WO1993014789A1 - Therapie par lymphocytes t cytotoxiques (ltc) specifiques d'un pathogene - Google Patents

Therapie par lymphocytes t cytotoxiques (ltc) specifiques d'un pathogene Download PDF

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WO1993014789A1
WO1993014789A1 PCT/US1993/000619 US9300619W WO9314789A1 WO 1993014789 A1 WO1993014789 A1 WO 1993014789A1 US 9300619 W US9300619 W US 9300619W WO 9314789 A1 WO9314789 A1 WO 9314789A1
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cell
cells
pathogen
virus
antigen
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PCT/US1993/000619
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Judy Lieberman
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New England Medical Center Hospitals, Inc.
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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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/05—Immunological preparations stimulating the reticulo-endothelial system, e.g. against cancer
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to therapies involving administration of cytotoxic T lymphocytes (or cells) .
  • Cytotoxic T cells that specifically lyse HIV- 1 infected autologous target cells have been found to occur at uncommonly high frequency in the blood of HIV-infected individuals; killing by such cells is predominantly mediated by CD3 + CD8 + effector cells although cytotoxic CD4 + cells and natural killer cells also play a role (Walker et al.. Nature 128:345, 1987; Plata et al.. Nature 128:348, 1987; Walker et al.. Science 240:64, 1988; Sethi et al., Nature 335:178. 1988; Koenig et al., Proc. Natl . Acad. Sci . USA 8j5:8638, 1988; Nixon et al..
  • CTLs Cytotoxic T cells
  • CD8 + T cells recognize antigenic peptides presented by MHC class I molecules.
  • a peptide must be properly processed, be capable of binding to MHC strongly enough to compete with other peptides, and be recognized as a peptide-MHC complex by T cells in the repertoire.
  • CTL specific for these epitopes dominate the lytic response (Braciale et al., Immunol. Rev. 8:95, 1987; Whitton et al., J. Virol . j62:687, 1988; Klavinskis et al., J. Virol. 61:4311, 1989; Whitton et al.. J. Virol . 62.:687, 1988; Braciale et al. , Proc. Natl . Acad. Sci . USA 86:277, 1989; Townsend et al.. Cell 44:959, 1986).
  • the invention features a method of treating a mammal infected with an intracellular pathogen.
  • the method involves (i) isolating a cell from the mammal; (ii) expressing in the cell a purified nucleic acid which encodes a pathogen-specific polypeptide, whereby the polypeptide is processed by the cell and an antigenic portion of the polypeptide is presented on the surface of the cell; (iii) contacting the antigen-presenting cell with a sample of the mammal's peripheral blood mononuclear cells (PBMCs) to produce a sub-sample enriched for cytotoxic T lymphocytes which recognize and which are capable of lysing the pathogen-infected cells of the mammal; and (iv) administering to the mammal an infection-reducing amount of the cytotoxic T lymphocyte sub-sample.
  • PBMCs peripheral blood mononuclear cells
  • the antigen-presenting cell is an autologous peripheral blood mononuclear cell, preferably an autologous monocyte or an autologous B cell; the nucleic acid is expressed from a vaccinia vector or a Bacillus Calmette-Guerin vector; the intracellular pathogen is a virus (for example, an immunodeficiency virus, a T cell leukemia virus, a Herpes virus, or an Epstein-Barr virus) , a mycobacterium, a protozoan, a mycoplasma, or a fungus; and the mammal is a human.
  • a virus for example, an immunodeficiency virus, a T cell leukemia virus, a Herpes virus, or an Epstein-Barr virus
  • the virus is a human immunodeficiency virus
  • the nucleic acid encodes a human immunodeficiency virus env, gag, or pol protein.
  • intracellular pathogen is meant a disease- causing organism which resides, during at least a part of its life cycle, within a host cell.
  • enriched for cytotoxic T lymphocytes is meant that the sub-sample has a substantially greater number of pathogen-specific cytotoxic T lymphocytes (i.e., T lymphocytes which recognize and destroy cells bearing foreign antigens, in this case, pathogen-specific antigens, on their surfaces) than a freshly isolated sample of the patient's peripheral blood mononuclear cells.
  • a purified nucleic acid is meant a nucleic acid which encodes (as used herein) the pathogen- specific polypeptide (and which may also include upstream or downstream regulatory elements) but which is free of the genes that, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene encoding the pathogen-specific polypeptide.
  • pathogen-specific polypeptide is meant any chain of amino acids which includes an antigen specific to the pathogen; as used herein, such an antigen is recognized (i.e., responded to as foreign) by the cells, in this case, cytotoxic T cells, of the patient's immune system.
  • processed is meant converted into an antigenic peptide (e.g., by proteolysis or denaturation) and displayed on the cell's surface in association with an MHC class I antigen.
  • lyse is meant to destroy or disintegrate, for example, a host cell harboring a pathogen.
  • pathogen- infected cells is meant those host cells harboring a pathogen, either in an active or a latent state.
  • antigen-presenting cell is meant any cell capable of displaying on its cell surface an antigen, or an immunogenic fragment thereof.
  • autologous is meant occurring in the same patient.
  • immunodeficiency virus is meant, without limitation, HIV-1 and HIV-2; by “T cell leukemia virus” is meant, without limitation, HTLV-I and HTLV-II; by “Herpes virus” is meant, without limitation. Herpes simplex type 1 and type 2, Herpes zoster. and cytomegalovirus as well as Epstein-Barr virus.
  • virus is also meant, without limitation, Papillomavirus, Hepatitis virus, Creutzfeldt- Jakob virus, and feline leukemia virus.
  • mycobacterium is meant, without limitation, Mycobacterium leprae or Mycobacterium tuberculosis.
  • protozoan is meant, without limitation, Toxoplasma ⁇ ondii. Giardia Iambiia, Trypanosoma cruzi. organisms of the genus Leishmania r and organisms of the genus Plas odium which cause malaria.
  • fungus is meant, without limitation, Pneumocvstis carinii., Candida albicans. and Candida tr ⁇ picalis.
  • the CTLs of the enriched sub-sample recognize and selectively target for lysis pathogen-infected cells. Because such pathogen- infected cells represent a small percentage of the total cell population, this method minimizes side effects, such as immunosuppression, which may result from other forms of therapy such as those which destroy or impair the function of all host cells at risk of pathogen infection. Moreover, the pathogen-specific CTL population may be administered to the mammal free of (or with a low dose of) lymphokines, thereby avoiding the vascular-leak syndrome which generally accompanies the superphysiologic doses of lymphokines associated with such therapies, at least in humans and mice.
  • the CTL sub-sample is produced by contact with an antigen-presenting cell derived from the same mammal.
  • antigens capable of inducing an effective CTL response i.e., inducing significant CTL proliferation
  • the antigen(s) which are immunogenic (and, very likely, immunodominant) for that particular individual are naturally selected and presented on the cell surface.
  • this antigen-presenting cell is used for the preparation of pathogen-specific CTLs, such CTLs target infected host cells which display that same immunogenic or immunodominant antigen on their surface, thereby maximizing the efficacy of the therapy.
  • FIG. 1 is a graph showing HIV-1-specific cytotoxicity as a function of the effector:target cell ratio.
  • the effector cells recognize an HIV-1 envelope antigen.
  • FIG. 2 is a graph showing HIV-1-specific cytotoxicity as a function of the effector:target cell ratio.
  • the effector cells recognize an HIV-1 envelope antigen.
  • FIG. 3 is a graph showing HIV-1-specific cytotoxicity as a function of the effector:target cell ratio.
  • the effector cells recognize an HIV-1 reverse transcriptase antigen.
  • FIG. 4A is a graph showing HIV-1-specific cytotoxicity as a function of effector:target ratio before stimulation with an autologous HIV-1 antigen presenting cell.
  • FIG. 4B is a bar graph showing that effector cells recognize a gag-specific antigen following stimulation.
  • FIG. 5 is a series of three graphs showing HIV-l- specific cytotoxicity as a function of effector:target ratio.
  • the effector cells (from a single HIV-1-infected subject) recognize an HIV-1 envelope antigen (A) , an HIV-1 reverse transcriptase antigen (B) , and an HIV-1 gag antigen (C).
  • cytotoxic T lymphocytes CTL which recognize and lyse cells displaying an HIV-1-specific antigen on their cell surfaces.
  • the method generally involves introducing into a patient's cells (specifically, the patient's peripheral blood mononuclear cells) a vector expressing all or a part of one or several HIV-1 protein(s) .
  • the protein is processed by the patient's cells, and an antigenic fragment is naturally presented on the cell surface.
  • Such cells are then used as antigen- presenting cells to stimulate the proliferation of an HIV- 1-specific sample of cytotoxic T lymphocytes.
  • PBMC peripheral blood mononuclear cells
  • Adherent cells were infected at approximately 2 pfu/adherent cell with either VPE16 (a recombinant vaccinia vector which expresses the HIV-1__ 8 gpl60 envelope protein; available from the National Institute of Allergy and Infectious Disease AIDS Depository, Bethesda, MD) or vSC8 (a recombinant vaccinia vector which expresses the lacZ protein; available from the National Institute of Allergy and Infectious Disease AIDS Depository, Bethesda, MD) .
  • VPE16 a recombinant vaccinia vector which expresses the HIV-1__ 8 gpl60 envelope protein; available from the National Institute of Allergy and Infectious Disease AIDS Depository, Bethesda, MD
  • vSC8 a recombinant vaccinia vector which expresses the lacZ protein; available from the National Institute of Allergy and Infectious Disease AIDS Depository, Bethesda
  • APCs autologous B cells as antigen-presenting cells
  • CRL 1612 CRL 1612
  • a l ⁇ g/ml solution of cyclosporin i.e., RPMI 1640 medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, KS) , 2 mM glutamine, 2mM HEPES, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 50 ⁇ M /3-mercaptoethanol] .
  • the cells were then incubated, with biweekly changes of medium, until they enlarged, formed clumps, and divided at an exponential rate.
  • the cells were considered to be immortalized (see, e.g., Blumberg et al., J. Infect. Dis . 155:877, 1987) and were infected overnight with 2 pfu/cell of either vSC8 or vPE16 (as described above) .
  • the vaccinia recombinant virus vector was inactivated by 30 minutes of UV-irradiation and 5000 rad of ⁇ -irradiation.
  • HIV-vaccinia-infected autologous B cells were used as target cells at an effector:target (E:T) ratio of 20:1.
  • E:T effector:target
  • Results are expressed as percent specific cytotoxicity and were calculated from the average cpm as [(average cpm - spontaneous release)/(total release - spontaneous release) ] x 100.
  • CTL sub-samples prepared by the above method specifically target and lyse Epstein-Barr virus-infected host cells. Such CTL sub- samples are particularly useful for the treatment of patients doubly-infected with a human immunodeficiency virus and an Epstein-Barr virus, who are at risk of developing B cell lymphomas.
  • peripheral blood mononuclear cells were freshly isolated from a patient with AIDS-defining constitutional symptoms.
  • the PBMC at 5xl0 5 cells/well in a 24 well microtiter plate
  • vSC8 at 5xl0 5 cells/well in a 24 well microtiter plate
  • vPE16 at 5xl0 5 cells/well in a 24 well microtiter plate
  • vDKl a recombinant vaccinia vector which expresses HIV-l H ⁇ B2 gag protein
  • 2xl0 6 freshly isolated PBMC were then added to each well in the same medium.
  • the medium in the wells was replaced biweekly and, after 17 days, the gag-stimulated line was restimulated with autologous B cells that were infected with 2 pfu/ml vDKl (as described above) .
  • Twelve days post- stimulation, a 4 hour 51 Cr-release assay was performed to assess envelope-specific or gag-specific cytotoxicity. Experiments were carried out using a series of effector:target ratios, and results were expressed as percent specific cytotoxicity (as described above) .
  • the nonspecifically-stimulated T cells (i.e., as derived from the patient) had a low level of cytotoxicity directed against gag-expressing autologous target cells. (This line also exhibited a low level of cytotoxicity against reverse transcriptase-expressing autologous target cells) .
  • the gag-selected T cells developed substantial cytotoxicity against gag-expressing target cells. At an E:T ratio of 6:1, cytotoxicity against gag-expressing target cells was comparable to that measured for unselected T cells at an E:T ratio of 50:1. Envelope- stimulated and lacZ-stimulated T cells did not grow well and were not assayed.
  • Figs. 1 and 2 demonstrate that HIV-1-specific CTLs were produced from the PBMCs of two independent HIV-1- infected individuals following stimulation with autologous envelope-expressing cells (i.e., autologous B cells which were infected with vaccinia vector vPE16, described above).
  • autologous envelope-expressing cells i.e., autologous B cells which were infected with vaccinia vector vPE16, described above.
  • the percent specific cytotoxicity exhibited by envelope- selected CTLs ( —•— ) was greater than that exhibited by an unselected (--•—) cell line. Background cytotoxicity was determined using lacZ-expressing target cells (—o—) .
  • Fig. 3 demonstrates that an HIV-1-specific T cell line may be generated from the PBMCs of an HIV-1-infected individual following stimulation with autologous reverse transcriptase-expressing cells (i.e., autologous B cells which were infected with vaccinia vector VCF21; available from the National Institute of Allergy and Infectious Disease AIDS Depository, Bethesda, MD) .
  • autologous reverse transcriptase-expressing cells i.e., autologous B cells which were infected with vaccinia vector VCF21; available from the National Institute of Allergy and Infectious Disease AIDS Depository, Bethesda, MD
  • the percent specific cytotoxicity exhibited by the reverse transcriptase-selected CTLs ( —•— ) was greater than that exhibited by an unselected (--•--) cell line. Background cytotoxicity was assayed using lacZ-expressing target cells (—0— ).
  • Fig. 4 demonstrates that an HIV-1-specific T cell line may be generated from the PBMCs of an HIV-1-infected individual following stimulation with autologous gag- expressing cells (i.e., autologous B cells which were infected with vaccinia vector vDKl; available from the AIDS Research and Reference Program, Bethesda, MD) .
  • autologous gag- expressing cells i.e., autologous B cells which were infected with vaccinia vector vDKl; available from the AIDS Research and Reference Program, Bethesda, MD
  • This patient had no significant HIV-specific cytotoxicity before selection (Fig. 4A) , and, again, the percent specific cytotoxicity exhibited by the gag-selected CTLs ("vDKl”) was greater than that exhibited by the unselected ("vSC8”) cell line (Fig. 4B) .
  • Fig. 5 demonstrates that different HIV-1-specific T cell lines may be generated from the PBMCs of a single HIV- 1-infected individual following stimulation with either autologous envelope-expressing cells (Fig. 5A) , autologous reverse-transcriptase-expressing cells (Fig. 5B) , or autologous gag-expressing cells (Fig. 5C) (specifically, by selecting the T cell line against autologous B cells infected with either vaccinia vector VPE16, VCF21, or vDKl, respectively) .
  • Fig. 5A autologous envelope-expressing cells
  • Fig. 5B autologous reverse-transcriptase-expressing cells
  • Fig. 5C autologous gag-expressing cells
  • Each of the T cell lines was tested for cytotoxicity against target cells expressing an envelope antigen from vPE16 (— ⁇ —), a reverse transcriptase antigen from vCF21 (—0—), a gag antigen from vDKl ( — ⁇ — ) , or a background lacZ antigen control from vSC8 (— ⁇ — ) .
  • the percent specific cytotoxicity exhibited by the HIV-1 antigen-selected CTLs was greater than that exhibited by an unselected cell line.
  • a T cell line is first established by incubating 5 X 10 5 previously frozen or freshly isolated PBMCs (isolated as described above) in T cell medium supplemented with phytohemagglutinin [i.e., RPMI 1640 medium supplemented with 15% fetal calf serum (JRH
  • T cells may be frozen for storage after 7-10 days of culture (as described above) by aliquoting 2 X 10 7 cells/vial in 95% calf serum/5% DMSO and freezing in liquid nitrogen using a programmed cell freezer (Cryomed) .
  • Such an extended culture system may also be used to expand a CTL sub-sample following pathogen- specific CTL selection.
  • antigen-presenting cells were derived from either total peripheral blood mononuclear cells, the adherent layer of a sample of peripheral blood mononuclear cells, or autologous B cells. Therapy
  • Sub-samples enriched for pathogen-specific CTLs are administered to a pathogen-infected patient as follows. Cells are washed twice with PBS to remove culture medium, resuspended in 200 ml of the same medium, and infused back into the patient by the standard techniques developed for cancer therapy by Rosenberg (see, e.g., Rosenberg et al., N. Eng. J. Med. 319:1676. 1988). Typically, infusion is performed intravenously using 10 7 - 10 cells, and the procedure takes approximately 30 minutes. If necessary, treatment can be repeated, preferably at 2-3 week intervals. Therapy can be administered soon after pathogen infection or upon onset of symptoms.
  • one or more PBMC samples isolated from a pathogen-infected, asymptomatic individual, or a CTL-enriched sub-sample prepared following pathogen infection may be stored, frozen in liquid nitrogen (as described above) , until such time as that patient requires therapy.
  • the CTLs of the enriched sub-sample recognize and selectively target pathogen-infected cells and because such pathogen-infected cells represent a small percentage of the total cell population, this method minimizes side effects resulting from generalized cell damage.
  • the enriched CTL sub-sample would target HIV-infected CD4 lymphocytes, monocytes and macrophages, leaving other cells of the immune system (including uninfected CD4-bearing lymphocytic and monocytic cells) intact and thus reducing the risk of im unosuppression.
  • This method also avoids the side effects, e.g., the vascular-leak syndrome associated with high dosage lymphokine therapy.
  • lymphokines such as IL-2 or IL-4 may be co-administered with a sub-sample of pathogen- specific CTL-enriched lymphocytes to further enhance lymphocyte proliferation.
  • a patient may be treated with antihistamines, aspirin, or acetaminophen prior to administration of lymphokines.
  • a patient may be treated with cyclophosphamide or other cytotoxic drugs prior to administration of pathogen-specific CTL- stimulatory peptides or pathogen-specific CTLs.
  • Pathogen-specific CTLs can combat pathogen infection by recognizing and lysing cells infected with the pathogen, thereby preventing further spread of infection.
  • certain pathogen-specific CTLs e.g., CTLs specific for Epstein-Barr virus, can be used to prevent or to treat a virus-induced lymphoma in a patient infected with EBV alone or in a patient infected with EBV and a human immunodeficiency virus.
  • Embodiments The general methods described herein may be modified, e.g., to simplify production of the antigen- presenting cells, to simplify production of the pathogen- specific CTL sub-sample, or to extend the use of the method to the treatment of other pathogenic infections.
  • vaccinia vectors may be utilized for antigen expression.
  • Such vectors include vPE8 (a vaccinia-HIV-l BH10 gpl20 vector) , vPE5 (a vaccinia-HIV-l ⁇ g gpl60 vector) , vPE6 (a vaccinia- HIV-l BHg gpl20 vector) , vSC40 (a vaccinia-HIV-l BH10 gag-pol vector) , VCF21 (a vaccinia-HIV-l H ⁇ B2 reverse transcriptase vector) , vVKl (a vaccinia-HIV-l H ⁇ B2 gag-pol vector) , W:gag (a vaccinia-HIV- 1 HXB gag vector) , VMN462 (a vaccinia-HIV-l ⁇ gpl60 vector) , VRF222 (a vaccinia-HIV-1 ⁇ gpl60 vector) , pl
  • vaccinia vectors include vPE17, vPE18, vPE20, vPE21, and vPE22 (available from the National Institute of Allergy and Infectious Disease AIDS Depository, Bethesda, MD) which encode truncated envelope proteins and VCF32, VCF33, vCF34, vCF35, VCF36, and VCF37 (available from National Institute of Allergy and Infectious Disease AIDS Depository, Bethesda, MD) which encode truncated reverse transcriptase proteins.
  • Methods for constructing other vaccinia expression vectors are provided, e.g., in Chakrabarti et al.
  • any cell may act as an antigen-presenting cell.
  • Cells of the immune system e.g., monocytes, macrophages, B cells, or T cells
  • other useful cells include fibroblasts or neurons.
  • cells infected with Epstein Barr virus represent preferred antigen presenting cells. Appropriate vectors for antigen expression in these (and other) cell types are provided, e.g., in Cloning Vectors : A Laboratory Manual (P.H. Pouwels et al., 1985, Supp.
  • preferred vectors include a poxvirus, avipox virus, SV40, retrovirus, Epstein-Barr virus, or bovine papilloma virus sequence.
  • An HIV mutant for example, a packaging or fra eshift mutant, may also be used to infect an appropriate antigen-presenting cell for HIV-specific CTL enrichment (see, e.g., Aldovini and Young, J. Virol . 64:1920, 1990).
  • hepatocytes infected with an antigen-expressing hepatitis B vector may be used as an antigen-presenting cell.
  • peripheral blood lymphocytes are transduced with an antigen- presenting retroviral vector (e.g., T4-pMV7, T8-MV7, or pHIVlacZ; available from the AIDS Research and Reference Reagent Program, Bethesda, MD) .
  • an antigen- presenting retroviral vector e.g., T4-pMV7, T8-MV7, or pHIVlacZ; available from the AIDS Research and Reference Reagent Program, Bethesda, MD
  • Expression vectors may be introduced by stable or transient transfection or by viral infection. Transfection methods are provided, e.g., in Current Protocols in
  • expression vectors may be inactivated by UV- irradiation (as described above) , ⁇ f -irradiation (as described above), exposure to mutagens (e.g., mitomycin C) , treatment with glutaraldehyde, or inactivation of a gene(s) required for infectivity (e.g., by site directed mutagenesis or deletion) .
  • CTL-stimulatory antigens may be included in H ⁇ V-1- encoded proteins other than env (i.e., gpl60) and gag. For example, they may be included in the reverse transcriptase, tat, rev, or nef proteins. DNA encoding such proteins or fragments thereof may be inserted into a vaccinia expression vector (or any appropriate expression vector as defined above) , and the resultant antigen-presenting cells used to produce an enriched sub-sample of HIV-1-specific CTLs as described in the above example.
  • a vaccinia expression vector or any appropriate expression vector as defined above
  • antigen-presenting cells may be prepared by infection of cells (preferably, monocytes, macrophages, T cells, or B cells) with one of the following vectors: vPE8, vPE5, vPE6, VSC40, vVKl, W:gag, VMN462, VRF222, plllenv3-l, pDOLHIVenv, pHenv, or pNL4-3dPst (all described above) ; or pCVl (a pCV- HIV-1 tat and rev vector) , pSV2tat72 (a pSV2-dhfr-HIV-l tat vector) , or pU3R-III CAT (a pSV2CAT-HIV-l LTR vector) (all available from the AIDS Research and Reference Reagent Program, Bethesda, MD) .
  • vPE8 vPE5, vPE6, VSC40, vVKl, W:gag, VMN462, VRF222
  • Any isolate of HIV may be used as a source of viral-specific polypeptide genes (including virus isolated from an HIV-infected patient's lymphocytes).
  • a patient infected with any isolate of HIV e.g., HIV-I JJ JJ
  • Genes coding for proteins homologous to HIV-1-encoded proteins may also be useful in this invention if such proteins elicit an HIV-1-specific CTL response; the proteins are generally coded for by related primate lentiviruses, e.g., HTLV-I and HTLV-II as well as the simian immunodeficiency viruses.
  • the antigen-presenting cell may be infected with HIV itself (e.g., virus isolated from the subject to be treated) , although the level of antigen expression is generally lower than that obtained using expression vectors such as those described herein.
  • HIV itself
  • the instant methods can be used to treat human patients or mammals infected with other pathogenic viruses including, but not limited to, human T-cell leukemia viruses.
  • Herpes viruses e.g., Epstein-Barr virus
  • Hepatitis virus as well as any intracellular disease-causing mycobacterium, mycoplasma, protozoan, or fungus.
  • antigen-presenting cells may be prepared by infection of cells (preferably, monocytes, macrophages, T cells, or B cells) with one of the following HIV-2 vectors: vSC50 (a vaccinia-HIV-22 SBL . Is ⁇ gpl60 vector) , rW/ROD (a vaccinia-HIV-2 R0D gpl60 vector) , or rW/ST (a vaccinia-HIV- 2 ST gpl60 vector) ; all available from the AIDS Research and Reference Program (Bethesda, MD) .
  • HIV-2 vectors preferably, monocytes, macrophages, T cells, or B cells
  • the mycobacterium Bacillus Calmette-Guerin may be engineered to encode foreign proteins and used to infect, e.g., monocytes or macrophages, to produce antigen- presenting cells; such mycobacterium and methods for infection are described in Aldovini and Young (Nature 351:479. 1991).
  • mycobacterium and methods for infection are described in Aldovini and Young (Nature 351:479. 1991).
  • the sub-samples of pathogen- specific CTLs would be prepared as described above for HIV- 1.
  • the PBMC sample may be presented simultaneously with more than one pathogen-specific CTL-stimulatory epitope, e.g., by expressing more than one pathogen-specific gene in the antigen-presenting cell or by contacting the PBMC sample with multiple APCs, each displaying a different pathogen- specific antigen.
  • a sample of PBMCs or a T cell line is presented with two or more different autologous antigen presenting cells at the same time — for example, a first APC expressing the HIV-1 envelope gene, a second APC expressing the HIV-1 gag, and a third APC expressing the HIV-1 reverse transcriptase gene; such APCs may be produced, e.g., by infection of the autologous cell with an HIV-1-vaccinia vector (e.g., as described herein) .
  • Any pathogen-infected mammal may be treated using a pathogen-specific CTL sub-sample of the invention.

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Abstract

On décrit un procédé de traitement concernant les patients infectés par un pathogène intracellulaire. A cette fin, il convient d'administrer aux patients un sous-échantillon de leurs propres cellules sanguines mononucléaires périphériques enrichies en lymphocytes T cytotoxiques (LTC) spécifiques du pathogène. On prépare ce sous-échantillon contenant lesdits LTC: (i) en isolant une cellule de mammifère, (ii) en exprimant dans cette cellule un acide nucléique qui code pour un polypeptide, spécifique du pathogène, qui est alors produit par la cellule, laquelle présente ainsi à sa surface une portion antigénique dudit polypeptide; et (iii) en mettant cette cellule présentant ledit antigène en contact avec un échantillon des cellules sanguines mononucléaires périphériques du mammifère pour stimuler la production du sous-échantillon enrichi en LTC qui reconnaissent et peuvent lyser les cellules du mammifère infectées par le pathogène.
PCT/US1993/000619 1992-01-22 1993-01-22 Therapie par lymphocytes t cytotoxiques (ltc) specifiques d'un pathogene WO1993014789A1 (fr)

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US824,009 1992-01-22

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5738852A (en) * 1993-04-20 1998-04-14 Solis Therapeutics, Inc. Methods of enhancing antigen-specific T cell responses
US6013660A (en) * 1996-10-02 2000-01-11 The Regents Of The University Of California Externally targeted prophylactic and chemotherapeutic method and agents
US6752993B1 (en) 1993-11-23 2004-06-22 The Regents Of The University Of California Abundant extracellular product vaccines and methods for their production and use
US6761894B1 (en) 1993-11-23 2004-07-13 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US7300660B2 (en) 1993-11-23 2007-11-27 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation

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US5081029A (en) * 1985-09-25 1992-01-14 Oncogen Methods of adoptive immunotherapy for treatment of aids

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US5081029A (en) * 1985-09-25 1992-01-14 Oncogen Methods of adoptive immunotherapy for treatment of aids

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5738852A (en) * 1993-04-20 1998-04-14 Solis Therapeutics, Inc. Methods of enhancing antigen-specific T cell responses
US6752993B1 (en) 1993-11-23 2004-06-22 The Regents Of The University Of California Abundant extracellular product vaccines and methods for their production and use
US6761894B1 (en) 1993-11-23 2004-07-13 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US7300660B2 (en) 1993-11-23 2007-11-27 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US6013660A (en) * 1996-10-02 2000-01-11 The Regents Of The University Of California Externally targeted prophylactic and chemotherapeutic method and agents
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation

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