WO2002027027A2 - Criblage de substances therapeutiques en rapport avec des maladies infectieuses - Google Patents

Criblage de substances therapeutiques en rapport avec des maladies infectieuses Download PDF

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WO2002027027A2
WO2002027027A2 PCT/US2001/030334 US0130334W WO0227027A2 WO 2002027027 A2 WO2002027027 A2 WO 2002027027A2 US 0130334 W US0130334 W US 0130334W WO 0227027 A2 WO0227027 A2 WO 0227027A2
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cells
gene products
host cell
cell gene
infected
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PCT/US2001/030334
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WO2002027027A3 (fr
WO2002027027A8 (fr
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Maurice Zauderer
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University Of Rochester
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Priority to EP01975512A priority patent/EP1335990A2/fr
Publication of WO2002027027A2 publication Critical patent/WO2002027027A2/fr
Publication of WO2002027027A3 publication Critical patent/WO2002027027A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS

Definitions

  • the present invention relates to methods of identifying therapeutics useful for infectious diseases. More specifically, the present invention relates to methods of identifying antigens which are produced by infected cells, and the use of such antigens in immunogenic compositions or vaccines to treat or prevent infection. .
  • the immune system is the primary biological defense of the host (self) against potentially pernicious agents (non-self). These agents may be pathogens, such as bacteria or viruses, as well as modified self cells, including virus-infected cells, tumor cells or other abnormal cells of the host. Collectively, these targets of the immune system are referred to as antigens.
  • antigens Collectively, these targets of the immune system are referred to as antigens.
  • the recognition of antigen by the immune system rapidly mobilizes immune mechanisms to destroy that antigen, thus preserving the sanctity of the host environment.
  • Antigens may provoke antibody-mediated responses and/or cell-mediated responses.
  • Cells of the immune system termed B lymphocytes, orB cells, produce antibodies that specifically recognize and bind to the foreign substance.
  • Other lymphocytes termed T lymphocytes, or T cells, both effect and regulate the cell- mediated response resulting eventually in the elimination of the antigen.
  • T cells are involved in the cell-mediated response. Some induce particular B cell clones to proliferate and to produce antibodies specific for the antigen. Others recognize and destroy cells that present foreign antigens on their surfaces. Certain T cells regulate the response by either stimulating or suppressing other cells. Prospects for development of broadly effective tumor vaccines have been advanced by evidence that several self -proteins can be recognized as tumor antigens by immune T cells (Van den Eynde et al. , J. Exp. Med. 173: 1373 (1991); Bloom et al, J. Exp. Med. 185:453 (1997); Nan Der Bruggen et al, Science 254:1643 (1991); Gaugler et al, J. Exp. Med. 179:921 (1994); Boel et al,
  • Infected cells sometimes express self -proteins that are not expressed in uninfected cells.
  • the present invention provides a method of screening for therapeutics for infectious diseases, comprising identifying host cell gene products which are differentially expressed in infected cells, screening the differentially expressed gene products for immunogenicity, and determining which gene products are immunogenic.
  • the present invention also provides a method comprising identifying host cell gene products which are differentially expressed in infected cells, identifying which of the differentially expressed gene products are expressed embryonically, screening the differentially- and embryonically-expressed gene products for immunogenicity, and determining which gene products are immunogenic.
  • the differentially expressed gene products may be identified using subtractive hybridization, representational difference analysis, differential display, or ordered microarrays of nucleic acids.
  • Immunogenicity includes cytotoxic T lymphocyte responses, T helper responses, and B cell responses, such as antibody production.
  • Figure 1 shows the results of hybridization to an array of 24 cDNA clones selected following subtractive hybridization of cDNA from HN-inf ected THP- 1 monocytic cell line minus uninfected THP-1 cD ⁇ A + HTV D ⁇ A.
  • Figure 2 shows hybridization to Northern blots of poly-A RNA from uninfected and HTV-infected cells.
  • Altered features of an infected cell which are recognized by the immune system as non-self may be the basis for development of treatments or vaccines against infectious diseases. Since many pathogens elude immune surveillance by frequent reproduction and mutation, it is of considerable value to develop a vaccine that targets host gene products that are not likely to be subject to mutation. Thus, the present invention relates to a method of identifying potential therapeutics useful for the treatment or prevention of infectious diseases.
  • treatment is meant reduction in symptoms, reduction in pathogen load, 5 reduction in the rate of pathogen replication, and/or no worsening of symptoms, pathogen load, or pathogen replication over a specified period of time.
  • Host gene products that are overexpressed in infected cells are identified. Those that are shown to be overexpressed by a factor of 9 or greater in infected cells as compared to uninfected cells are the most likely to be immunogenics.
  • relative gene expression is then determined in a broad panel of normal tissues. It is expected that immune tolerance will be induced to gene products expressed at relatively high levels in any normal tissue. Such gene products are excluded from further analysis. Immunogenicity is then directly assayed.
  • a method comprising identifying host cell genes which are differentially expressed in infected cells, screening the gene products of the differentially expressed host cell genes for immunogenicity, and determining which differentially expressed host cell gene products are immunogenic.
  • a method comprising identifying host cell genes which are differentially expressed in infected cells, identifying which of the differentially expressed genes are expressed in embryonic tissue, screening the gene products of said differentially- and embryonically-expressed genes for immunogenicity, and determining which differentially expressed host cell gene
  • a method comprising identifying host
  • a method comprising identifying host cell genes which are differentially expressed in infected cells, identifying which which of the differentially-expressed genes are not expressed in other adult tissues, screening the gene products of said differentially-expressed genes which are not expressed in adult tissue for immunogenicity, and determining which differentially expressed host cell gene products are immunogenic.
  • Suitable cells include, but are not limited to, mammalian cells, including animal (rodents, including mice, rats, hamsters and gerbils), primates, and human cells, including cells of all types, including breast, skin, lung, cervix, colorectal, leukemia, brain, etc.
  • Cells include dividing cells, non dividing cells, terminally differentiated cells, pluripotent stem cells, committed progenitor cells and uncommitted stem cells.
  • Cells and cell types also include muscle cells such as cardiac muscle cells, skeletal muscle cells and smooth muscle cells, myofibrils, intrafusal fibers and extrafusal fibers; skeletal system cells such as osteoblasts, osteocytes, osteoclasts and their progenitor cells.; and epithelial cells such as squamous epithelial cells, including endothelial cells, cuboid epithelial cells and columnar epithelial cells.
  • muscle cells such as cardiac muscle cells, skeletal muscle cells and smooth muscle cells, myofibrils, intrafusal fibers and extrafusal fibers
  • skeletal system cells such as osteoblasts, osteocytes, osteoclasts and their progenitor cells.
  • epithelial cells such as squamous epithelial cells, including endothelial cells, cuboid epithelial cells and columnar epithelial cells.
  • Cells that can be used in the method of the present invention also include nervous system cells such as neurons, including cortical neurons, inter neurons, central effector neurons, peripheral effector neurons and bipolar neurons; and neuroglia, including Schwann cells, oligodendrocytes, astrocytes, microglia and ependyma.
  • nervous system cells such as neurons, including cortical neurons, inter neurons, central effector neurons, peripheral effector neurons and bipolar neurons
  • neuroglia including Schwann cells, oligodendrocytes, astrocytes, microglia and ependyma.
  • endocrine and endocrine-associated cells may also be used such cells as pituitary gland cells including epithelial cells, pituicytes, neuroglia, agranular chromophobes, granular chromophils (acidophils and basophils); adrenal gland cells including epinephrine-secreting cells, non-epinephrine- secreting cells, medullary cells, cortical cells (cells of the glomerulosa, fasciculata and reticularis); thyroid gland cells including epithelial cells (principal and parafoUicular); parathyroid gland cells including epithelial cells (chief cells and oxyphils); pancreas cells including cells of the islets of Langerhans (alpha, beta and delta cells); pineal gland cells including parenchyma!
  • pituitary gland cells including epithelial cells, pituicytes, neuroglia, agranular chromophobes, granular chromophils (
  • thymus cells including parafollulicular cells
  • cells of the testes including seminiferous tubule cells, interstitial cells ("Leydig cells"), spermatogonia, spermatocytes (primary and secondary), spermatids, spermatozoa, Sertoli cells and myoid cells
  • cells of the ovary including ova, oogonia, oocytes, granulosa cells, theca cells (internal and external), germinal epithelial cells and follicle cells (primordial, vesicular, mature and atretic).
  • Circulatory system cells are also included such cells as heart cells (myocardial cells); cells of the blood and lymph including erythropoietin- sensitive stem cells, erythrocytes, leukocytes (such as eosinophils, basophils and neutrophils (granular cells) and lymphocytes and monocytes (agranular cells)), thrombocytes, tissue macrophages (histiocytes), organ-specific phagocytes (such as Kupffer cells, alveolar macrophages and microglia), B-lymphocytes, T- lymphocytes (such as cytotoxic T cells, helper T cells and suppressor T cells), megaloblasts, monoblasts, myeloblasts, lymphoblasts, proerythroblasts, megakaryoblasts, promonocytes, promyelocytes, prolymphocytes, early normoblasts, megakaryocytes, intermediate normoblasts, metamyelocytes (such as juvenile metamyelocytes, segment
  • Respiratory system cells are also included such as capillary endothelial cells and alveolar cells; as are urinary system cells such as nephrons, capillary endothelial cells, granular cells, tubule endothelial cells and podocytes; digestive system such as simple columnar epithelial cells, mucosal cells, acinar cells, parietal cells, chief cells, zymogen cells, peptic cells, enterochromaffin cells, goblet cells, Argentaffen cells and G cells; and sensory cells such as auditory system cells (hair cells) ; olfactory system cells such as olfactory receptor cells and columnar epithelial cells; equilibrium/vestibular apparatus cells including hair cells and supporting cells; visual system cells including pigment cells, epithelial cells, photoreceptor neurons (rods and cones), ganglion cells, amacrine cells, bipolar cells and horizontal cells are also included.
  • urinary system cells such as nephrons, capillary end
  • mesenchymal cells stromal cells, hair cells/follicles, adipose (fat) cells
  • cells of simple epithelial tissues squamous epithelium, cuboidal epithelium, columnar epithelium, ciliated columnar epithelium and pseudostratified ciliated columnar epithelium
  • cells of stratified epithelial tissues stratified squamous epithelium (keratinized and non-keratinized), stratified cuboidal epithelium and transitional epithelium
  • goblet cells endothelial cells of the mesentery, endothelial cells of the small intestine, endothelial cells of the large intestine, endothelial cells of the vasculature capillaries, endothelial cells of the microvasculature, endothelial cells of the arteries, endothelial cells of the arterioles, endothelial cells of the veins, endothelial
  • the method of the present invention can be used to screen for antigens which are differentially expressed in cells infected with any infectious agent, including viruses, fungal agents, mycobacteria, bacteria or parasitic agents.
  • the cells are infected with human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • This method of vaccine development is broadly applicable to any infectious agent but especially to infectious agents that, like HIN, replicate or mutate rapidly, for example, hepatitis C virus and many R ⁇ A viruses (because they depend on R ⁇ A polymerases which are more error prone since they do not have a "proof-reading" function).
  • the cells are infected with infectious agents causing chickenpox, shingles, rubella, influenza, rubeola, mumps, yellow fever, mononucleosis, rabies, acute viral gastroenteritis, poliomyelitis, subacute sclerosing panencephalitis, encephalitis, Colorado tick fever, pharyngitis, croup, bronchiolitis, viral pneumonia, pleurodynia, aseptic meningitis, keratitis, conjunctivitis, viral leukemias, rabies, polio, myocarditis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E; and any infections caused by adeno viruses, coxsackieviruses, parainfluenza viruses, respiratory syncytial virus, reovirus, cytomegalo virus, Epstein-Barr virus, herpes simplex viruses, herpes
  • viruses include, but are not limited to the following D ⁇ A and R ⁇ A viral families: Arbovirus, Adenoviridae, Arenaviridae Arteri virus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Arbovirus, Adenoviridae, Arenaviridae Arteri virus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Arbovirus, A
  • Paramyxoviridae Morbilli virus, Rhabdoviridae
  • Orthomyxoviridae e.g. Influenza
  • Papovaviridae e.g., Parvoviridae
  • Picornaviridae e.g., Poxviridae (such as Smallpox or Vaccinia)
  • Reoviridae e.g., Rotavirus
  • Retroviridae HTLV-I, HTLN-II, Lentivirus
  • Togaviridae e.g., Rubivirus
  • Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
  • arthritis bronchiollitis, encephalitis
  • eye infections e.g., conjunctivitis, keratitis
  • chronic fatigue syndrome hepatitis (A, B, C, E, Chronic Active, Delta)
  • meningitis
  • parasitic agents include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, andTrichomonas.
  • These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis.
  • Fungal pathogens include, but are not limited to Candida albicans and pneumocystis carnii.
  • Mycobacterial pathogens include, but are not limited to, M. tuberculosis, M. avium.
  • Host cell gene products which are "differentially expressed" in infected cells include gene products which are upregulated during infection, i.e., expressed in a cell during both infection and non-infection but at higher levels during infection; and those which are expressed in a cell only during infection.
  • differential expression is determined by subtractive hybridization.
  • Methods of subtractive hybridization are known in the art. See, for example, U.S. Patent Nos. 5,827,658; 5,700,644; and 5,525,471.
  • differential expression is determined by representational difference analysis (RDA).
  • RDA is a subtractive hybridization basedmethod applied to "representations" of total cellular DNA (Lisitsyn, N. and N., M. Wigler. 1993. Science 259: 946-951).
  • the differential display methods of Liang and Pardee (1992, Science 257:967-971) employ an arbitrary 10 nucleotide primer and anchored oligo-dT to PCR amplify an arbitrary subset of fragments from a more complex set of DNA molecules.
  • differential expression is determined by the modified differential display described below.
  • differential expression is determined using microarrays.
  • differential expression is determined using ordered microarrays of nucleic acids. Two color differential hybridization may be used.
  • Gene expression in embryonic tissues is known to be more complex than in adult tissues. Many of these genes are downregulated in the adult and would, therefore, not be expected to induce tolerance in newly arising lymphocytes of the adult. If expression of any of these gene products is again upregulated in infected cells, as is known to happen for some such genes in cells that undergo tumor transformation, then these would encode antigens that could be targeted for immunotherapy.
  • An ordered library of cDNA clones expressed during early embryonic development can be made. See, e.g., Tanaka et al, PNAS 97:9127-9132 (2000). Microarrays representing the ordered library can be produced and be employed to efficiently identify developmentally regulated genes that are overexpressed in infected cells.
  • RNA and Gene PoolTM cDNA which is either total RNA or first strand cDNA prepared from over 30 different human normal adult or fetal tissues, is screened for expression of the differentially expressed gene. Those sequences that are consistently expressed in infected cells but which have low or undetectable expression in diverse normal tissues especially thymus are more likely to be immunogenic.
  • Immuno response encompasses humoral and cell-mediated immune responses, including, but not limited to, antibody response, cytotoxic T lymphocyte response, T helper response, inflammation, cytokine production, and complement.
  • a gene product is immunogenic if it induces one or more of these immune responses.
  • the ability of a differentially expressed gene product to modulate an immune response can be readily determined by an in vitro assay.
  • T cells for use in the assays include transformed T cell lines, such as T cell hybridomas, or T cells which are isolated from a mammal, e.g., from a human or from a rodent such as a mouse. T cells can be isolated from a mammal by known methods. See, for example, Shimonkevitz et al, J. Exp. Med. 158:303 (1983).
  • a suitable assay to determine if a gene product is capable of modulating the activity of T cells is conducted by coculturing T cells and antigen presenting cells.
  • the most effective antigen presenting cells for stimulation of a primary immune response are dendritic cells (DC).
  • DC dendritic cells
  • recombinants of vaccinia, retroviral, or adenoviral vectors are generated for the same gene product and employed DC infected with these vectors to alternately stimulate autologous human T cells. Alternate cycles of stimulation with different vectors minimize selection of a vector specific response and focus immune reactivity on the recombinant gene.
  • the differentially expresssed gene product may be added to the culture medium. Production of IL-2 is measured. An increase in IL-2 production over a standard indicates the compound can stimulate an immune response and is immunogenic.
  • immunogenicity is determined by cultivating T cells with antigen-presenting cells, adding a differentially expressed gene product to the cell culture, and measuring IL-2 production.
  • immunogenicity is determined by cultivating T cells with antigen-presenting cells, transfecting the antigen-presenting cells with a vector expressing the differentially expressed gene product, and measuring IL-2 production.
  • T cell activation include secretion of other cytokines (IFN- ⁇ , TNF- ⁇ , GM-CSF) measured by ELISA, ELISpot, or flow cytometric detection (Luminex bead system). Many of these methods are described in Current Protocols in Immunology (John Wiley & Sons, New York).
  • modulation of T cell activation can be suitably determined by changes in antigen-dependent T cell proliferation as measured by radiolabelling techniques or colorimetric MTT assay as are recognized in the art.
  • nucleotide may be introduced to an assay culture medium.
  • T cell proliferation serves as a measure of T cell proliferation.
  • This assay is not suitable for T cells that do not require antigen presentation for growth, e.g., T cell hybridomas.
  • a difference in the level of T cell proliferation following contact with the compound of the invention indicates the complex modulates activity of the T cells .
  • An increase in T cell proliferation indicates the compound can stimulate an immune response.
  • cytotoxic T lymphocyte (CTL) activity can be detected using a standard 4 hour 51 Cr release assay, as well known in the art.
  • CTL cytotoxic T lymphocyte
  • In vivo assays also may be suitably employed to determine the ability of a compound of interest to activate T cells.
  • a compound of interest can be assayed for its ability to inhibit immunoglobulin class switching (i.e. IgM to IgG). See, e.g., Linsley et al, Science 257:192-195 (1992)).
  • IgM immunoglobulin class switching
  • a compound of interest can be administered to a mammal such as a mouse, blood samples obtained from the mammal at the time of initial administration and several times periodically thereafter (e.g. at 2, 5 and 8 weeks after administration). Serum is collected from the blood samples and assayed for the presence of antibodies raised by the immunization. Antibody concentrations may be determined.
  • the differentially expressed genes may be screened for complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). See U.S. Pat. No. 5,500,362 for ADCC and CDC assays.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • a defect in antigen processing is not readily corrected with present methods, but a deficiency in T cell clonal expansion can be overcome by the most fundamental of all immunological manipulations - vaccination. Rather than examine what is immunogenic in an infected cell, it may be more profitable to evaluate what can become immunogenic if the representation of specific T cells is first augmented by vaccination.
  • a method comprising identifying genes which are differentially expressed in infected cells, followed by immunization with a the differentially expressed gene product or a recombinant expression vector comprising the differentially expressed gene, and isolation of CTL specific for one or more of these gene products.
  • the immunogenicity of these peptide targets in man can be established by highly efficient in vitro stimulation of human T cells with autologous peptide-pulsed dendritic cells.
  • Cellular peptides derived by degradation of endogenously synthesized proteins are translocated into a pre-Golgi compartment where they bind to class I MHC molecules for transport to the cell surface.
  • class I MHC:peptide complexes are the target antigens for specific CD8+ cytotoxic T cells. Since all endogenous proteins turn over, peptides derived from any cytoplasmic or nuclear protein may bind to an MHC molecule and be transported for presentation at the cell surface. This allows T cells to survey a much larger representation of cellular proteins than antibodies which are restricted to recognize conformational determinants of only those proteins that are either secreted or integrated at the cell membrane.
  • Class I-bound peptides are generally 8-10 residues in length and accommodate amino acids side chains of restricted diversity at certain key positions that match pockets in the MHC peptide binding site. These key features of peptides that bind to a particular MHC molecule constitute a peptide binding motif.
  • HLA class I A major concern for the development of broadly effective human vaccines is the extreme polymorphism of HLA class I molecules. Extensive characterization of peptide binding motifs of different human class I MHC molecules has suggested that there are four major subtypes of HLA- A and HLA-
  • mice which are transgenic for human CD8 and a human HLA antigen can be used to determine whether a particular differentially expressed gene product is immunogenic in humans.
  • the invention will be better understood by reference to the specific embodiments detailed in the examples which follow.
  • cDNA is synthesized from both a tracer (represented by infected cell mRNA) and a driver (represented by parental, non-infected cell mRNA). "Representations" of both tracer and driver cDNA are created by digestion with Rsal which cuts the four-base recognition sequence GTAC to yield blunt end fragments. Adaptors, which eventually serve as primer sites for PCR, are ligated to the 5' ends of only the tracer cDNA fragments. Two aliquots of tracer representation are separately ligated with two different adapters. A series of two hybridizations are carried out.
  • each adapter ligated tracer sample is denatured and hybridized with a ten fold excess of the denatured representation of driver cDNA for 8 hours. Under these conditions re-annealing of all molecules is incomplete and some of both the high and low copy molecules remain single stranded. Since re-annealing rates are faster for more abundant species, this leads to normalization of the distribution through relative enrichment of low copy number single stranded molecules .
  • the two hybridization reactions with each of the different adapter ligated tracer cDNA representations are then combined without fractionation or further denaturation but with addition of more freshly denatured driver in a second hybridization reaction that is allowed to proceed further to completion, approximately 20 hours.
  • Pardee (1992, Science 257:967-971) employs an arbitrary 10 nucleotide primer and anchored oligo-dT to PCR amplify an arbitrary subset of fragments from a more complex set of DNA molecules.
  • differences among the fragments generated from normal and infected cells should reflect differences in gene expression in the two cell types.
  • this method sometimes works well but often gives rise to numerous false positives. That is, bands which appear to be differentially displayed are, upon further characterization, found not to be differentially expressed. This is presumably due to variable PCR amplification of individual species in complex populations and a relatively high background that can obscure less prominent bands. Since considerable effort is required to establish differential expression, these endemic false positives are costly in terms of efficiency and productivity.
  • a single arbitrary primer may also be used for differential display, as described by Welch et al.. Use of single primers does, however, require synthesis of a much larger set of independent primers to achieve the same coverage of a complex cDNA population.
  • a second arbitrary primer is substituted for the anchored oligo-dT employed in PCR amplification. This results in fewer DNA products in each PCR reaction so that individual DNA fragments can be more reliably resolved on sequencing gels.
  • each subset of fragments generated in this modified differential display protocol is a smaller representation of total cDNA, more primer pairs are required for adequate sampling.
  • Employing the negative binomial distribution it can be predicted that if 12 independent primers are utilized in all 66 possible primer pair combinations there is a greater than 85% probability that for an average size eukaryotic cDNA at least one primer pair will amplify a representative PCR fragment of size >70bp.
  • PCR primers are used for cDNA synthesis to avoid the 3' bias imposed by oligo-dT primed cDNA synthesis.
  • the relative orientation of the two primers in cDNA is randomized by carrying out a separate synthesis with each primer.
  • These cDNA can be mixed in the same combinations as the primers chosen for PCR amplification.
  • PCR amplified cDNA fragments are resolved on 6% acrylamide gels and dried for autoradiography. Those bands which are differentially displayed in at least 2 infected cells samples samples and not in the parental cells are cut out and rehydrated.
  • This section presents methods for facilitating selection of corresponding full length cDNAs from fragments of differentially expressed genes identified by representational difference analysis or by modified differential display.
  • a single stranded biotinylated probe is synthesized from isolated cDNA fragments and is used to select the longer cDNA that contain a complementary sequence by solution hybridization to single stranded circles rescued from a phagemid infected cell cDNA library.
  • This method is especially well-suited to the use of DNA fragments isolated by the modified differential display method employing two arbitrary primers. The same arbitrary primers employed for PCR amplification of a given fragment in differential display can be modified to generate a single stranded hybridization probe from that fragment.
  • a key manipulation to achieve the efficient ligation necessary for construction of a high titer cDNA library is to insure that cDNA inserts are 5' phosphorylated by treating with T4 polynucleotide kinase prior to ligation.
  • the biotin-labeled single stranded probe generated from the differential display fragment is hybridized in solution to the ssDNA circles of the phagemid library.
  • the biotin-labeled hybridization complexes can then be separated from unrelated ssDNA on streptavidin magnetic beads and the ss circles eluted for further analysis.
  • Example 4 The Use of Ordered Microarrays to Identify Gene Expression
  • the microarrays maybe used to determine differential gene expression in infected and uninfected cells.
  • the microarrays may also be used to determine expression patterns of genes in adult and embryonic tissue.
  • the probe for the microarrays is prepared as follows:
  • One reaction is made for two sets of membranes (one set contains 7 blots, A-G) or two reactions for 3 sets of membranes.
  • 190 ⁇ l is aliquoted per 0.5 ml tube. Tubes are put in thermocycler: 65°C for 10 minutes; 1°C for 75 seconds; repeat for 23 cycles; and cooled down to 42°C. 10 ⁇ l Superscipt ⁇ Reverse transcriptase/tube (Gibco) is added. The tubes are incubated at 42 °C for 45 minutes. Another 10 ⁇ l Superscipt II Reverse transcriptase/tube (Gibco) is added. The tubes are incubated another 45 minutes at 42°C.
  • each tube 25 ⁇ l Superscript II Reverse transcriptase; 25 ⁇ l 0.5M EDTA; and 50 ⁇ l 1M NaOH; the tubes are incubated at 65°C for 20 minutes. 12.5 ⁇ l 1 M Tris-HCl, pH 7.5 is added. 1 ⁇ l is removed for "Total cpm", and added to 3 mL ECO-scinct (Bio-Rad). The remaining probe is purified on Bio-spin 6 columns, using 8 columns per probe. The caps are snapped from bottom, then the top of column is removed. The column is drained by gravity, and the flowthrough is discarded. The column is spun at lOOOxg, for 2 min. The column is transferred to a fresh collection tube, add 70 ⁇ l/ column and spun lOOOxg, 2 minutes. The flow through is pooled from all columns, and 1 ⁇ l is counted in 3 ml ECO-scint.
  • Mycrohybe (Research Genetics) is warmed to hybridization temperature. For two sets: 43 ml Microhybe, 0.5 ml denatured salmon sperm DNA 5 mg/ml stock. Membranes are wet in 2X SSC for 5 minutes. Membranes are placed in 15 ml Falcon screw cap conical with 3 ml/tube and incubated 3-4 hours at 55- 65 ° C f or prehybridization.
  • CoT DNA, yeast tRNA, and probe is denatured for 5 minutes at 99 °C, then placed on ice.
  • 0.5 ml 50 mg/ml Poly A(Pharmacia) is mixed to a final concentration of 1 mg/ml; 0.5 ml 1 mg/ml human CoTl DNA (Gibco), to a final concentration of 17 ⁇ g/ml; 0.5 ml 50 mg/ml yeast tRNA (Sigma), to a final concentration of 1 mg/ml; 6 ml 50% dextran sulfate, to a final concentration of 10%; 22.5 ml Microhybe; and 0.6 ml probe. Pre-hybe is discarded, and 2 mL hybe solution tube is added and incubated at 55-65 °C overnight.
  • the membranes are rinsed lx at room temperature with 250 ml 2X SSC/0.5% SDS altogether in one container, followed by incubation 2X for 25 minutes at room temperature with 250 ml 2X SSC/0.5% SDS altogether in one container.
  • the membranes are then incubated 2X for 30 minutes at 65 °C with 2X SSC/0.5% SDS, 3 ml/tube (one membrane/tube).
  • the membranes are incubated 2X for 30 minutes at 65 °C in O.lXSSC/0.5% SDS 3 ml/tube (one membrane/tube).
  • the membranes are wrapped in plastic wrap and exposed to phosphorimager for 10 days.
  • mice are immunized intravenously with 5 x 10 6 pfu of each recombinant vaccinia virus which express the differentially expressed gene of interest (Bennink and Yewdell, 1990, Current Topics in Microbiol. and Immunol. 163: 153-178). After at least two weeks, mice are sacrificed and CD8+ splenic T cells are enriched on anti-CD8 coated magnetic beads.
  • CD8+ cytolytic precursors are restimulated in vitro with parental SV-HUC cells that are transfected with the recombinant differentially expressed gene previously isolated in the pcDNA3.1/Zeo(+) plasmid expression vector. Substitution of the plasmid recombinant in place of the vaccinia vector for restimulation in vitro is necessary to avoid a large vaccinia vector specific response. After five days in vitro culture, cytolytic activity is determined by 51 Cr release from SV-HUC target cells transfected with either the specific recombinant plasmid or a control ovalbumin gene recombinant.
  • Dendritic cells are the most potent stimulators of T cell responses identified to date. To test immunogenicity of differentially expressed gene products, DC are incubated with the relevant gene products and assayed for the ability to activate human autologous T lymphocytes. Immature dendritic cells are prepared from healthy donors according to the method of Bhardwaj and colleagues (Reddy, A. et al, . Blood 90:3640-3646 (1997)). Briefly, PBMC are incubated with neuraminidase-treated sheep erythrocytes and separated into rosetted T cell (ER+) and non-T cell (ER-) fractions. The ER+ fraction is cryopreserved for later use. The ER- fraction
  • Mature (or immature) DC are pulsed with the gene products of the interest for a short period followed by cocultivation with autologous T cells in 24-well plates for a period of 7-14 days.
  • these may be total T lymphocytes, but it may also be desirable to fractionate CD4 and CD8 cells using magnetic separation systems (Miltenyi Biotech).
  • Total T lymphocytes are incubated with the appropriate antibody-magnetic bead conjugates to isolate total CD4, CD8, naive CD4+CD45RA+, naive CD8+CD45RA+, memory CD4+CD45RO+ or memory CD8+CD45RO+ lymphocytes.
  • CD8 lymphocytes a cytokine cocktail consisting of IL-2 (20 U/ml), IL-12 (20 U/ml), IL-18 (10 ng/ml), IFN-gamma (1 ng/ml) and a monoclonal antibody specific for IL-4 (50 ug/ml) is especially potent in enhancing DC activation of cytotoxic T cells in vitro.
  • a cytokine cocktail consisting of IL-2 (20 U/ml), IL-12 (20 U/ml), IL-18 (10 ng/ml), IFN-gamma (1 ng/ml) and a monoclonal antibody specific for IL-4 (50 ug/ml) is especially potent in enhancing DC activation of cytotoxic T cells in vitro.
  • the DC are washed and cultured in maturation conditions (1000 U/ml GM-CSF, 1000 U/ml
  • IL-4 1% autologous plasma and 12.5% monocyte-condition medium
  • Cells manipulated in this manner are viable and have morphological and surface characteristics (CD83 + ) of mature DC.
  • CTL activity is assessed in a 4 hour 51 Cr release assay.
  • An efficient means to express a specific gene product for presentation by dendritic cells is through infection with a recombinant viral vector.
  • Human DC infected with either retro viral, vaccinia, or adeno viral vectors recombinant for the same foreign, gene are employed to alternately stimulate autologous human T cells.
  • Example 7 Differential expression of host gene products in HIV-1 infected monocytic cells.
  • deregulated gene expression in HIV-1 infected cells is identified that might give rise to novel antigens encoded by the host rather than the virus.
  • host encoded antigens are expected to be a relatively stable target for protective immune responses and would not have any of the risks associated with immunization with attenuated virus.
  • Subtractive hybridization was employed to identify genes differentially expressed in HIV-1 infected vs. uninfected cells. HTV-1 genes are naturally differentially expressed in HIV-1 infected cells. To eliminate HIV genes from consideration and control for subtraction efficiency, the subtraction driver was spiked with 1% HIN sequences.
  • Figure 1 shows the results of hybridization to an array of 24 cD ⁇ A clones selected following subtractive hybridization of cD ⁇ A from infected THP-1 monocytic cell line minus uninfected THP- 1 cD ⁇ A + HIN D ⁇ A.
  • Three different probes were employed to test for differential gene expression: HIV genomic D ⁇ A from a plasmid vector, cD ⁇ A from the normal uninfected THP-1 monocytic cell line, and cD ⁇ A from HIV-1 infected THP-1. Only one clone in this set, Bl, hybridized to the HIV probe (top panel). Seven clones gave detectable hybridization to the uninfected cD ⁇ A probe (middle panel). These included one,
  • Clones 85a and 49 represent known genes, CTP synthetase (bands at approximately 7 kb and 4kb) and tricarboxylate carrier (approximately 5 kb), that are significantly overexpressed in the infected cells.
  • Clone 89 (2.5 kb) is a novel sequence of unknown function, but, by normalizing the RNA loaded in each lane to the relative expression of housekeeping genes (actin and G3PDH), clone 89 was found to be overexpressed in infected cells by only a factor of three relative to uninfected cells.
  • Example 8 Immunogenicity of Gene Products Differentially Expressed in HIV-infected Cells
  • a vaccine that targets deregulated host gene products that are not expressed in normal uninfected cells would not have any of the risks associated with use of an attenuated viral vaccine (for example, that its activity might be reconstituted by recombination with another viral genome).
  • Tat stimulates HIN-1 gene expression during transcription initiation and elongation. Tat functions primarily through specific interactions with TAR, the transactivation response element downstream of the transcriptional start site, and several cellular cofactors to increase the processivity of R ⁇ A polymerase ⁇ complexes during HTV-l transcription elongation.
  • Suggestive evidence for potential regulatory interactions of tat with host genes include at least two human mR ⁇ A that have been reported to contain TAR-like sequences as well as the existence of a cellular protein, TRP-2, which has been shown to bind to the functional tat-binding trinucleotide bulge on TAR.
  • TRP-2 a cellular protein
  • a number of different cellular proteins, including transcription factors Sp-1 and TFHF, have been found to bind directly to tat protein.
  • Other cellular proteins have been shown to bind to either the Rev Response Element, RRE, or directly to the rev protein.
  • Such interactions may lead directly or indirectly to deregulated expression of host genes and that some of these host gene products may give rise to immunogenic targets in HIV-1 infected cells that could be the basis for immunotherapeutic intervention.
  • the rev(-) mutants express early regulatory genes of HIN-1 that have been shown to also be expressed in some latently infected cell lines. If immunogenic molecules can be identified among genes expressed early in infection, then this might make it possible to target the reservoir of latently infected cells that appears to escape other forms of therapy.
  • the rev(+) virus expresses, in addition to rev, late HIV- 1 accessory genes, vpu, vif, vpr and gag that may induce further quantitative or qualitative changes in host gene expression.
  • the most efficient and reliable way to determine immunogenicity is to employ human dendritic cells pulsed with immunodominant peptides for stimulation of autologous human T cells in vitro.
  • T cells in order to identify immunodominant peptides, it is necessary to first induce specific T cells. It is first determined whether a gene product is potentially immunogenic by the ability to induce specific CTL in HLA-A2 and human CD8 double transgenic mice. The murine T cells selected are then be tested for crossreactivity on HIN-1 infected and uninfected targets. If differential reactivity is confirmed, then the same T cells can be employed to identify which of the peptide sequences that express an HLA binding motif in that gene product are immunodominant. It is then be possible to determine whether human T cells are capable of responding to these identified peptides presented by mature autologous dendritic cells or whether they may have been rendered tolerant perhaps due to expression of related gene products in other normal tissues.
  • THP-1 monocytic cell line ATCC, TIB 202
  • This cell line shares more phenotypic and functional markers with normal mature monocytes than most other available monocytic cell lines.
  • THP-1 is Fc receptor positive and phagocytic and provides costimulator activity for T cell responses to Con A.
  • THP-1 expresses several other histologic and enzymatic markers of monocytes, most notably HLA-DR, and treatment with
  • THP-1 160 nM phorbol diester (TPA) induces THP-1 to differentiate into cells with the functional characteristics of mature macrophage.
  • HTV-l infection of THP-1 has been previously reported. Shattock, etal , J. Virol 67:3569-3515 (1993). It is especially useful that THP-1 expresses HLA-A2 (haplotype: HLA-A2, A9, B5, DRWl, and DRW2).
  • HLA-A2 haplotype: HLA-A2, A9, B5, DRWl, and DRW2
  • HTV-l en (-) mutants that have been pseudotyped with the vesicular stomatitis virus envelope glycoprotein G (NSN-G) are employed.
  • NS V pseudotype has the advantage that the interaction of viral and host genes can be studied independently of membrane CD4 and chemokine receptor expression in the target cell. There is, however, a concern that some effects on gene expression may be mediated by VS V envelope interactions. Two controls are incorporated to identify possible effects of the VSV envelope alone or in concert with the HIV genome.
  • THP- 1 cells are also infected with a VS V-G pseudotyped defective MuLV based vector that expresses Thy- 1.2 under the Moloney murine sarcoma virus LTR.
  • This VS V-G pseudotyped defective MuLV vector is packaged by triple co- transfection of COS cells with the defective MuLV based plasmid (pSRLthy), together with the packaging and env(-) deficient MuLV gag and pol expression construct pS V(-)env(-)MLV, and with the VS V-G expression construct pHCMV-
  • a number of cell lines have been modified to express high levels of CD4 and CCR5 co-receptor so that a high frequency of HTV-l infection is feasible.
  • Viral mutants was constructed by deletion of the envelope gene sequences between two bgi ⁇ restriction endonuclease sites. Planelles et al., J. Virol. 69:5883-5889 (1995).
  • a related clone, HTV-l NL4 . 3 Thy-1.2env(-) has the murine thymocyte Thy-1.2 surface antigen introduced into the nef open reading frame by deletion of the nef gene sequences between Xhol and Kpnl sites. This clone is especially useful to determine the frequency of infected cells in a population.
  • This extra BamHI site was removed by digesting DBQN-3nef with Xmal (position 9149) and Xbal (position 9131), blunt ending with pfu polymerase, and religating the D ⁇ A.
  • the ligated D ⁇ A was transformed into XL- 10 Gold bacteria and clones were identified that contained a unique BamHI site in the Rev gene. This clone is designated DHIN-3nef-BamHI(-).
  • NSN-G pseudotyped env-deficient HIV- 1 are produced by electroporation of COS-7 cells with p H-TV-ln ⁇ env(-) (or p HTV-I ⁇ ⁇ env(-)rev(-)) and pHCMV-G, which expresses the VSV-G gene under the control of the CMV promoter (39).
  • the rev(-) plasmid triple cotransfection with pcRev provides the necessary rev functions in trans.
  • Viral supematants are harvested at 48 and 72 hrs and are titred by activation of the b-Galactosidase gene in the MAGI cell assay (40).
  • Expression of specific genes previously identified as differentially expressed in HTV-l infected THP-1 cells are determined by Northern blot and RNAse protection assays with RNA extracted from infected and uninfected primary monocyte cultures .
  • THP- 1 cells can be induced by GM-CSF as well as by TPA, an interesting variation on this experiment is to compare HTV- 1 infected, GM-CSF induced THP- 1 and GM- CSF activated primary monocytes (monocyte derived macrophage).
  • Discovery LineTM RNA and Gene PoolTM cDNA (Invitrogen) is used. Those sequences that are consistently expressed in HTV-l infected monocytic cells but which have low or undetectable expression in diverse normal tissues especially thymus are more likely to be immunogenic.
  • vEL/tk a new direct ligation vector was constructed, vEL/tk, that incorporates unique Notl and Apal restriction sites downstream of the early/late 7.5k vaccinia promoter.
  • This vector gives higher levels of expression of the recombinant gene, permits directional cloning of DNA, and largely eliminates the background of non-recombinant virus following ligation. Merchlinsky et al, 1997. Virology, 238: 444-451 (1997).
  • the avidity of interaction between the cytolytic T cell receptor and MHC:peptide complex on the target cell must, in general, be enhanced by a parallel interaction between the CD8 molecule on the T cell membrane and MHC class I of the target. Since murine CD 8 does not interact efficiently with human HLA class I, induction of HLA-restricted T cell responses in HLA-transgenic mice requires that either a second transgene for human CD 8 be introduced or that the HLA molecule be modified to permit interaction with murine CD 8.
  • HLA- A2.1 the latter can be accomplished by construction of a chimeric HLA molecule, HLA-A2/K , with the al and a2 domains of HLA-A2.1 and the a3 domain of murine H-2K b .
  • HLA-A2/K b x huCD8 Fj hybrid mice are therefore used for induction of HLA-A2.1 restricted murine T cell responses.
  • HLA transgenic mice have been previously employed to characterize peptide epitopes of HTLV-1 in association with HLA-B35 (Schonbach et al, Virology. 226: 102-12 (1996)), and epitopes of Hepatitis C Virus ( Shirai et ⁇ /., J. Immunol. 154: 2733-42 (1995)), Human Papilloma Virus type 16 (Ressing et al, J. Immunol. 154:5934-43 (1995)), and circumsporozoite protein of Plasmodiumfalciparum (Blum-Tirouvanziamet ⁇ /., J. Immunol. 154: 3922-31) in association with HLA- A2.1.
  • mice are immunized intravenously with 5 x 10 6 pfu of vaccinia virus recombinant for a differentially expressed gene. Bennink et al, Current Topics in Microbiol. and Immunol. 163: 153-178 (1990). After at least two weeks, mice are sacrificed and CD8+ splenic T cells are enriched on anti-CD8 coated magnetic beads.
  • CD8+ cytolytic precursors are restimulated in vitro with THP-1 monocytic cells that are transfected with the recombinant differentially expressed gene previously isolated in the pcDNA3.
  • l/Zeo(+) plasmid expression vector Substitution of the plasmid recombinant in place of the vaccinia vector for restimulation in vitro is necessary to avoid a large vaccinia vector specific response.
  • cytolytic activity is determined by 51 Cr release from THP- 1 target cells transfected with either the specific recombinant plasmid or a control ovalbumin gene recombinant.
  • This same cytolytic assay can be readily applied to determine whether CTL epitopes are also presented by other HLA compatible HTV- 1 infected cells .
  • HLA compatible targets include cells that either express native HLA-A2.1 or that have been transfected with HLA-A2.1 (or HLA-A2/K b ).
  • human dendritic cells pulsed with immunodominant peptides for presentation to autologous T cells in vitro are used.
  • a two-phase strategy can be used in which it is first determined whether a gene product is immunogenic by the ability to induce specific CTL in HLA-A2 and human CD8 double transgenic mice.
  • the T cells selected will then be tested for crossreactivity on HTV-l infected, HLA compatible tumors that express the corresponding mRNA and, if tumor reactivity is confirmed, will be used to identify which of the peptide sequences that express an HLA binding motif in that gene product are immunodominant. It will then be possible to determine whether human T cells are capable of responding to these identified peptides or whether they may have been rendered tolerant.

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Abstract

Cette invention a trait à un méthode de criblage de produits géniques, dérivés de cellules hôtes, se révélant des plus utiles dans le cadre d'une thérapie de maladies infectieuses. La méthode consiste à identifier des gènes exprimés de manière différentielle dans des cellules infectées et à procéder à un criblage de ces produits géniques exprimés aux fins de la détermination de leur pouvoir antigénique.
PCT/US2001/030334 2000-09-29 2001-10-01 Criblage de substances therapeutiques en rapport avec des maladies infectieuses WO2002027027A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7135278B1 (en) 2000-09-29 2006-11-14 University Of Rochester Method of screening for therapeutics for infectious diseases
EP1786934A1 (fr) * 2004-08-13 2007-05-23 Athlomics Pty Ltd Diagnostic assiste par micro-puce de l'infection par le virus de l'herpes par monitoring de l'expression du gene differentiel de l'hote lors de l'infection
US9371352B2 (en) 2013-02-08 2016-06-21 Vaccinex, Inc. Modified glycolipids and methods of making and using the same
US9603922B2 (en) 2007-02-21 2017-03-28 Vaccinex, Inc. Modulation of NKT cell activity with antigen-loaded CD1d molecules
US9809654B2 (en) 2002-09-27 2017-11-07 Vaccinex, Inc. Targeted CD1d molecules

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5721351A (en) * 1995-03-03 1998-02-24 Millennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders
WO1998046638A1 (fr) * 1997-04-17 1998-10-22 Millennium Pharmaceuticals, Inc. Nouveau gene specifique de th-2
WO2002031117A2 (fr) * 2000-10-13 2002-04-18 Arbor Vita Corporation Proteines transmembranaires clasp-2

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5721351A (en) * 1995-03-03 1998-02-24 Millennium Pharmaceuticals, Inc. Compositions and methods for the treatment and diagnosis of immune disorders
WO1998046638A1 (fr) * 1997-04-17 1998-10-22 Millennium Pharmaceuticals, Inc. Nouveau gene specifique de th-2
WO2002031117A2 (fr) * 2000-10-13 2002-04-18 Arbor Vita Corporation Proteines transmembranaires clasp-2

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7135278B1 (en) 2000-09-29 2006-11-14 University Of Rochester Method of screening for therapeutics for infectious diseases
US9809654B2 (en) 2002-09-27 2017-11-07 Vaccinex, Inc. Targeted CD1d molecules
EP1786934A1 (fr) * 2004-08-13 2007-05-23 Athlomics Pty Ltd Diagnostic assiste par micro-puce de l'infection par le virus de l'herpes par monitoring de l'expression du gene differentiel de l'hote lors de l'infection
EP1786934A4 (fr) * 2004-08-13 2008-02-27 Athlomics Pty Ltd Diagnostic assiste par micro-puce de l'infection par le virus de l'herpes par monitoring de l'expression du gene differentiel de l'hote lors de l'infection
US9603922B2 (en) 2007-02-21 2017-03-28 Vaccinex, Inc. Modulation of NKT cell activity with antigen-loaded CD1d molecules
US9371352B2 (en) 2013-02-08 2016-06-21 Vaccinex, Inc. Modified glycolipids and methods of making and using the same
US10111950B2 (en) 2013-02-08 2018-10-30 Vaccinex, Inc. Modified glycolipids and methods of making and using the same

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