EP1157102A1 - Inhibition du chemin de presentation de l'antigene du complexe majeur d'histocompatibilite de classe ii et presentation aux cellules cd4+ - Google Patents

Inhibition du chemin de presentation de l'antigene du complexe majeur d'histocompatibilite de classe ii et presentation aux cellules cd4+

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Publication number
EP1157102A1
EP1157102A1 EP00905939A EP00905939A EP1157102A1 EP 1157102 A1 EP1157102 A1 EP 1157102A1 EP 00905939 A EP00905939 A EP 00905939A EP 00905939 A EP00905939 A EP 00905939A EP 1157102 A1 EP1157102 A1 EP 1157102A1
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Prior art keywords
protein
cells
vector
mhc
class
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EP00905939A
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German (de)
English (en)
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EP1157102A4 (fr
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David C. Johnson
Roman Tomazin
Jessica Boname
Nagendra R. Hegde
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Oregon Health Science University
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Oregon Health Science University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • FIELD This invention relates to an immunosuppressive agent that can be used to improve persistence of viral vectors and the transgene used in gene therapy, sustain transplanted cells and organs, or to treat autoimmune disorders.
  • non-self antigens e.g. , such as antigens present on transplanted tissues
  • T lymphocytes T cells
  • MHC major histocompatibility
  • T cells have antigen-binding molecules on their cell surfaces, called T-cell receptors, that react with MHC proteins bearing peptide antigens on the APCs surface-
  • MHC class I molecules collect peptides derived primarily from proteins synthesized in the cytosol, and are thus able to display fragments of viral and cellular proteins, normally found inside cells, on the cell surface.
  • MHC class II molecules bind peptides derived from proteins that are often taken up by cells into phagocytic or endocytic vesicles by macrophages and B cells. Internal antigens, produced inside MHC class II-expressing cells, can also be presented by MHC class II proteins.
  • CD8+ T cells are often cytotoxic T lymphocytes that recognize and remove virus-infected, tumor, or transplanted cells.
  • CD4 + T cells often provide "help" in the form of cytokines and costimulation that is necessary for expansion and activation of CD8+ T cells, and also B cells that produce antibodies.
  • vectors such as viruses
  • Both the vector and the protein or transgene can be recognized by the immune system, and cause an immune response to be mounted.
  • the immune response often involves CD8+ and CD4+ T cells and these cells interfere with the function of the vector, and reduce both the effectiveness and duration of the therapy.
  • U.S. Patent No. 5,750,398 discloses a method of the treatment and prevention of autoimmune disease by administering or expressing the Herpes Simplex Virus (HSV) immediate early protein ICP47.
  • HSV Herpes Simplex Virus
  • This protein inhibits the presentation of viral and cellular antigens associated with MHC I proteins to CD8+ T cells. This inhibition was said to be useful to improve the effectiveness of viral gene therapy vectors, by increasing infective persistence.
  • PCT publication WO 97/32605 disclosed a recombinant human cytomegalovirus nucleic acid encoding a US1 1 or US2 protein. These proteins, which were said to have specific binding domains for MHC I molecules, downregulated expression of class I MHC genes.
  • the invention included mutating the binding domain of US2 to recognize MHC II but not MHC I molecules, to change the binding specificity and immune function of the protein.
  • no evidence was provided that a mutated form of HCMV US2 could bind to or downregulate MHC II.
  • specific downregulation of MHC I or II mediated immunity would be helpful in some situations, it would also be useful to have immune modulators that would downregulate both arms of the cellular immune reaction.
  • the present invention concerns use of the human cytomegalovirus (HCMV) protein, US2, or soluble fragments or variants thereof, to inhibit inappropriate immune responses, for example immunity directed at vectors or the transgenes expressed by such vectors, immunity to transplanted cells or tissues, or in autoimmune disorders.
  • HCMV human cytomegalovirus
  • US2 effectively inhibits the MHC class II antigen presentation pathway by causing degradation of two components of this pathway, namely MHC-II- ⁇ and DM- ⁇ . When these pathway components are destroyed or disabled, cells that normally present antigens via the MHC II pathway can no longer present these antigens to CD4+ T cells.
  • US2 when US2, or a biologically active variant thereof (e.g., a soluble active fragment of US2), is expressed in cells or in tissues, it will have a dual effect of blocking recognition by both CD4+ and CD8+ T cells.
  • a biologically active variant thereof e.g., a soluble active fragment of US2
  • the CD4+ T cell response is dominant, or is required in order for CD8+ T cells to become activated and expand. Therefore, the effects of US2 on the MHC II pathway are important and essential for the protein to function in an effective manner.
  • PCT WO 97/32605 described mechanistic details of how US2 inhibits the MHC-I antigen presentation pathway.
  • the invention to include a method for inhibiting cell recognition by both CD8+ and CD4+ cells, by introducing into a mammalian cell an isolated nucleotide sequence encoding US2, or a variant thereof which maintains the native activity of US2 (e.g., a soluble variant), and wherein the binding domain recognizes both MHC I and MHC II molecules.
  • the binding domain of US2 may be a native binding domain, and not a binding domain that has been mutated to recognize MHC II molecules.
  • the nucleotide sequence may be introduced through the use of a viral vector, such as an adeno virus or a retrovirus.
  • the invention includes a recombinant expression vector that includes a promoter operably linked to a nucleotide sequence encoding a US2 protein, or a biologically active variant thereof, wherein a binding domain of the US2 protein or variant has not been mutated to recognize MHC II molecules.
  • the expression vector inhibits the ability of the cell to present antigens associated with MHC class II proteins to T cells. Inhibition of this ability is useful in the treatment of autoimmune diseases in which CD4+ T cells recognize self MHC II proteins, or in tissue and organ transplantation where CD4+ T cells recognize foreign MHC II proteins.
  • the vector includes an isolated nucleotide sequence that encodes a protein having the activity of native US2.
  • the vector can be used in methods of treating autoimmune disease, or improving gene therapy, by administering the vector to a subject.
  • the vector also includes a nucleotide sequence that codes for a therapeutic product.
  • Another embodiment is a purified soluble protein having US2 protein biological activity.
  • Such proteins include the US2 binding site for MHC II, and for instance may include the amino acid sequence as shown in about residues 28 through about 143 of SEQ ID NO: 3. Also encompassed in the invention are isolated nucleic acid molecules that encode these purified soluble US2 proteins, and recombinant nucleic acid molecules and cells that include them.
  • MHC class II expression is reduced in HCMV-infected cells
  • Fig. 1 A U373 cells were treated with IFN- ⁇ for 72 hours then left uninfected or infected with wildtype (Wt) HCMV (strain AD 169) or a mutant HCMV lacking the IRS 1 -US 11 genes.
  • MHC class II complexes were immunoprecipitated from cell extracts using MAb DA6.147, specific for class Il- ⁇ or HCMV immediate early (IE1) protein 86, precipitated using a rabbit polyclonal. Samples were separated by using 14% polyacrylamide gels.
  • Fig. IB U373 cells transfected with the MHCII transactivator gene CIITA (U373- CIITAHis) to induce constitutive expression of MHC class II were left uninfected or infected with wildtype HCMV or HCMV mutants lacking the genes indicated for 36 hours. Cells were radio- labeled and class II or IE immunoprecipitated as described in A.
  • Fig. lC U373CIITAHis cells were left uninfected or infected with wildtype HCMV or the HCMV mutant unable to express IRS 1 -US 11 for 36 hours. Total RNA was harvested and separated using agarose gels containing formaldehyde. Cells were disrupted using the TRIZOL reagent
  • RNA was separated using electrophoresis with a 1% agarose gel containing 2.2 M formaldehyde, transferred to Hybond- N+ membranes and the membranes probed with linearized, 32 P-labeled plasmids containing the HLA- DR-alpha, HLA-DR-beta, invariant chain, CIITA, HCMV glycoprotein B (gB), or beta-actin gene.
  • the RNA was transferred to membranes and probed with 32 P-labeled plasmids containing the class II- DR- ⁇ , DR- ⁇ , Ii, CIITA, glycoprotein B or ⁇ -actin genes.
  • Fig. 2A U373CIITA-H ⁇ s cells were left uninfected or infected with an AdtetUS2 and Adtet- trans using two quantities of virus 10 PFU/cell of Adtet-trans and 50 PFU/cell of AdtetUS2, or 20 PFU/cell of Adtet-trans and 100 PFU/cell of AdtetUS2 for 12 hours
  • the cells were radio-labeled with 35 S-meth ⁇ omne/cyste ⁇ ne for 1 minute, then the label chased with excess methiomne for 0, 15 or 30 minutes before cell extracts were prepared Immunoprecipitations were then performed under non- denaturing conditions for antibodies specific for the class II complex (DR- ⁇ -specific MAb DA6 147), US2 (rabbit ant ⁇ -US2N), class I heavy chain (rabbit anti-HC), transfer ⁇ n receptor (TfR) (MAb B3/25), calnexm (rabbit anti-Cxn/c), or grp-
  • DM- ⁇ and DM- ⁇ were denatured and immunoprecipitated using the MAb 5C1 and MaP DMB-C, respectively
  • US2 induces degradation of class Il- ⁇ chains by proteosomes without a cytosohc intermediate Fig. 3A.
  • U373-CIITAH ⁇ s cells were left unmfected or infected with Adtet-trans and
  • U373CIITA-H1S cells were infected, treated with 35 ⁇ M ZL3VS, and then radio- labeled as described m 3 A
  • the cells were then subjected to subcellular fractionation, so that 1,000 X g, 10,000 X g and 100,000 X g pellets and 100,000 X g supernatants were prepared
  • Radio-labeled cells were homogenized and subjected to subcellular fractionation, as previously described (Wiertz et al , Nature 384 432-438, 1996) with the following exceptions
  • Subcellular pellets were resuspended and denatured in SDS lysis buffer SDS to a concentration of 1% was added to the 100,000 x g supernatant before heating, then 1% Triton X-l 00 was added, and the SDS removed by using SDS- Out (Pierce Chemical Company, Rockford. IL).
  • Membrane pellets or supernatant fractions were denatured using SDS then MHC class Il-ct, M
  • Fig. 4A US2 was immunoprecipitated using a rabbit polyclonal antibody, then proteins denatured with SDS, the SDS diluted and samples immunoprecipitated using anti-US2, anti-class Il- ⁇ , anti-class II- ⁇ , anti-Ii, anti-class I HC or anti- transferrin receptor (TfR) antibodies.
  • Fig. 4B Extracts were immunoprecipitated using anti-class Il- ⁇ , anti-class II- ⁇ , anti-Ii, anti- class I HC or anti-transferrin receptor antibodies, samples denatured and reprecipitated using anti- US2 antibodies or antibodies directed to ⁇ , ⁇ , Ii, HC, or TfR.
  • CIITA-HIS16 cells were left uninfected or were infected with Adtet-trans, an adenovirus control without US2, or using AdUS2 and Adtet-trans, so that HCMV US2 was expressed in the cells, for 18 hours. The cells were washed and mixed with a CD4+ T cell clone that is specific for tuberculous antigen, and at the same time, with tuberculous antigen TbH9. The ability of the MHC class II antigen presenting CIITA-HIS16 cells to present TbH9 to the CD4+ T cells was measured.
  • Fig. 6 is a schematic diagram of plasmid p ⁇ ElsplB and insert used to construct the Adtet vector of the present invention.
  • the mapped sequence at the bottom of the figure was ligated into plasmid p ⁇ ElsplB (Microbix Biosystems, Inc., Toronto, Ontario, CA) at the Hindlll and Xhol restriction endonuclease sites to produce the plasmid p ⁇ ElsplB-tet.
  • the p ⁇ El splB-tet plasmid possesses a core HCMV promoter with flanking tet operator sequences, as well as an SV40 poly- adenylation site.
  • the complex of tet operator sequences linked to the HCMV core promoter is called the tet responsive element. This element cannot effect transcription without an exogenous transcription factor (ETF); the ETF can be expressed using a recombinant adenovirus (E1 -). Furthermore, this transcription system is inactivated by tetracycline.
  • Fig. 7 is a comparison of the amino acid sequences of US2, US3, and US1 1 , all of which bind to MHC class I. However, as shown in this specification, only US2 binds to MHC class II. By comparing all three related proteins, the sequences that are unique in US2 contain the regions responsible for, or involved in, binding to class II. Fig. 7 shows that residues 28-48 and 122-143 of US2 (underlined and marked with asterisk * in drawing) are less conserved regions between these proteins, and thus are likely to contribute to the binding domain(s) for MHC class II.
  • Soluble US2 blocks antigen presentation by T cell clones.
  • Fig. 8 is a graph showing that the soluble US2t-DL6 inhibits interferon-g secretion by T cells. This demonstrates that the soluble US2 protein blocks antigen presentation to the T cell clones.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 is the reverse complement of a cDNA sequence of the open reading frame that encodes US2.
  • SEQ ID NO: 2 is a cDNA sequence and corresponding amino acid sequence of the open reading frame that encodes US2.
  • SEQ ID NO: 3 is an amino acid sequence of US2.
  • SEQ ID NO: 4 is the nucleotide and amino acid sequence of the soluble US2 fusion US2t- DL6.
  • This chimeric sequence contains the bee mellitin signal sequence (residues 1-63), a portion of the US2 cDNA (residues 64-471, corresponding to residues 58 to 465 of SEQ ID NO: 2), the sequence for a single glycine spacer (residues 472-474), a DL6 epitope (residues 475-531), and a stop codon (residues 532-534).
  • SEQ ID NO: 5 is the amino acid sequence of the soluble US2 fusion US2t-DL6.
  • the bee mellitin signal sequence (corresponding to amino acid residues 1-21) is removed during translation to yield the final soluble US2 protein.
  • SEQ ID NOs: 6 and 7 are oligonucleotide primers that can be used to amplify the US2 cDNA sequence.
  • CMV Cytomegalovirus
  • HCMV Human Cytomegalovirus MHC: Major histocompatibility complex
  • Animal Living multicellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • Binding domain (in a US2 molecule) for class I MHC The region on the US2 protein that normally interacts (by binding, complexing or associating) with the class I MHC molecule.
  • Specific amino acids of the US2 protein that are responsible for MHC chain binding can be determined through standard point-mutation and binding experiments (see, for instance, Fleury et al., Cell 66: 1037-1091, 1991 ; Olson et al, J. Biol. Chem. 274:36905-36911, 1999).
  • point mutations e.g., amino acid substitutions or short deletions
  • US2 protein can be made in well-known recombinant techniques, and the resultant US2 proteins assayed for MHC class I-linked US2 biological activity using methods described herein.
  • Binding domain (in a US2 molecule) for class II MHC The region on the US2 protein that normally interacts (by binding, complexing or associating) with the class II MHC molecule.
  • MHC class I binding domain of US2 specific amino acids of the US2 protein that are responsible for MHC II binding can be determined through standard point-mutation and binding experiments.
  • US2 activity assays may be those that examine MHC class II-linked US2 activity, as discussed herein.
  • the binding domain within US2 for binding to class II MHC may share at least some residues with the US2 binding domain for class I MHC, and vice versa.
  • Binding domain (in a US2 molecule) for DM- ⁇ The region on the US2 protein that normally interacts (by binding, complexing or associating) with the DM- ⁇ molecule.
  • MHC class I and MHC class II binding domain of US2 specific amino acids of the US2 protein that are responsible for DM- ⁇ binding can be determined through standard point-mutation and binding experiments. Examples of US2 activity assays are those that examine DM- ⁇ -linked US2 activity, as discussed herein.
  • the binding domain within US2 for binding to DM- ⁇ may share at least some residues with the US2 binding domain for class I MHC and/or class II MHC, and vice versa.
  • CD4+ T cell mediated immunity An immune response implemented by presentation of antigens to CD4- T cells.
  • CD8+ T cell mediated immunity An immune response implemented b ⁇ presentation of antigens to CD8+ T cells
  • DNA molecules that hybridize DNA molecules and nucleotide sequences which are derived from the disclosed DNA molecules as described above may also be defined as DNA sequences which hybridize under stringent conditions to the DNA sequences disclosed, or fragments thereof
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used Generally, the temperature of hybridization and the ionic strength (especially the Na + concentration) of the hybridization buffer will determine the stringency of hybridization
  • a hybridization experiment may be performed by hybridization of a DNA molecule (for example, a variation of the US2 cDN A of SEQ ID NO 2) to a target DNA molecule (for example, the US2 cDNA itself) which has been electrophoresed in an agarose gel and transferred to a nitrocellulose membrane by Southern blotting, a technique well known m the art and described in (Sambrook et al , In Molecular Cloning A Laboratory Manual, Cold Spring Harbor, New York, 1989)
  • Hybridization with a target probe labeled with [ 32 P]-dCTP is generally carried out in a solution of high ionic strength such as 6 x SSC at a temperature that is 20-25° C below the melting temperature, T m , described below For such
  • stringent conditions may be defined as those under which DNA molecules with more than 25% sequence variation (also termed "mismatch") will not hybridize.
  • stringent conditions are those under which DNA molecules with more than 15% mismatch will not hybridize, and more stringent conditions are those under which DNA sequences with more than 10% mismatch will not hybridize.
  • Very stringent conditions are those under which DNA sequences with more than 6% mismatch will not hybridize.
  • the degeneracy of the genetic code further widens the scope of the present invention as it enables major variations in the nucleotide sequence of a DNA molecule while maintaining the amino acid sequence of the encoded protein.
  • the nucleotide sequence of the US2 cDNA could be changed without affecting the amino acid composition of the encoded protein or the characteristics of the protein.
  • the genetic code and variations in nucleotide codons for particular amino acids is known.
  • variant DNA molecules may be derived from the cDNA molecules disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. DNA sequences that do not hybridize under stringent conditions to the cDNA sequences disclosed, by virtue of sequence variation based on the degeneracy of the genetic code, are herein also comprehended by this invention.
  • a pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g. a US2 protein or variant, for instance a soluble variant.
  • the active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like.
  • non-toxic auxiliary substances such as emulsifying agents, preservatives, and pH buffering agents and the like.
  • Isolated An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Nucleic acid sequence DNA or RNA molecule, and includes polynucleotides encoding full length proteins and/or fragments of such full length proteins which can function as a therapeutic agent.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • ORF open reading frame
  • ORF A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.
  • Parenteral Administered outside of the intestine, e.g., not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
  • compositions and formulations suitable for pharmaceutical delivery of the US2 proteins and variants herein disclosed are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the US2 proteins and variants herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
  • Reco binant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e g , by genetic engineering techniques
  • Sequence identity the similarity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity Sequence identity is frequently measured in terms of percentage identity (or similarity or homology), the higher the percentage, the more similar are the two sequences
  • sequence identity is frequently measured in terms of percentage identity (or similarity or homology), the higher the percentage, the more similar are the two sequences
  • Methods of alignment of sequences for comparison are well-known in the art Various programs and alignment algorithms are described in Smith and Waterman, Adv Appl Math 2 482, 1981 , Needleman and Wunsch, J Mol Bio 48 443, 1970, Pearson and Lipman, Meth Mol Btol 24 307-331, 1988, Higgins and Sharp, Gene 73 237-244, 1988, Higgins and Sharp, CABIOS 5 151-153, 1989, Corpet et al Nucleic Acids Res 16 10881-90, 1988, Huang et al Computer Applications in BioSciences 8 155-65,1992,
  • BLAST The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al , J Mol Btol 215 403-410, 1990) is available from several sources, including the National Center for Biological Information (NBCI, Bethesda, MD) and on the Internet, for use m connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx It can be accessed at http //www ncbi nlm nih gov/BLAST/ A description of how to determine sequence identity using this program is available at http //www ncbi nlm nih gov/BLAST/blast_help html
  • Homologs of US2 are typically characterized by possession of at least 70% sequence identity counted over the full length alignment with the disclosed ammo acid sequence of either the human, hamster, mouse, sheep, cow or other ammo acid sequences using the NCBI Blast 2 0, gapped blastp set to default parameters
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1)
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties)
  • Such homologous peptides may possess at least 75%, at least 80% or at least 90% or 95% sequence identity determined by this method When less than the entire sequence is bemg compared for sequence identity, homologs will possess at least 75%, at least 85%, at least 90% or 95% sequence identify over short windows
  • the present invention provides not only the peptide homologs that are described above, but also nucleic acid molecules that encode such homologs.
  • Therapeutic agent Used in a generic sense, it includes treating agents, prophylactic agents, and replacement agents.
  • Therapeutically effective amount of a US2 protein A quantify of US2 protein (or variant thereof) sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit an immune response, for instance an autoimmune response or an immune response to a graft, transplant, or gene therapy treatment. In general, this amount is sufficient to measurably inhibit a CD4+ mediated immune response.
  • an effective amount of US2 protein may be administered in a single dose, or in several doses, for example daily, during a course of treatment.
  • the effective amount of US2 will be dependent on the specific protein applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the fusion protein.
  • a therapeutically effective amount of US2 protein can vary from about 0.01 mg/kg body weight to about 1 g/kg body weight.
  • US2 proteins and US2 protein variants disclosed in the present invention have equal application in medical and veterinary settings. Therefore, the general term "subject being treated” is understood to include all animals (e.g. humans, apes, dogs, cats, horses, and cows) that are or may experience an immune response that is susceptible to US2 protein-mediated inhibition.
  • a transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • US2 protein A viral protein that is the translation product of the US2 gene of a cytomegalovirus, the DNA sequence of which is shown in SEQ ID NO: 2.
  • the translated amino acid sequence is shown in SEQ ID NO: 3, and it is 180 amino acids in length after cleavage by signal peptidase, and 199 amino acids in length before cleavage of the signal sequence.
  • the US2 protein includes a native cytomegalovirus US2 protein, which down-regulates expression of MHC I and MHC II, interferes with CD8+ and CD4+ T cell mediated immunity, and particularly interferes with the expression of the MHC class Il ⁇ subunit and the DM- ⁇ subunit to a greater extent than it interferes with expression of class Il ⁇ or the invariant chain li.
  • the US2 protein can be a translation product of a unique short open reading frame of the DNA sequence of SEQ ID NO: 2.
  • US2 binding domain A region of the US2 protein that binds to a specific molecule, for instance a MHC class II, MHC class I, or DM- ⁇ polypeptide chain. US2 binding domains for each of these proteins may share at least some residues, and are found in the region of about amino acid residue 20 to about amino acid residue 155 of SEQ ID NO: 3. In certain examples, US2 binding domains may be contained in about amino acid residues 28 to 48 and/or 122 to 143 of SEQ ID NO: 3, although all of these residues are not necessarily essential to binding.
  • Variant US2 polypeptides A US2 protein (or peptide fragment) having one or more amino acid substitutions, one or more amino acid deletions, and/or one or more amino acid insertions, so long as the peptide retains the property of inhibiting CD4+ mediated immunity.
  • Conservative amino acid substitutions may be made in at least 1 position, for example 2, 3, 4, 5 or even 10 positions, as long as the peptide retains inhibition of CD4+ mediated immunity.
  • Such variant peptides or proteins include amino acid sequences with greater than about 60%, 70%, 80%, 90%, 95%, 98% or even 99% homology with the native US2 protein, or a subsequence thereof.
  • alterations of US2 made to create an US2 variant will be entirely outside of the MHC II binding site of the US2 protein.
  • variant US2 polypeptide may also refer to a shortened fragment of the native US2 protein (SEQ ID NO: 3), so long as the fragment retains the property of inhibiting CD4+ mediated immunity.
  • Appropriate fragments include, for instance, the entire soluble portion of the US2 protein (i.e., residues about 1 through about residue 170), or shorter fragments, such as about residue 20 through about residue 155. Even shorter fragments may also be appropriate, for instance residues 20 through 165, through 160, through 155, or through 150 or thereabouts.
  • soluble US2 proteins or other US2 variants, are also beneficially expressed as fusion proteins with, for instance, a stabilizing or solubilizing protein domain joined to the US2 fragment or variant. Construction of soluble forms of proteins (e.g., immunoglobulin fusions) that can bind to cells and inhibit immune responses is known (see, e.g., Murphy et al, J. Exp.
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • the term "vector” includes viral vectors, such as adenoviruses, adeno-associated viruses, vaccinia, and retrovirus vectors.
  • the present invention relates to a method of inhibiting immunity to either a viral vector or a transgene.
  • This inhibition of immunity is useful for a number of purposes, including study of the immune response to vectors (or inhibition such a response), study of autoimmunity (or inhibition thereof), delivery of genes to cells, and in gene therapy.
  • a general strategy for transferring genes into donor cells is disclosed in U.S. Patent No. 5,529,774, which is incorporated by reference.
  • a gene encoding a protein having therapeutically desired effects is cloned into a viral expression vector, and that vector is then introduced into the target organism.
  • the virus infects the cells, and produces the protein sequence in vivo, where it has its desired therapeutic effect. See, for example, Zabner et al, Cell 75:207-216, 1993.
  • one major limitation of this use of viral vectors for gene therapy is that the infected cells are recognized and killed by the CD4+ and CD8+ T lymphocytes, which eventually results in the loss of the virus and its beneficial therapeutic effect.
  • CD4+ and CD8+ T lymphocyte responses are frequently directed to the transgene. Therefore, even if the virus vector can be made to be non-immunogenic, e.g. with "third generation" helper-dependent adenoviruses, T cell responses specific for foreign transgenes will frequently reduce or inhibit expression of that transgene.
  • the present invention offers a method by which the persistence of viral gene therapy vectors and expression of foreign transgenes might be improved, thereby enhancing the therapeutic value of the vectors.
  • This method entails adding to the vector sequence a DNA sequence that encodes the US2 protein, or a US2 protein variant, or a substantially identical or substantially homologous protein, that inhibits CD4+ T cell recognition of infected cells.
  • An added advantage of this method is that the US2 protein (and its variants and homologs) also effectively inhibits CD8+ T cell recognition of infected cells. Expression of this protein is designed to enhance the length of time the gene of interest is expressed, by inhibiting both CD4+ and CD8+ mediated immune responses that would otherwise eliminate the vector.
  • Example 5 One embodiment of this invention is shown in Example 5, wherein the US2 gene has been isolated and inserted into AdtetUS2, a replication defective adenovirus vector that was constructed by coupling the US2 gene to a tetracycline inducible promoter. This vector was thereafter able to cause degradation of both MHC I and MHC II proteins as well as MHC II DM proteins (Fig. 2), and to inhibit both CD8+ and CD4+ mediated immune responses (Fig. 5).
  • This example illustrates that a vector or vector element could be used to protect cells in which they reside from a CD4+ T cell mediated immune response, whether the gene therapy is carried out with that or another vector. Since it is the US2 product that causes the inhibition, this technique is generally applicable to the expression of US2 for improving the persistence of any virus in which such persistence may be desired.
  • sequences encoding US2 or a homologous protein to the DNA of a virus
  • the sequence may be introduced into infected or uninfected cells by the use of a secondary virus, for example a virus that is not the virus that is expected to produce the immune response.
  • a secondary virus may be introduced which carries the US2 coding sequence, prior to the introduction of a viral gene therapy vector.
  • Example 2 in which the gene for US2 is carried on an adenovirus vector, and is able to inhibit a T cell mediated immune response specific for CMV, with which the cells were co-infected.
  • a second vector e.g. a virus carrying a therapeutic gene, could be used to infect the same subject.
  • the first vector would then inhibit recognition by both CD4+ and CD8+ T cells.
  • a second use of the present invention is to prevent or inhibit organ rejection following transplantation of heterologous cells into a host.
  • Rejection could be avoided or treated by supplying a therapeutically sufficient amount of US2 to the site of transplantation, either by exogenous administration or expression from cells in or near the organ that have been transformed to express US2.
  • Yet another use of the invention is in the treatment of autoimmune diseases, particularly autoimmune diseases in which therapeutic amounts of US2 are administered or provided to the patient.
  • a sequence that codes for US2 may be introduced into the cells of individuals suffering from autoimmune diseases, either by introducing the sequence into the somatic DNA or into a viral gene therapy vector, or alternatively, to introduce the protein US2 or its homologue into the subject's cells. The purpose of this is to limit display of both MHC I and II complexes, and particularly MHC II complexes, and thereby limit autoimmune responses and symptoms of the disease.
  • T cell immunity such as tissue and organ transplant rejection, diabetes, multiple sclerosis and arthritis.
  • T cell immunity By reducing the recognition of cells of involved tissue by T cells, the symptoms of the disorder will be reduced.
  • This therapy should also be of value in subjects at high risk for developing autoimmune disorders, even if the symptoms have not yet appeared.
  • the genetic or protein elements may be introduced only into certain cells or tissues.
  • the elements may be introduced only into the tissue or organ being transplanted.
  • it may be more therapeutically effective and simple to treat all of the patients cells, or more broadly disseminate the vector, for example by intravascular administration.
  • US2 could be delivered selectively to specific cells or organs and would more specifically block immune mediated damage to those specific cells.
  • Nucleic acid sequences encoding therapeutic agents that may be placed into the vector include, but are not limited to, nucleic acid sequences encoding tumor necrosis factor (TNF) genes, such as TNF- ⁇ ; genes encoding interferons such as Interferon- ⁇ , Interferon- ⁇ , and Interferon- ⁇ ; genes encoding interleukins such as IL-1 , IL-l ⁇ , GMCSF.
  • TNF tumor necrosis factor
  • the method of the present invention may be employed to treat diseases or disorders of a variety or organs, including the brain and central nervous system.
  • diseases and disorders include, but are not limited to, ischemic strokes, angiogenesis, metabolic diseases of the brain, axonal injury, Alzheimer's disease, Parkinson's disease, Huntington's disease, central nervous system infections, mucopolysaccharidoses (MPS, types I-VII), lipidoses (such as, for example, Gaucher's disease), Lesch- Nyhan syndrome, X-linked ADL, metachromatic leukodystrophy, Krabbe's disease, Charcot-Marie- Tooth disease, Fragile X, stroke, epilepsies, Down's syndrome, phenylketonuria, degenerative disorders, mental disorders, and a variety of disorders that can be affected by introducing a new compound or modifying the levels of existing proteins in the brain or nervous system.
  • a vector including a gene encoding an endothelial growth factor (such as vascular endothelial growth and permeability factor) or fibroblast growth factor (FGF) may be administered to a subject suffering from an ischemic stroke or with severely occluded arteries in the leg.
  • a vector including a gene encoding nerve growth factor, or a gene encoding choline acetyl transferase may be administered to a subject suffering from Alzheimer's disease.
  • genes which may be contained in the vectors include, but are not limited to, genes encoding glial derived neurotrophic factor (GDNF), or tyrosine hydroxylasation for the treatment of Parkinson's disease; genes encoding resistance to central nervous system infections; the PMP-22 gene for treatment of Charcot-Marie-Tooth disease; the FMR-1 gene for treatment of Fragile X; neuroprotective genes to prevent further neuronal damage from stroke; and genes encoding inhibitory signals that reduce brain excitability for treatment of epilepsy.
  • GDNF glial derived neurotrophic factor
  • tyrosine hydroxylasation for the treatment of Parkinson's disease
  • genes encoding resistance to central nervous system infections include, but are not limited to, genes encoding resistance to central nervous system infections; the PMP-22 gene for treatment of Charcot-Marie-Tooth disease; the FMR-1 gene for treatment of Fragile X; neuroprotective genes to prevent further neuronal damage from stroke; and genes encoding inhibitory signals that reduce brain excit
  • the nucleic acid sequence encoding at least one therapeutic agent is under the control of a suitable promoter.
  • suitable promoters which may be employed include, but are not limited to, the gene's native promoter, retro viral LTR promoter, or adeno viral promoters, such as the adeno viral major late promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMTV promoter; the metallothionein promoter; heat shock promoters; the albumin promoter: the histone promoter; the ⁇ -actin promoter; TK promoters; B19 parvovirus promoters; and the ApoAI promoter.
  • CMV cytomegalovirus
  • RSV Rous Sarcoma Virus
  • inducible promoters such as the MMTV promoter
  • the metallothionein promoter such as the MMTV promoter
  • heat shock promoters such as the MM
  • the recombinant nucleic acid can be administered to the animal host by any method that allows the recombinant nucleic acid to reach the appropriate cells. These methods include injection, infusion, deposition, implantation, intra-arterial, oral ingestion or topical administration. Injections can be intramuscular, intravenous, intra-dermal, subcutaneous or intraperitoneal. Intravascular injections may use vectors that will transduce endothelial cells of blood vessels, such as those located in the brain, and produce the therapeutic agent in the endothelium.
  • the recombinant nucleic acid can be delivered as part of a viral vector, such as a pox virus, recombinant vaccinia virus, a he ⁇ es virus, replication-deficient adenovirus strains or poliovirus, or as a non-infectious form such as naked DNA or liposome encapsulated DNA.
  • a viral vector such as a pox virus, recombinant vaccinia virus, a he ⁇ es virus, replication-deficient adenovirus strains or poliovirus, or as a non-infectious form such as naked DNA or liposome encapsulated DNA.
  • the dose range When administered as an intramuscular injection in humans, the dose range may be about 10 3 to 10 13 infectious particles per injection, for example about 10 5 to 10 ⁇ infectious particles per injection, depending on the virus vector used. Single or multiple doses can be administered over time. With adenoviruses, one can inject as much as 10 ' ' PFU kg into monkeys; but for he ⁇ es viruses or pox viruses this can be 10 5 PFU total.
  • the vector used to express US2 (or a biologically active variant thereof, e.g., a soluble US2 protein) and downregulate MHC-II is a viral vector.
  • Viral vectors which may be employed include RNA viral vectors (such as retroviral vectors), and DNA virus vectors (such as adenoviral vectors, adeno-associated virus vectors, a he ⁇ esvirus, and vaccinia virus vectors).
  • RNA viral vectors such as retroviral vectors
  • DNA virus vectors such as adenoviral vectors, adeno-associated virus vectors, a he ⁇ esvirus, and vaccinia virus vectors.
  • the polynucleotide encoding the therapeutic agent is in the form of RNA.
  • DNA virus vector the polynucleotide encoding the therapeutic agent is in the form of DNA.
  • Adenoviral vectors may include essentially the complete adenoviral genome (Shenk et al, Curr. Top. Microbiol. Immunol. 1 1 1 : 1-39, 1984).
  • the adenoviral vector may be a modified adenoviral vector in which a portion of the adenoviral genome has been deleted.
  • the vector could be a so-called "first generation" Ad vector with deletions encompassing the El region or encompassing the El and E3 region of the Ad genome.
  • Such vectors can be propagated on 293 cells that supply El gene products, as in Hitt et al. (Meth. Mol. Gen. 7:13-30, 1995).
  • the vector could also be a so-called "second generation" adenovirus, which has deletions in the El region as well as in the E2 and/or E4 regions of the Ad genome.
  • Such vectors can be propagated in 293 cells that have been transfected with E2 and/or E4 genes.
  • the vector is a third generation adenovirus vector that includes an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence encoding a therapeutic agent: and a promoter for expressing the DNA sequence encoding a therapeutic agent.
  • the vector could also be free of all Adenovirus ORFs and is propagated using 293-cre cells and a helper adenovirus that has loxP sequences flanking the DNA packaging sequences
  • Such third generation vectors do not engender anti AD immune responses, although they do engender anti-transgene responses See Parks et al , Proc Natl Acad Sci USA 93 13565 ( 1996) and Scheider et al , Nat Gen , 18 180 (1998)
  • the gene in the E2a region that encodes the 72 kilodalton (kDa) binding protein is mutated to produce a temperature sensitive protein that is active at 32° C, the temperature at which viral particles are produced, but is mactive at 37° C, the temperature of the animal or human host
  • This temperature sensitive mutant is described in Ensmger et al , J Virol , 10 328-339, 1972, Van der V et et al , J Virol , 15 348-354, 1975, and F ⁇ efeld et al , Virol, 124 380-389, 1983, Englehardt et al , Proc Nat Acad Sc ⁇ , 9 ⁇ 6196-6200, 1994, Yang et al , Nat Gen 1 362-369, 1994
  • Such a vector may be constructed according to standard techniques, usmg a shuttle plasmid which contains, beginning at the 5' end, an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence, a promoter (which may be an adenoviral promoter or a foreign promoter), a tripartite leader sequence, a multiple cloning site (which may be as herein described), a poly A signal, and a DNA segment which corresponds to a segment of the adenoviral genome
  • the DNA segment serves as a substrate for homologous recombmation with a modified or mutated adenovirus, and may encompass, for example, a segment of the adenovirus 5' genome no longer than from base 3329 to base 6246
  • the plasmid may also mclude a selectable marker and an origin of replication
  • the o ⁇ gm of replication may be a bacterial origin of replication
  • the plasmid may be used to produce an adenoviral vector by homologous recombmation with a modified or mutated adenovirus m which at least the majority of the El and E3 adenoviral DNA sequences have been deleted
  • Homologous recombmation may be effected through co-transfection of the plasmid vector and the modified adenovirus mto a helper cell line, such as 293 cells, by CaP0 4 precipitation
  • the homologous recombination produces a recombinant adenoviral vector which mcludes DNA sequences derived from the shuttle plasmid between the Not I site and the homologous recombmation fragment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombmation fragment and the 3' ITR
  • a vector may be formed which mcludes an adenoviral 5' ITR.
  • the vector may then be transfected into a helper cell line, such as the 293 helper cell line (ATCC No CRL1573, available from Microbix Biosystems, Inc , Toronto, Ontario, CA), which will include the Ela and Elb DNA sequences, which are necessary for viral replication, to generate replication defective viral vector particles
  • the vector may include a multiple cloning site to facilitate the insertion of DNA sequence(s) encoding therapeutic agent(s) into the cloning vector
  • the multiple cloning site includes "rare" restriction enzyme sites, for example sites which are found in eukaryotic genes at a frequency of from about one in every 10,000 to
  • the adenovirus may be constructed by using a yeast artificial chromosome (or YAC) containing an adenoviral genome accordmg to the method described m Ketner et al (Proc Natl Acad Sci USA, 91 6186-6190, 1994), in conjunction with the teachmgs contained herein
  • the adenovirus yeast artificial chromosome is produced by homologous recombination in vivo between adenoviral DNA and yeast artificial chromosome plasmid vectors carrying segments of the adenoviral left and right genomic termini
  • a DNA sequence encodmg a therapeutic agent then may be cloned mto the adenoviral DNA
  • the modified adenoviral genome then is excised from the adenovirus yeast artificial chromosome m order to be used to generate adenoviral vector particles as heremabove described
  • the adenoviral particles are administered in an amount effective to produce a therapeutic effect in a host
  • the particles may, for example, be admmistered IM, IP, IV or even intra-arterially (for example mto the hepatic artery)
  • the adenoviral particles are admmistered mtravascularly (for example through a catheter mto an artery proximal to a target organ such as the brain) m an amount of at least 1 x 10 8 plaque formmg units (pfu) per kg of body weight, and in general such amount does not exceed about 5 x lO 11 pfu per kg of body weight, and generally is from about 1 x 10 9 pfu to about 1 x 10 n pfu per kg of body weight
  • the exact dosage of adenoviral particles to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient to be treated, and the nature and extent of the disease or disorder to be treated
  • the viral vector is a retroviral vector
  • retroviral vectors which may be employed include, but are not limited to, Moloney Murme Leukemia Virus (MoMuLV), spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloprohferative sarcoma virus, and mammary tumor virus
  • the vector is generally a replication defective retrovirus particle Retroviral vectors are useful as agents to effect retroviral-mediated gene transfer mto eukaryotic cells
  • Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest Most often, certain viral structural genes (.
  • gag, pol, and env are removed from the retroviral backbone usmg genetic engineering techniques known in the art This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments contaming appropriate portions of the packaging signal
  • New genes may be mco ⁇ orated into proviral backbones in several general ways
  • the structural genes of the retro virus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences withm the long termmal repeat (LTR)
  • LTR long termmal repeat
  • Retroviral vectors have also been constructed which can introduce more than one gene mto target cells
  • one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter
  • two genes may be expressed from a single promoter by the use of an Internal Ribosome Entry Site
  • the retroviral vector may be one of a series of vectors based on the N2 vector contammg a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems
  • the retroviral vector may be a MoMuLV of the LN series of vectors, such as those herem mentioned above, and described further in Bender et al (J Virol 61 1639-1646, 1987) and Miller et al (Biotechntques, 1 980-990, 1989)
  • Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
  • the term "mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
  • the retroviral vector may include up to or at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; for example, the restriction product has an average DNA size of at least 10,000 base pairs.
  • Advantageous cloning sites are selected from the group consisting of Notl, SnaBI, Sail, and Xhol.
  • the retroviral vector includes each of these cloning sites.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of Notl, SnaBI, Sail, and Xhol located on the retroviral vector.
  • the shuttle cloning vector may also include at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
  • the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites.
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems, and may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
  • the shuttle cloning vector may be derived from plasmids such as pBR322 or pUC 18.
  • the vector includes one or more promoters.
  • Suitable promoters that may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al. (Biotechniques, 7:980-990, 1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters).
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B 19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the vector then is employed to transduce a packaging cell line to form a producer cell line.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, PA12, T19-14X, VT-19-17-H2, GP+E-86, GP+envAml2, and DAN cell lines, as described in Miller (Hum. Gene Ther., 1 :5-14, 1990).
  • the vector containing the nucleic acid sequence encoding the therapeutic agent may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 precipitation.
  • the producer cells generate retroviral vector particles, which are administered to a host, whereby the retroviral vector particles transduce cells of the host, and produce the therapeutic agent.
  • the retroviral vector particles may be administered to the host in an amount effective to produce a therapeutic effect in the host.
  • the retroviral vector particles are administered in an amount of at least 1 x 10 8 colony forming units (cfu), and in general not exceeding 1 x 10 10 cfu, and often about 10 9 cfu.
  • the exact dosage of retroviral vector particles is dependent upon the factors already mentioned with respect to the adenoviral particles.
  • the retroviral vector particles are administered as part of a preparation having a titer of retroviral vector particles of at least 1 x 10 cfu/ml and in general not exceeding 1 x 10 9 cfu/ml.
  • T cells CD8+ and CD4+ T lymphocytes (T cells) are the principal immune defense against viral infections. These T cells recognize small peptides derived from viral proteins, and respond by rapidly killing virus infected cells, or orchestrating specific immune responses against the virus.
  • Major histocompatibilify (MHC) class I proteins are loaded with peptides in the endoplasmic reticulum (ER), where they are loaded with peptides that are subsequently presented to CD8+ T cells (York and Rock, Ann. Rev. Immun. 14:369-396, 1996). MHC class I proteins are ubiquitous, which enables CD8+ T cells to monitor the inside world of cells and detect viral proteins.
  • MHC class II proteins which present to CD4+ T cells, are primarily expressed by professional antigen presenting cells, B cells, macrophages and dendritic cells, which take up extracellular proteins by endocytosis or phagocytosis and present on their surfaces peptides derived from the proteins.
  • B cells professional antigen presenting cells
  • macrophages macrophages and dendritic cells, which take up extracellular proteins by endocytosis or phagocytosis and present on their surfaces peptides derived from the proteins.
  • CD8+ T cell responses directed to viruses are usually weak, as in the case of AIDS.
  • Class II proteins are first synthesized in the endoplasmic reticulum (ER) as a complex of ⁇ and ⁇ chains, bound to a third component, the invariant chain Ii, which protects the peptide-binding site (Wolfand Ploegh, -4 «. Rev. Cell & Dev. Biol 11 :267-306, 1995; Cresswell, Cell 84:505-507, 1996).
  • the ⁇ / ⁇ /Ii complexes are transported to a lysosomal-like compartment known as the MHC class II compartment (MIIC), where peptide loading occurs.
  • MIIC MHC class II compartment
  • HLA-DM Another class II component, HLA-DM, binds to the class II complex and catalyzes exchange of a remnant of Ii for antigenic peptides produced by protease degradation of proteins in the MIIC (Denzin and Cresswell, Cell 82:155-165, 1995; Sloan et al, Nature 375:802-806, 1995). From the MIIC, peptide-loaded class II complexes move to the cell surface for recognition by CD4+ T cell receptors. To study the effects of HCMV infection on MHC class II molecules, a transformed glioblastoma cell line U373 was used, which is permissive for HCMV infection and can be induced to express MHC class II by IFN- ⁇ treatment (Basta et al, J.
  • U373-derived cell line denoted U373-CIITAHis
  • U373-CIITAHis a U373-derived cell line that constitutively expresses MHC class II proteins, by transfecting the cells with a class II transactivator gene, CIITA (Steimle et al, Science 265: 106-109, 1994).
  • Infection of U373-CIITAHis cells with mutant viruses lacking various genes within the IRSI- US1 1 region Jones et al, J. Virol. 69:4830-4841 , 1995
  • mutants lacking both US2 and US3 were unable to cause this effect (Fig. IB).
  • the reduced expression of class II was not due to inhibition of IFN -mediated transcription of MHC class II genes, as described (Miller et al, J. Exp. Med. 187:675-683, 1998), because mRNAs specific for the MHC class II ⁇ , ⁇ and invariant chains were not reduced after 36 hours of HCMV infection (Fig IC), yet diminished expression of class II proteins was observed after only 12 hours.
  • EXAMPLE 5 The HCMV US2 Protein Inhibits MHC II Expression To assess whether US2, US3, or both were responsible for this inhibition, and whether US2 or US3 could be sufficient to downregulate class II proteins, we studied transfected cells expressing US2 or US3 and found that US2 but not US3 expression leads to diminished expression of class II.
  • a replication-defective adenovirus (Ad) vector, AdtetUS2, expressing US2 was constructed by coupling the US2 gene to the tetracycline inducible promoter.
  • US2 was expressed in U373 CIITAHis cells by co-infecting the cells with AdtetUS2 and a second virus, Adtet-trans, that expresses the tetracycline inducible element (Massie et al, Virol. 72:2289-2296, 1998).
  • Adtet-trans a virus that expresses the tetracycline inducible element
  • Monocyte/macrophages play a pivotal role in the lifecycle of HCMV and the cells express
  • US2 expression in macrophages blocks the MHC class II pathway, and presentation of antigens to CD4+ T cells. When infecting cultured macrophages with HCMV, only 10-15% of the macrophages exhibited evidence of infection.
  • US2 was introduced into macrophages by using AdtetUS2. The macrophages were incubated with tetanus toxin, an antigen that can be processed and presented by the macrophages, then the macrophages were irradiated and mixed with tetanus toxin- specific CD4+ T cells. Proliferation of the CD4+ T cells (in response to the tetanus toxin) was measured.
  • HCMV US2 is the first viral inhibitor of the MHC class II pathway to be described, and is the only reported specific inhibitor of the MHC class II antigen presentation pathway.
  • US2 should confer resistance to CD4+ cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • CD4+ T cells can provide costimulation for CD8+ T cells by binding to the same antigen presenting cell (Schoenberger et al, Nature 393:480-483, 1998), US2 may indirectly reduce CD8+ CTL responses.
  • the downregulation of both class I and class II pathways in professional antigen presenting cells would allow global reductions in cellular and humoral immunity, and sustained replication in antigen presenting cells.
  • US2 can be used to block inappropriate immune responses in autoimmunity, transplantation or against gene therapy vectors.
  • the US2 gene (the US2 open reading frame of SEQ ID NO: 2) was first described by Weston and Barrell (J. Mol. Biol. 192: 177, 1986), who sequenced the unique short component of the human cytomegalovirus genome. The entire US2 gene was subcloned by using PCR primers or oligonucleotides: US2 forward (SEQ ID NO: 6: 5' CGC GGA TCC ATG AAC AAT CTC TGG
  • the 600-nucleotide PCR product generated with these two o gonucleotides was cut with EcoRI and Xbal and inserted between the EcoRI and Xbal sites of p ⁇ E 1 sp 1 B-tet (Fig 6)
  • p ⁇ E 1 sp 1 B-tet was produced previously by altering p ⁇ E 1 sp 1 B (Microbix Biosystems, Inc , Toronto, Ontario, CA, Brett et al , Proc Natl Acad Set USA 91 8802-8806, 1994), by inserting a tetracyc ne-responsive promoter cassette between the Hindlll and Xhol sites (see map in Fig 6)
  • the tetracychne-responsive promoter consists of a tetracycline operator sequence coupled to a HCMV core promoter element (see map in Fig 6)
  • p ⁇ ElsplB-tet was cotransfected into 293 cells along with a second
  • Adtet-trans expresses a transactivator protein, composed of the tetracycline repressor fused to the herpes simplex virus VP16 transactivating domain (Gossen and Bujard, Proc Natl Acad Sci , USA 89 5547-5551, 1992)
  • This tet transactivator protein binds to tet operator sequences in the promoter of AdtetUS2 and activates expression of the US2 gene Therefore, to obtain expression of US2, cells are comfected with AdtetUS2 and Adtet-trans
  • Adenovirus vectors such as Adtet US2 or others in which the US2 was coupled to other promoters could be used to express US2 as well as other therapeutic transgenes
  • US2 would be expected to sustain long term expression by these adenovirus vectors
  • Both CD4+ and CD8+ T cell responses reduce and shorten expression of transgenes by Ad vectors
  • adenoviral vectors could also be used to express the US2 in conjunction with other inhibitors of the immune system
  • adenoviral vectors could include but not be limited to Ad vectors, expressing US2 as well as ICP47, the human CMV UL18 gene product that blocks NK cells, HCMV USII, or pox virus proteins that inhibit inflammation
  • Ad vectors could also mclude third-generation or helper-dependent Ad vectors, as described in Parks et al , Proc Natl Acad Sci USA 93 13565-13570, 1996, and vectors expressmg a variety of therapeutic transgenes, such as the cystic fibrosis transporter, human alpha- l-ant ⁇ tryps ⁇ n, leptin, citrulhnaemia, and others
  • US2 could be used to treat rejection following organ transplantation
  • the liver would be removed from the donor, and AdtetUS2 introduced into the organ, for example by perfusing the portal vein with 10 12 pfu of AdtetUS2 (as well as 5-fold less Adtet-trans) Alloge c transplantation of the liver would then be performed into a heterologous host
  • the recipient would be lmmunosuppressed with cyclospo ⁇ ne for a period of time (for example, a few days to two weeks)
  • expression of US2 and perhaps ICP47
  • hepatocytes prepared from liver biopsies or livers removed from donors by collagenase treatment could be infected with
  • pancreatic islets In the treatment of pancreatic disorders, isolated pancreatic islets could be targets for gene therapy, as in Csete et al (Transplantation 59 263-268, 1995)
  • the pancreatic islet cells could be infected with AdtetUS2/Adtet-trans or other Ad vectors carrying immunosuppressive genes The cells could then be reintroduced into an allogenic recipient to restore partial pancreatic function (as in Platt et al , Nat Med 3 26-27, 1997, and Gunsulas et al , Nat Med 3 48-53, 1997)
  • EXAMPLE 11 Conditions Associated Primarily with CD4 + T Cell Immunity, or MHC II CD4 + mediated immunity (via MHC II presentation) is associated with the immune response provoked by gene therapy vectors, as well as transplantation and autoimmune disorders
  • Soluble US2 can be constructed using standard genetic engineermg techniques Additionally, it is sometimes advantageous to express the soluble US2 as a fusion protem with, for instance a stabilizing or solubihzing protein domain functionally joined to the US2 fragment or variant
  • the chimeric domain can be, for instance, an lmmunoglobulm domain (see, e g , Mu ⁇ hy et al , J Exp Med 180 233-231, 1994, McKnight et al , J Immuno 152 5220-5225, 1994, Lmsley et al , Science 257 792-795, 1992, Lenschow et al , Science 257 789-792, 1992)
  • the construction of fusion proteins, and more particularly soluble fusion proteins is well known to one of ordinary skill m the art Merely by way of example, below is a discussion of the construction of one useful soluble US2 fragment, which includes the majority of the
  • a recombinant baculovirus vector was used to infect the cells and cause high level expression of this US2 recombinant protein.
  • the bBM-US2-DL6 baculovirus was made by using the polymerase chain reaction to produce a DNA sequence encoding the US2 protein fused to an antibody epitope (the DL6 epitope of he ⁇ es simplex virus glycoprotein D) at the extreme C-terminus. This epitope was added merely to facilitate study of this soluble protein, and is not required for the functionality of the soluble US2.
  • a signal sequence can be included in the construct.
  • the bee mellitin signal sequence was added at the N- terminus of the recombinant protein, replacing the native (US2) signal sequence.
  • the resultant amplified PCR product (containing bee mellitin, US2, and DL6 sequences) was inserted into a shuttle vector pFASTBACl (GIBCO, Gaithersburg, MD) at BamHI and EcoRI restriction sites.
  • pFASTBACl was recombined with a baculovirus genome cloned into a bacterial artificial chromosome (BAC) present in E. coll cells strain DH10BAC (GIBCO, Gaithersburg, MD). Recombination between the soluble US2 construct and the BAC produced a BAC containing the US2 sequences.
  • This BAC was extracted from the DH10BAC bacteria and transfected into Tn5 insect cells to obtain a baculovirus, bBM-US2-DL6, capable of expressing recombinant US2 (US2t-DL6).
  • the expressed and secreted protein contains the following amino acid sequences: US2 amino acids 20-155 (from SEQ ID NO: 3), followed by a single glycine residue and DL6 epitope (amino acid residues 159-177, SEQ ID NO: 5).
  • the bee mellitin signal sequence (amino acid residues 1-21, SEQ ID NO: 5) is removed during the synthesis of the protein.
  • the protein preparations were immediately neutralized to pH 7.4 with 3 M sodium acetate, pH 5.0 and dialyzed extensively against phosphate buffered saline, pH 7.2. Inhibition of antigen presentation to CD4+ T cells by US2t-DL6.
  • Epstein-Barr Virus- transformed lymphoblastoid cells or LCLs which express relatively high levels of MHC class II proteins and can present exogenous antigen to MHC class II-restricted, CD4+ T cell clones
  • LCLs which express relatively high levels of MHC class II proteins and can present exogenous antigen to MHC class II-restricted, CD4+ T cell clones
  • U373 glioma cells that express little or no MHC class II protein and cannot present antigens to CD4+ T cells were used as a negative control. These cells were incubated with various concentrations of the US2t-DL6 protein for 4 hours. Following this incubation the cells were incubated with US2t-DL6 and an antigenic protein, Mtb39, derived from Mycobacterium tuberculosis.
  • Mtb39 can be recognized by Mtb39-specific, CD4+ T cell clones, after the Mtb39 proteins is taken by endocytosis, digested into peptides and presented on the appropriate class II proteins present on the antigen presenting cells, in this case, LCL or U373 cells. Simultaneously, the cells were incubated with CD4+ T cells clones specific for Mtb39. This incubation was for 18 hours and subsequently cell culture supematants were harvested and analyzed for the presence of the cytokine interferon- ⁇ which is released by the T cells after the cells recognize antigen (Mtb39) presented by the appropriate MHC class II proteins.
  • the US2t-DL6 protein was able to block antigen presentation to the T cell clones as shown by the inhibition of the interferon- ⁇ secretion by the T cells (Fig. 8). Half-maximal inhibition of the T cells was observed with 4-1 1 ⁇ g/ml of the US2--DL6 protein.
  • this soluble form of US2 can bind to class II proteins on the surfaces of antigen presenting cells (LCL) and can effectively block recognition of these class II proteins by CD4+ T cells.
  • a soluble, truncated form of US2 can effectively inhibit recognition of antigen presenting cells by CD4+ T cells in an in vitro assay.
  • soluble chimeric proteins derived from native cellular proteins to inhibit CD8+ and CD4+ T cells responses in vivo.
  • CTLA4 a soluble form of CTLA4 has been used to block various types of immunity in experimental animals and in human patients (Lenschow et al, Science 257:789-792, 1992).
  • CTLA-4 is a membrane protein found on T lymphocytes, and binds B7-1 and B7-2 costimulatory molecules on antigen presenting cells (Schwartz, Cell 71 : 1065-1071 , 1992).
  • CTLA-4- IgG is a fusion protein that contains a soluble, truncated form of CTLA-4 fused to the immunoglobulin G (IgG) constant domain.
  • the CTLA-4-IgG protein is able to block the interaction between the CTLA-4 molecules on T cells and B7-1 or B7-2 on antigen presenting cells and, by doing so can effectively inhibit T cell responses.
  • CTLA-4-IgG can be injected into animals or humans and blocks T cells responses in vivo. This soluble form of CTLA-4 acts as a immunosuppressive agent, and can be used to reduce immunity directed to transplanted tissues (Lenschow et al.
  • This invention encompasses soluble, truncated forms of US2, including the one described herein and others where the US2 MHC binding domain is fused to the immunoglobulin G (IgG) constant domain or other domains, that can be used to block inappropriate immune responses in vivo.
  • US2-based proteins can be injected into the bloodstream or directly into specific tissues.
  • the presence of soluble forms of US2 in the body or in specific tissues may block recognition of antigen presenting cells by CD8+ or CD4+ T lymphocytes, as indicated by Fig. 8. This inhibition could be useful to block inappropriate T cell immunity that occurs in auto immunity (e.g., diabetes, arthritis) or during tissue or cell transplantation or to inhibit T cell responses directed to foreign transgenes or virus vectors in gene therapy.
  • the US2 proteins of this invention may be administered to humans, or other animals on whose cells they are effective, in various manners such as topically, orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, and subcutaneously.
  • Treatment may involve daily or multi-daily doses of US2 protein(s) over a period of a few days to months, or even years.
  • US2 proteins and US2 variants are particularly useful in the inhibition of inappropriate or detrimentally extreme immune responses, such as those that may occur in the course of an autoimmune disease, or be associated with organ or tissue transplantation, or gene therapy.

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Abstract

La protéine du cytomégalovirus humain, qui s'est déjà avérée capable de bloquer le chemin de présentation de l'antigène du complexe majeur d'histocompatibilité (CMH) de classe I, s'est ici avérée capable de bloquer le chemin de présentation du CMH de classe II. Cela est surprenant car les protéines de classe I et de classe II ne sont pas homologues. La protéine US2 provoque la dégradation de protéines alpha de classe II et également de protéines DM-alpha de classe II, partie d'un complexe enzymatique nécessaire au chargement de peptides antigéniques. En ce sens, la protéine US2 a un double effet inhibiteur sur le chemin du CMH de classe II. L'expression de US2 bloque efficacement la présentation d'antigènes aux lymphocytes T CD4+. La protéine US2 ou des variantes solubles de cette dernière peuvent être utilisées pour réduire des réponses immunitaires inappropriées dirigées contre des vecteurs ou des transgènes exprimés. De plus, de telles molécules peuvent être utilisées pour bloquer l'immunité vis-à-vis de cellules ou d'organes transplantés ou lors de maladies auto-immunes.
EP00905939A 1999-02-02 2000-02-02 Inhibition du chemin de presentation de l'antigene du complexe majeur d'histocompatibilite de classe ii et presentation aux cellules cd4+ Withdrawn EP1157102A4 (fr)

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US20120020938A1 (en) * 2010-07-26 2012-01-26 Searete Llc, A Limited Liability Corporation Of The State Of Delaware MHC- less cells
KR101263103B1 (ko) * 2011-01-18 2013-05-09 고려대학교 산학협력단 ＣＤ１ｄ 발현을 감소조절하는 ＵＳ２ 단백질 유전자를 포함하는 자연 살해 Ｔ 세포활성 억제용 조성물 및 이를 이용한 자연 살해 Ｔ 세포 활성 억제 방법
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WO1997032605A1 (fr) * 1996-03-08 1997-09-12 Massachusetts Institute Of Technology Acides nucleiques de cytomegalovirus codant pour des proteines ayant une liaison mhc de classe i normale ou modifiee et utilises dans le traitement de certaines maladies

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JONES T R ET AL: "MULTIPLE INDEPENDENT LOCI WITHIN THE HUMAN CYTOMEGALOVIRUS UNIQUE SHORT REGION DOWN-REGULATE EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I HEAVY CHAINS" JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 69, no. 8, 1 August 1995 (1995-08-01), pages 4830-4841, XP000770462 ISSN: 0022-538X *
MILLER DANIEL M ET AL: "Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/stat pathway" JOURNAL OF EXPERIMENTAL MEDICINE, vol. 187, no. 5, 2 March 1998 (1998-03-02), pages 675-683, XP002280345 ISSN: 0022-1007 *
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