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WO1997046573A1 - Procede d'inhibition du traitement des antigenes, proteines chimeres intervenant dans ce procede, et leurs procedes d'expression - Google Patents

Procede d'inhibition du traitement des antigenes, proteines chimeres intervenant dans ce procede, et leurs procedes d'expression Download PDF

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Publication number
WO1997046573A1
WO1997046573A1 PCT/US1997/009294 US9709294W WO9746573A1 WO 1997046573 A1 WO1997046573 A1 WO 1997046573A1 US 9709294 W US9709294 W US 9709294W WO 9746573 A1 WO9746573 A1 WO 9746573A1
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Prior art keywords
protein
gly
dna
epitope
ala
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PCT/US1997/009294
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English (en)
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WO1997046573A9 (fr
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Michael Kurilla
Maria Masucci
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The University Of Virginia Patent Foundation
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Publication date
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Priority to AU32916/97A priority Critical patent/AU3291697A/en
Publication of WO1997046573A1 publication Critical patent/WO1997046573A1/fr
Publication of WO1997046573A9 publication Critical patent/WO1997046573A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—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
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention pertains to chimeric nuclear proteins, and methods of expression of the same, which proteins do not trigger a conventional major histocompatibility complex reaction where inserted into a cell of a patient.
  • proteins, particularly nuclear proteins, provided with a Gly-Ala repeat domain appear to suppress recognition by cytotoxic T lymphocytes (CTL) and inhibit an induced immune response, interfering with antigen presentation.
  • CTL cytotoxic T lymphocytes
  • Epstein- Barr virus is a well-studied herpes virus, which transforms cells through expression of episomal DNA in the host cell.
  • the same avenue is the basic modality employed in gene therapy, where foreign genetic material is inserted into the cells of the target patient, to achieve expression of that genetic material, producing the therapeutic protein(s) .
  • the immune response discussed above includes attack by cytotoxic T lymphocytes (CTL) mediated by the major histocompatibility complex (MHO .
  • CTL cytotoxic T lymphocytes
  • MHO major histocompatibility complex
  • cytotoxic T cells are drawn to, and attack, the foreign material by recognition of the MHC proteins present in response to the inserted foreign or exogenous material.
  • proteins, and nuclear proteins in particular are susceptible to MHC Class I- mediated CTL immune response, given the cell surface glycoprotein pattern associated therewith.
  • Class II-mediated immune response is also associated with this phenomenon, but not necessarily directly targeted to nuclear proteins.
  • the EBV-encoded nuclear antigen EBNA1 is a viral protein regularly detected in all malignancies associated with EBV. See, e.g., Masucci et al . , Trends in Microbiology 2, 125-130 (1994) . No MHC-I mediated CTL response has been demonstrated in connection with this nuclear protein. Rickinson et al .. A New Look at Tumor Immunology, 53-80 (Cold Spring Harbor Laboratory Press, 1992) . If the method by which the MHC-I mediated CTL response to this nuclear protein is inhibited could be adopted for therapeutic proteins, a wide class of proteins could be better utilized for the therapies described.
  • Gly-Ala repeat domain is intended herein to mean a series of three tripeptides, wherein each tripeptide consists of Gly and Ala residues. Three triplets define a nine amino acid sequence, which is then repeated. Each triplet contains one Gly, one Ala and a third ammo acid, either Ala or Gly, in any order. The sequence of 3 triplets is repeated in the domain. A total of about 35-50 amino acids appear effective suppressing MHC-I mediated CTL response to the proteins, in "infected cells".
  • the domain may be so small as to not sufficiently inhibit toxicity. While the domain may be incorporated m the chimeric protein at any point, it is preferably incorporated downstream of the epitope triggering the
  • the Gly-Ala repeat present occupies or "redirects" the mechanism responsible for MHC mediated CTL response to such a degree the epitope-t ⁇ ggered response does not occur possibly due to failure to express foreign protein-induced MHC glycoprotein fragments on the cell surface.
  • t intended that the DNA sequence inserted into the sequence encoding the antigen must be inserted so as to encode the repeat itself. That is, the inhibition of the CTL response is a protein-associated phenomenon, not a DNA-associated phenomenon.
  • Gly-Ala repeats may be used, if desired. Repeats encoded by DNA segments of up to 700 base pairs have been demonstrated to be effective. Optimal repeat size can be determined by empirical methods, but it appears that 100-130 amino acid domains may be effective in MHC Class-I and perhaps Class-II mediated CTL immune response.
  • Figure 1 is a schematic outline of chimaera prepared according to the invention, including a (full-length) EBNA1 chimaera.
  • the position of insertion of the EBNA4 416-424 epitope (E4) and the EBNA1 internal repeat region (IR) are indicated by arrows pointing to the amino-acid number in the B95.8 sequence.
  • the Gly-Ala repeats are indicated in solid marking, while the nuclear localization signal is dark-shaded, and DNA binding and dimerization domains are indicated by cross-hatching.
  • Applicants' invention resides in the discovery that the "infection" by an antigenic protein, of a cell, which would otherwise induce MHC-mediated CTL immune response, can be expressed while inhibiting that response if the expression includes a Gly-Ala repeat domain within the protein.
  • the antigenic presentation of the protein, incorporating the Gly-Ala repeats is somehow suppressed, such that CTL response is not triggered.
  • the Gly-Ala repeat domain should be on the order of 30 amino acids-150 amino acids. Optimally, it appears that an insertion domain of 50-120 amino acids provides significant inhibition of CTL response.
  • the Gly-Ala repeat segment is inserted downstream of that segment of DNA encoding the CTL epitopic portion of the protein, where such is known. Restriction, insertion, ligation, plasmid preparation and cell insertion or "infection" are all conventional technologies familiar to those of ordinary skill in the art, and are not detailed herein, saved for their expression in terms of the examples set forth below.
  • the nuclear antigen EBNAl encoded by the EBV is expressed in latent EBV-infected B lymphocytes that persist for life in healthy virus characters, is the only viral protein regularly detected in all malignancies associated with EBV, and not bee demonstrated to be subject to a MHC-mediated CTL response. Mapping of the antigenic protein demonstrated the presence of a Gly-Ala repeat domain, which interferes with antigenic processing and image Class - I, and perhaps Class - II presentation. By fixing the epitope, and inserting the DNA encoding the Gly-Ala repeat downstream of that epitope, CTL recognition of otherwise CTL-recognized epitopes was inhibited.
  • EBNAl is a phosphoprotein composed of unique amino and carboxy-terminal domains (amino acids 1-89 and 327-641, respectively, in the prototype B95.8 EBV strain) joined by a repetitive sequence of Arg-Gly-containing motifs, surrounding an internal Gly-Ala repeat. While Gly-Ala repeats of different le-.-gths are present in all EBV isolates and represent the major target of EBNA-specific antibody responses, the specific function of these repeats has not been previously described.
  • EBV induces CTL responses.
  • Caucasian leukocyte antigen- (HLA)All-positive individuals are often dominated by All- restricted reactivities to peptide epitopes corresponding to residues 399-408 and 416-424 of the EBNA4 protein.
  • Dittmar et al Proc. Natl. Acad. Sci. USA, 81,4652-4656 (1984) .
  • recombinant vaccinia viruses expressing chimeric genes that contain the EBNA4 416-424 epitope inserted in-frame within the attached EBNAl sequence, or within EBNAl deletion mutants that did not contain the Gly-Ala repeats were prepared.
  • EBNAl deletion the native protein with the Gly-Ala repeat domain excised
  • EBNA4 416-424 epitope inserted at the His 39, Pro 446 or Lys 520 positions relative to the B95.8 sequence sensitized HLA All-positive fibroblasts to lysis by EBV-specific CTL.
  • the level of killing was in each case comparable to that observed after infection with a vaccinia recombinant expressing EBNA4.
  • fibroblasts expressing a chimeric full-sized EBNAl with the EBNA4 416-424 epitope inserted at His 39 (E1N-E4) were not recognized by the CTL.
  • E1N-E4 and E1 ⁇ GAN-E4 were expressed with slower kinetics and at lower levels compared to the positive orientation E1 ⁇ GAN-E4 whereas the orientation of insertion did not affect the expression of E4IR. It should be noted that the expression of E1N-E4 and E4IR is probably overestimated because of the presence of Gly-Ala-specific antibodies in the human serum.
  • Vaccinia recombinants carrying the coding sequences of EBNAl and EBNA4 also known as EBNA-3B
  • EBNA-3B Vaccinia recombinants carrying the coding sequences of EBNAl and EBNA4 (also known as EBNA-3B) from B95.8 were used.
  • EBNAl (El) or an EBNAl deletion mutant that lacks the Gly-Ala repeat region (E1 ⁇ GA) were produced by inserting an oligonucleotide corresponding to the E4 epitome in the Ncol ,
  • PffMl or Bsu361 site of the EBNAl coding sequence (genomic positions: 108,067, 109,291 and 109,510, respectively) .
  • the oligonucleotide pairs E1N-E4F/E1N-E4R (5' -CATGCCATAGTAACTGACTTT-
  • E4F/E1P-E4R (5' - CGATCGTAACTGACTTTAGTGTAATCAGG- 3 ' /5 ' -
  • TTACCTTGATTACACTAAGTCAGTTACGATCG-3 ' were annealed and ligated to appropriately digested pBS-El or pBS-El ⁇ GA at 100:1 ratio ⁇ .
  • the pBS-E4IR plasmid was constructed by inserting an
  • Ncol-Apal EBNAl fragment (IR, gp: 108,067-109,261) nto the scI site of EBNA4 (gp: 97,302) .
  • the E4IR open reading frame was excised by EcoRI and SstI digestion and ligated into pSCll. Insert containing plasmids were sequenced to determine correct orientation and reading frame alignment .
  • Recombinant viruses were generated by transfection into CV-I cells infected with WR strain wild- ype vaccinia virus, and recombinant viruses were selected on TK-143 cells. Viral stocks were prepared and titrated in CV-l cells.
  • Fibroblast lysis experiments were conducted by growing semiconfluent monolayers of fibroblasts from the donor. These were grown in 96-well plates. Infection was done in the assay wells in the presence of 3 ⁇ Ci sl NaCrOfact per well. CTL clones specific for the EBNA4 416-424 epitope were obtained by stimulation of lymphocytes from an EBV seropositive donor with the autologous B95.8 virus transformed LCL as described in Rickinson et al. supra. The cytotoxic activity was assayed in triplicate in standard four hour 51 Cr-release assays.
  • Cell lysis assays were conducted using semiconfluent monolayers of fibroblast, infected and labelled as described above. Aliquots of 5 x 10 6 cells were placed in 5-ml tubes and infected for one hour at 37°C with 1 ml concentrated virus before addition of 2 ml complete medium and further incubation for the indicated times. 100 ⁇ Ci 51 NaCr0 4 were added to each tube two hours before the initiation of the assay.
  • the invention is generally applicable to all MHC-mediated CTL response.
  • MHC Class-I restricted CTL responses are MHC Class II mediated responses may be similarly inhibited, but since the T cells implicated in such response are generally different from those in Class I responses, the inhibitory effects achieved may be of lower importance .
  • modifying proteins to include a Gly-Ala repeat by modifying the DNA encoding the same to include a Gly-Ala repeat domain segment encoding at least about 25-30 amino acids effectively inhibits presentation of the CTL-responsive epitope.
  • the repeat sequence is inserted downstream of the CTL-responsive epitope.
  • the chimeric proteins, and DNA encoding the same are effectively used in plasmids and other constructs effective in gene therapy, vaccine design and the like, where CTL response would interfere with attempts to employ exogenous proteins in therapy.
  • One interesting utility is the modification of autoantigens, or potential autoantigens, expressed by an individual. Individuals (mammals) who are either suffering from autoimmune disease, or at risk to develop autoimmune disease, may be treated by providing them with DNA expressing the autoantigen with a Gly-Ala repeat domain as described. This may be used to render the expressed protein non-i munogenic.
  • ADDRESSEE OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT
  • NAME OBLON, NORMAN F.

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  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

L'invention concerne des réponses immunes des lymphocytes T cytopathiques qui sont induites par la classe I du complexe majeur d'histocompatibilité, en réponse à l'expression dans une cellule de protéine étrangère, et inhibées par la présence, dans la cellule étrangère, d'un domaine de répétition Gly-Ala. Un minimum d'environ 30 acides aminés dans le domaine de répétition est nécessaire pour inhiber les réponses immunes des lymphocytes T cytopathiques (CTL). Des segments d'ADN codant la séquence de répétition sont de préférence insérés en aval de la séquence codant l'épitope de CTL de la protéine structurale. Les cellules hôtes, transfectées par l'ADN, lors de l'expression de la protéine chimère obtenue, ne déclenchent pas l'attaque des lymphocytes T cytopathiques.
PCT/US1997/009294 1996-06-07 1997-06-06 Procede d'inhibition du traitement des antigenes, proteines chimeres intervenant dans ce procede, et leurs procedes d'expression WO1997046573A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU32916/97A AU3291697A (en) 1996-06-07 1997-06-06 Method of inhibiting antigen processing, chimeric proteins reflecting this method, and methods of expressing those proteins

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US66027296A 1996-06-07 1996-06-07
US08/660,272 1996-06-07

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WO1997046573A1 true WO1997046573A1 (fr) 1997-12-11
WO1997046573A9 WO1997046573A9 (fr) 1998-04-16

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022577A1 (fr) * 1996-11-15 1998-05-28 Maria Grazia Masucci Proteines de fusion a demi-vie allongee
WO2001030378A1 (fr) * 1999-10-22 2001-05-03 The Walter And Eliza Hall Institute Of Medical Research Procede de prophylaxie et de traitement
AU767688B2 (en) * 1999-10-22 2003-11-20 Walter And Eliza Hall Institute Of Medical Research, The A method of prophylaxis and treatment

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NATURE, 22 June 1995, Vol. 365, LEVITSKAYA et al., "Inhibition of Antigen Processing by the Internal Repeat Region of the Epstein Barr Virus Nuclear Antigen-1", pages 685-688. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022577A1 (fr) * 1996-11-15 1998-05-28 Maria Grazia Masucci Proteines de fusion a demi-vie allongee
WO2001030378A1 (fr) * 1999-10-22 2001-05-03 The Walter And Eliza Hall Institute Of Medical Research Procede de prophylaxie et de traitement
AU767688B2 (en) * 1999-10-22 2003-11-20 Walter And Eliza Hall Institute Of Medical Research, The A method of prophylaxis and treatment

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