unhealthy cells analyzed by the epitope discovery process described herein can arise from virus infection or also from cancerous transformation. Unhealthy cells may also be produced following treatment of healthy cells with a cancer causing agent, such as but not limited to, nicotine, or by a disease state cytokine such as IL-4.
  • a cancer causing agent such as but not limited to, nicotine
  • a disease state cytokine such as IL-4.
  • the status of an unhealthy cell can also be mimicked by transfecting a particular gene known to be expressed during viral infection or tumor formation.
  • particular genes of HIV can be expressed in a cell line as described (Achour, A., et al., AIDS Res Hum Retroviruses, 1994.
  • epitope discovery with sHLA as described herein can be completed on cells infected with intact pathogens, cancerous cells or cell lines, or cells into which a particular cancer, viral, or bacterial gene has been transferred.
  • the sHLA described here will provide a means for detecting what changes in terms of epitope presentation and the source proteins for the epitopes.
  • the methods of the present invention have also been applied to identifying epitopes unique or upregulated in influenza infected cells as well as West Nile virus infected cells.
  • the methods for obtaining soluble HLA form cells infected with Influenza and West Nile Virus (WNV) are similar to those described hereinabove for HIV infection, except as described herein below.
  • WNV West Nile Virus
  • the viral infection was monitored to ensure that the cells secreting the HLA molecules were infected. For Influenza, this was accomplished by measuring intracellular infection using antibody staining combined with flow cytometry.
  • WNV West Nile virus
  • Table Il lists unique and upregulated peptide epitopes that have been identified by the A*0201 and B*0702 class I MHC molecules in cells infected with the PR8 strain of influenza A virus.
  • Table III lists unique peptide epitopes that have been identified by the A*0201 class I MHC molecules in cells infected with the West Nile virus. Both self and viral epitopes have been identified.

Landscapes

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

Abstract

La présente invention concerne de manière générale un procédé d'isolation, de purification et d'identification de ligands peptidiques présentés par des cellules positives vis-à-vis du MHC. Le procédé selon la présente invention concerne en particulier l'isolation, la purification et l'identification de cellules de ces ligands peptidiques dans des molécules solubles de MHC de classe I et de classe II qui peuvent provenir de cellules non infectées, infectées ou carcinogènes. Le procédé selon la présente invention permet d'identifier et d'utiliser largement ces ligands peptidiques et leurs protéines sources apparentées comme marqueurs qui permettent de distinguer les cellules infectées des cellules non infectées et/ou les cellules carcinogènes vis-à-vis des cellules non carcinogènes, ladite identification pouvant être utilisée pour marquer ou cibler une cellule dans un traitement thérapeutique ou pour amorcer la réponse immunitaire vis-à-vis de cellules infectées.
PCT/US2006/042549 2005-11-01 2006-11-01 Cartographie comparative des ligands de cellules positives vis-a-vis du mhc de classe i WO2007053644A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US73218305P 2005-11-01 2005-11-01
US60/732,183 2005-11-01
US80013406P 2006-05-12 2006-05-12
US60/800,134 2006-05-12

Publications (2)

Publication Number Publication Date
WO2007053644A2 true WO2007053644A2 (fr) 2007-05-10
WO2007053644A3 WO2007053644A3 (fr) 2007-09-20

Family

ID=38006475

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/042549 WO2007053644A2 (fr) 2005-11-01 2006-11-01 Cartographie comparative des ligands de cellules positives vis-a-vis du mhc de classe i

Country Status (1)

Country Link
WO (1) WO2007053644A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009074569A1 (fr) * 2007-12-11 2009-06-18 Bracco International Bv Composés de ciblage et thérapeutiques avec un espaceur comprenant de la polyproline et microvésicules remplies de gaz comprenant lesdits composés
WO2016127249A1 (fr) 2015-02-09 2016-08-18 Université de Montréal Nouveaux antigènes mineurs d'histocompatibilité et leurs utilisations

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002083903A2 (fr) * 2001-02-28 2002-10-24 Brown University Research Foundation Methodes de determination des epitopes du virus du nil occidental et methode d'utilisation correspondante
US20050267020A1 (en) * 2001-09-27 2005-12-01 Olivier Faure Polypeptides derived from inducible hsp70 and pharmaceutical compositions containing the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002083903A2 (fr) * 2001-02-28 2002-10-24 Brown University Research Foundation Methodes de determination des epitopes du virus du nil occidental et methode d'utilisation correspondante
US20050267020A1 (en) * 2001-09-27 2005-12-01 Olivier Faure Polypeptides derived from inducible hsp70 and pharmaceutical compositions containing the same

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009074569A1 (fr) * 2007-12-11 2009-06-18 Bracco International Bv Composés de ciblage et thérapeutiques avec un espaceur comprenant de la polyproline et microvésicules remplies de gaz comprenant lesdits composés
WO2016127249A1 (fr) 2015-02-09 2016-08-18 Université de Montréal Nouveaux antigènes mineurs d'histocompatibilité et leurs utilisations
JP2018505682A (ja) * 2015-02-09 2018-03-01 ウニヴェルシテ・ドゥ・モントリオール 新規マイナー組織適合抗原およびその使用
EP3256488A4 (fr) * 2015-02-09 2018-10-17 Université de Montréal Nouveaux antigènes mineurs d'histocompatibilité et leurs utilisations
US10414813B2 (en) 2015-02-09 2019-09-17 Université de Montréal Minor histocompatibility antigens and uses thereof

Also Published As

Publication number Publication date
WO2007053644A3 (fr) 2007-09-20

Similar Documents

Publication Publication Date Title
KR102488477B1 (ko) 신속하고 포괄적인 t-세포 면역 모니터링을 위한 세포 플랫폼
JP6718450B2 (ja) キメラ抗原受容体及びその使用方法
US20020156773A1 (en) Soluble HLA ligand database utilizing predictive algorithms and methods of making and using same
CN103249430A (zh) 抗原特异性t细胞受体和t细胞表位
JP7038703B2 (ja) Ｈｂｖ抗原特異的結合分子およびそのフラグメント
US20120301871A1 (en) Comparative ligand mapping from mhc positive cells
KR20210126073A (ko) 항원 특이적 t 세포의 식별을 위한 조성물 및 방법
US20030166057A1 (en) Method and apparatus for the production of soluble MHC antigens and uses thereof
US20120329670A1 (en) Epitope testing using soluble hla
Bioley et al. Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers
US20060034865A1 (en) Soluble MHC artificial antigen presenting cells
US12066430B2 (en) Trogocytosis mediated epitope discovery methods
KR20190133192A (ko) 야생형 항원 및 강력한 펩타이드 미모토프를 인식하는 환자 종양으로부터 단리된 t 세포 수용체에 대한 항원 발견
Sundaram et al. A novel multivalent human CTL peptide construct elicits robust cellular immune responses in HLA-A∗ 0201 transgenic mice: implications for HTLV-1 vaccine design
Wiendl et al. An autoreactive γδ TCR derived from a polymyositis lesion
US20050003483A1 (en) Comparative ligand mapping from MHC class 1 positive cells
US20190227063A1 (en) Methods and compositions for t-cell epitope screening
US20070099182A1 (en) Comparative ligand mapping from MHC class I positive cells
WO2007053644A2 (fr) Cartographie comparative des ligands de cellules positives vis-a-vis du mhc de classe i
US7521202B2 (en) Method and apparatus for the production of soluble MHC antigens and uses thereof
WO2004101607A2 (fr) Representation cartographique comparative de ligands a partir de cellules positives de classe i du cmh
KR20010043984A (ko) Ｈｌａ-ｂ35 분자에 결합하는 분리된 펩티드
JP2025501732A (ja) Ｄｃａｆ４ｌ２特異的ｔ細胞受容体
JP2013078325A (ja) Ｈｌａ結合性ペプチド、その前駆体、それをコードするｄｎａ断片および組み換えベクター
WO2002056908A2 (fr) Cellules de presentation d'antigene

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06836726

Country of ref document: EP

Kind code of ref document: A2