WO1997030590A1 - Immunotherapie cellulaire - Google Patents

Immunotherapie cellulaire Download PDF

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WO1997030590A1
WO1997030590A1 PCT/US1997/002309 US9702309W WO9730590A1 WO 1997030590 A1 WO1997030590 A1 WO 1997030590A1 US 9702309 W US9702309 W US 9702309W WO 9730590 A1 WO9730590 A1 WO 9730590A1
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cells
hiv
htv
serum
therapeutic agent
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PCT/US1997/002309
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Richard G. Olsen
John L. Ridihalgh
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Cira Technologies, Inc.
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Priority to EP97914765A priority Critical patent/EP0889693A1/fr
Priority to AU21898/97A priority patent/AU727865B2/en
Publication of WO1997030590A1 publication Critical patent/WO1997030590A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0651—Lymph nodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/59—Lectins

Definitions

  • the present invention relates to immune mediated diseases as typified by immunosuppressive virus infections exemplified by immunosuppressive virus infection
  • HIV both human, e.g., HIV, and animal, e.g., FIV
  • adoptive cellular therapy of such virus infections Much of the discussion will focus on HIV, although such discussion is by way of illustration and not by way of limitation.
  • cytokines are capable of inducing the replication of HTV; therefore, selective blocking of cytokines is an important strategy in the treatment of HTV disease. However, cytokines also are important for the expression and regulation of normal immune function.
  • the most direct and potentially most effective form of immunologic reconstitution is the replacement of cellular elements of the immune system, i.e., adoptive cellular therapy.
  • CD8 + cytolytic T cells CTL
  • Ho et al. conducted a phase I study to determine the safety and feasibility of infusing in vitro purified, activated, and expanded CD8 + cells in seven patients with AIDS-related complex or AIDS.
  • Ho, et al. "A phase I study of adoptive transfer of autologous CD8 + T lymphocytes in patients with acquired immunodeficiency syndrome (ATDS)-related complex or AIDS", Blood, 81:2093-2101 , 1993.
  • ATDS acquired immunodeficiency syndrome
  • CD8 + cells were first selectively isolated in monoclonal antibody-coated flasks from peripheral blood mononuclear cells (PBMC) recovered by leukopheresis. They were then cultured and expanded with phytohemagglutinin and IL-2 before infusion. Five cycles of isolations and infusions of increasing numbers of CD8 + T cells were achieved in five of seven subjects. Five cycles could not be completed in two subjects with AIDS whose CD4 + cell counts were ⁇ 48/ ⁇ L. Infusions of CD8 + cells alone were well tolerated. Four patients received IL-2 by continuous infusion for 5 days with their final cycle of CD8 + cells. All developed reversible adverse effects attributable to IL-2.
  • PBMC peripheral blood mononuclear cells
  • CD8 + cells After infusion, ⁇ In-labeled CD8 + cells quickly accumulated in the lungs, with less than 10% of the labeled cells remaining in the circulation. After 24 hours, labeled CD8 + cells were reduced in the lungs, but increased and persisted in liver, spleen, and bone marrow. Four of five patients who were treated with multiple infusions as CD8 + cells remained clinically stable, but a fifth developed Pneumocystis carinii pneumonia. Other attempts to reconstitute cellular elements under investigation include the transfusion of ex vivo IL-2 expanded HTV-specific CD8 + T cell clones, bone marrow transplantation, and thymic transplantation.
  • Virus-specific CD8 + CTLs have been demonstrated to cause immunopathology if a significant reservoir of actively infected cells is established in a vital organ as the response is elicited.
  • HTV-specific CTLs have been implicated in the pathogenesis of AIDS dementia and lymphocytic alveolitis (Riddell, supra), (iii) Peripheral blood-derived CD8 + do not appear to traffick to major sites of HIV activity, e.g., lymph nodes (Ho, et al., supra), (iv) There is evidence that HIV reactive CD8 + cells may contribute to the immunodeficiency by lysing HTV-infected CD4 cells. Grant, et. al., "Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic T lymphocytes from HTV-infected individuals", Clin Exp Immunol, 93:356-362, 1993.
  • the fundamental distinguishing feature between immunotherapy of HTV infection and that for other persistent viruses is that HIV undermines the ability of the host to appropriately respond to efforts to augment of elicit potentially protective host immune responses by inducing dysfunction and depletion of CD4+ TJ_J cells.
  • the presentation of an antigen (Ag) to CD4 + TJJ cells and the generation of T H activity are the central processes in any specific cellular immune response, such as the specific response to a HIV, upon which all the other elements depend.
  • Murine CD4 + Tu cells comprise at least three subsets (Powrie, et al., "Cytokine regulation of T-cell function: potential for therapeutic intervention", Immunol Today, 14:270-274, 1993): The T H 1 subset, which secretes TL-2 and TFN, but not TL-4, appears to be responsible for delayed type hypersensitivity (DTH) responses. T ⁇ 2 cells, which secrete TL-4 and TL- 5, but not IL-2, provide B-cell help. T cells produce TL-2, TFN, TL-4, and IL-5.
  • Several groups have now isolated human clones similar to the mouse Tul, Tu2, and TjrO phenotype.
  • Tjjl differentiation occurs in response to intracellular viruses and bacteria and is promoted by IFN, IFNa, and TL- 12.
  • Tu2 differentiation occurs in response to soluble allergens and some helminth components and is promoted by TL-4 and, in some studies, transforming growth factor (TGF) ⁇ .
  • TGF transforming growth factor
  • HTV-infected individuals recent reports suggest that there is a progressive imbalance immune systems with a selective defect in Tjjl responses and a predominance of Tu2 responses. Correcting this imbalance by the administration of Tul-type cytokines such as TL-2 or TL- 12 is under investigation. It is now evident that the initial host cellular immune response to HIV reduces the level of HIV in the peripheral blood. In the early, "immune activation" stage of infection, HTV is harbored in lymphatic tissues. Lymph nodes are characterized by germinal center hyperplasia and a CD4 + cell pleocytosis that includes a substantial number of TL-2 and TFN secreting cells suggesting that a TJJI differentiation has occurred. Emille, et.
  • CD4 + T cells by antigen presenting cells (APC), which normally occurs in the follicular center, renders uninfected cells susceptible to infection by the locally retained virus and results in the reactivation of HIV in latently infected cell, thus contributing to the gradual depletion of CD4 + Tfj cells observed during the course of HIV infection. Nonetheless, it does appear that early in the course of HTV infection an appropriate and potentially effective immune response is generated in lymph nodes. We are proposing to examine the possibility that cells that can provide appropriate TJJ function can be isolated and expanded ex vivo from the lymph nodes infected with HIV and that adoptive cellular therapy using the expanded lymph node lymphocytes (LNL) will generate antiviral immunity in vivo.
  • APC antigen presenting cells
  • HTV lymph nodes
  • CD4 + cells the central target of HTV infection
  • CD4 + cells the central target of HTV infection
  • the possibility does exist that under certain circumstances, and perhaps in association with the adrninistration of effective antiretroviral agents, stimulation of the immune system by CD4+.
  • LNL secreting appropriate cytokines might have a beneficial effect in reconstituting immune competence in HTV infected individuals. It should be noted that although CD8+ CTL suppress the replication of HTV in PBMC of patients with AIDS, Walker et al.
  • LNL locomotor capability of LNL, and, thus, the ability to traffick to lymph nodes, also may be greater than that of PBL. Triozzi, "Identification and activation of tumor-reactive cells for adoptive immunotherapy", Stem Cells, 11:204-211, 1993.
  • HTV infection a novel approach to the adoptive cellular therapy of immune mediated diseases exemplified by HTV infection which therapy exploits the potentially effective cellular immune response that is initially generated in HTV-infected individuals.
  • One aspect is a method for preparing cells having an immunorestorative effect on patients afflicted with human immunodeficiency virus (HIV), which includes subjecting cytokine-producing cells derived from lymph nodes excised from patients infected with HTV to mitogenic stimulation in serum-free media for their expansion.
  • HAV human immunodeficiency virus
  • the resulting therapeutic agent is effe e in inducing an immunorestorative effect on patients afflicted with human immunodeficiency virus (HIV) and includes in a pharmaceutically-acceptable carrier cytokine-producing cells in an amount effective for HTV reduction, the cytokine-producing cells having been produced by the step of subjecting cytokine-producing cells derived from lymph nodes excised from patients infected with HTV to mitogenic stimulation in serum-free media for their expansion.
  • HTV human immunodeficiency virus
  • the invention apparently is capable of inhibiting replication of HIV as determined by the viral load reductions exhibited by patients administered the therapeutic agent of the present invention.
  • HTV human immunodeficiency virus
  • Advantages of the present invention include a culture procedure that is easy, expedient, and reproducible. Another advantage is a therapeutic agent that is negative for HTV; yet, is effective in inhibiting the replication of HIV. A further advantage is that the capacity of the therapeutic agent to secrete cytokine may obviate the need for exogenous systemic cytokines to maintain their activity. A further advantage is that total lack of adverse side effects when using the novel therapeutic agent. A yet further advantage is the lack of adverse reactions between the novel therapeutic agent and other drugs taken by the patient.
  • Fig. 1 graphically depicts the influence of anti-CD3 MAb concentration on lymph node cell expansion.
  • Cells were cultured for 10 days with a range of concentrations of anti-CD2 MAb (OKT3) and constant concentrations of TL-2.
  • Data represent range and mean values for 5 different donors with peripheral blood CD4 counts from 46 to 210/mm 3 .
  • Figs. 2 and 3 graphically depict expansion of lymph node and peripheral blood CD4 and CD8 cells from 2 patients.
  • the peripheral blood CD4 count of the patient depicted in A was 23 and that of the patient depicted in B was 125/mm 3 .
  • Fig. 6 depicts HTV-1 mRNA expression of lymph node cells during ex vivo expansion with anti-CD3/TL-2, TL-2 alone, or with no additions (NA).
  • Fig. 7 graphically depicts the effect of culture supernatants generated with either
  • Fig. 8 graphically depicts proliferative responses of expanded cells to oligopeptides corresponding to HTV-1 envelope sequence. The drawings will be described in more detail below.
  • HTV- 1 -specific CTL generated by culturing peripheral blood lymphocytes with HTV-1 peptides
  • autologous cloned HTV- 1 -specific CTL transduced with a fusion marker/suicide gene and CD8 + cells transduced with genes coding for chimeric T-cell receptors that bind HTV- 1 -infected cells (reviewed by Bridges, et al, "Gene Therapy and Immune Restoration for HTV Disease", Lancet 345:427-432, 1995).
  • Dysregulation of the cytokine system plays a central role in the progression of HTV-1 infection and related immunosuppression.
  • the inventive approach to the cellular therapy of AIDS relies on lymph-node cell infusions as a means of modulating the cytokine system, rather than as a means of administrating HTV- 1- specific CTL.
  • the results reported herein indicate that significant numbers of cytokine-releasing cells with anti-HIV-1 activity can be expanded in short-term cultures from the lymph nodes of TTTV-1 infected individuals, even from individuals with advanced disease.
  • cytokines that could inhibit HTV-1 replication are potentially produced by the expanded cells, including: TL-8, IL-10, TNFa, and TGFb (Mackewicz, et al, "CD8 + Cell and Anti-HTV Activity: Nonlytic Suppression of Virus Replication” AIDS Res. Hum. Retrovirus 8:629-640, 1992).
  • Supernatants from the expanded cells were capable of suppressing HTV-1 mRNA expression in cells initially cultured in TL-2 alone.
  • CD8 cell anti-viral factor as described by Levy and coworkers (Mackewicz, et al, (supra); Mackewicz, et al, "Effect of Cytokines on HTV Replication in CDR + Lymphocytes: Lack of Identity with CD8 + Cell Antiviral Factor", Cell Immunol. 153:329-343, 1994; and Mackewicz, et al, "CD8 + T Cells Suppress Human Immunodeficiency Virus Replication by Inhibiting Viral Transcription", Proc. Natl Acad. Sci. USA 92:2308-2312, 1995), is present.
  • the cytokines produced would also be predicted to have a variety of immunorestorative effects.
  • the heterogeneity of HTV- 1 makes is difficult to select cells reactive with all strains present at the time of re-infusion.
  • Walker et al "Long-Term Culture and Fine Specificity of Human Cytotoxic T-Lymphocyte Clones Reactive with Human Immunodeficiency Virus Type 1", Proc. Natl. Acad. Sci. USA 86:9514-9518, 1989, have shown that MHC class I-restricted CTL specific for HIV-1 reverse transcriptase can be expanded from HTV- 1 -infected individuals. These CTL recognized multiple epitopes in conjunction with different host MHC molecules.
  • HTV- 1 -infected lymph nodes as a source of cells, rather than peripheral blood (PB).
  • PB peripheral blood
  • the relative frequencies of CTL specific for HIV-1 proteins from lymphoid organs differ at least one log from that of peripheral blood (Hadida, et al, "CTLs from Lymphoid Organs Recognize an Optimal HLA-A2-Restricted and HLA-B52-Restricted Nonpeptide and Several Epitopes in the C-Terminal Region of HTV-1", Nef. J.
  • the present invention uses the capacity of anti-CD3 MAb to mimic the normal pathways of T-cell activation and the capacity of TL-2 to expand multiple T-cell subpopulations from lymph nodes.
  • HTV- 1 -specific T-cell lines can be expanded by nonspecific stimulation with anti-CD3 MAb and TL-2 without the need for re-exposure to viral antigen (Walker, et al, (supra); Johnson, et al, "HIV-1 Gag Specific Cytotoxic T Lymphocyte Recognize Multiple Highly conserveed Epitopes. Fine Specificity of the Gag-Specific Response Defined by Using Unstimulated Peripheral Blood Mononuclear Cells and Cloned Effector Cells", J.
  • Gag Epitopes are Clustered in three Regions of the p218 a g Protein", J . Virol. 67:694-702, 1993). It is difficult to assess the spectrum of HIV-1 reactivities in vitro of cells cultured in the presence of polyclonal activators, such as anti-CD3 MAb and TL-2, particularly in short-term bulk cultures. A proliferative response to a variety of HTV-1 antigens was observed.
  • HTV-1 specific CTL and CTL precursors likely were present in culture and may have played a role in the anti-HIV-1 activity.
  • the inventive expansion regimen was not designed to maximize cytolytic activity, and cells were exposed to decreasing concentrations of TL-2 with the intent of reducing their dependency on TL-2. Although the possibility was not examined, cells with reactivities against other pathogens to complicate HTV-1 infection also may have been expanded.
  • Culture conditions were optimized to maintain the viability of the APCs present in the lymph nodes, so that at least theoretically, any HTV- 1 -associated antigens released in vitro could be presented by the APCs.
  • Cells were cultured in serum-free conditions using a media designed to maintain the viability of macrophages and dendritic cells, and not lymphocytes.
  • Cells expanded in serum-free conditions are potentially less toxic: the only significant toxicity observed in the study of Carter, et al, (supra), was hypersensitivity to the bovine serum proteins that were adherent to the expanded lymphocytes.
  • the concentration of anti-CD3 MAb influenced cell expansion.
  • a concentration of anti-CD3 MAb was used to elicit the effect of immobilized anti-CD3, by associating with the APCs present, and also elicit the effect of soluble anti-CD3 MAb and the possibility of in vitro immunization.
  • the concentration of anti-CD3 MAb is 0.5% of that used by Moran, et al (supra) to induce the effects of soluble anti-CD3.
  • the transient increase in HIV-1 mRNA observed would be a predicted consequence of exposure to soluble anti-CD3 (Moran, et al, supra).
  • TNFa and TL-6 in the culture supernatants would be a predicted effect of immobilized anti-CD3 MAb as would the presence of soluble factors that inhibit HTV-1 (Moran, et al, supra; Mackewicz, et al, 1994, supra).
  • Culture interval also influenced cytokine production; the greatest amounts of cytokines were produced by cells cultured for 8 to 10 days.
  • CD8+ cells are the major producers of TFNg, a THl-type cytokine in HTV- 1 -infected lymph nodes; they also produce TL-4, TL-5, and IL-10, TH2-type cytokines (Emilie, et al, supra; Manetti, et al, "CD30 Expression by CD8 + T. Cells Producing Type 2 Helper Cytokines. Evidence for Large Numbers of CD8 + CD30 + T Cell Clones in Human Immunodeficiency Virus Infection", J. Exp. Med.
  • CD8+ cells that expanded were CD1 lb-, which is the predominant phenotype of anti-HIV-1 CTL and CD8 + cells that suppress HTV-1 replication non-lytically (Mackewicz, et al, 1992, supra). Although expansion rates varied, the cytokine profiles of cells expanded from individuals with CD4 counts ⁇ 100 and those with CD4 counts >100 were comparable. These results are consistent with those of Graziosi, et al.
  • CD4+ cells present were comparable to the CD8 + cells.
  • adoptive transfer of a mixed population of CD4+ and CD8 + cells has been more effective than purified CD8+ cells, even when CD8 + cells are central to desired response (Byrne, et al, "Biology of Cloned Cytotoxic T Lymphocytes Specific for Lymphocyte choriomeningitis Virus: Clearance of Virus in vivo", J. Virol 51:682-686, 1984; Larsen, et al, "Role of T-Lymphocyte Subsets in Recovery from Herpes Simplex Virus Infection", J. Vriol.
  • CD4+ cells activated CDF + in particular, are the principal target for HIV- 1 and critical to the progression of the infection
  • CD8+ cells normally do not make enough IL-2 to support their own expansion and are dependent on TL-2, and possibly other cytokines from CD4 + cells for "help".
  • Antibodies have been able to protect against experimental retroviral infections under some circumstances (Vaslin, et al, "Induction of Humoral and Cellular Immunity to Simian Immunodeficiency Virus: What are the Requirements for Protection", Vaccine 12:1132-1140, 1994); correlative evidence suggests that some antibody may be associated with protection against progress of HTV- 1 infection (Salk, "Prospects for the Control of AIDS by Immunizing Seropositive Individuals", Nature 327:473-476, 1987); long-term survivors of HIV-1 have been characterized by a strong neutralizing-antibody response (Pantaleo, et al, "Studies in Subjects with Long-term Nonprogressive Human Immunodeficiency Virus Infection", N.
  • CD8+ T H cells are well- recognized. Thus, there is a potential advantage to the infusion of cells that can provide TH activity to B-cells. Some of the CD8+ cells were also CD45RA+ or CD30 + , suggesting the possibility of CD8 + TH function in vivo, including the induction of anti- HTV-1 antibody (Manetti, et al, supra) The release of THI cytokines, such as IFNg, suggests the possibility that DTH responses can be enhanced. The study of Carter, et al, (supra) has suggested the feasibility and safety of infusing a mixed population of uninfected CD4+ and CD8 + cells into HTV- 1 -infected individuals.
  • Cytokines can mediate both beneficial and detrimental effects depending on a variety of factors, including the nature of the responding cell, the stage of infection, and the concentration of the cytokine.
  • certain cytokines including TL-4 and IL-10, enhance the replication of HTV- 1 in lymphocytes but inhibit HIV-1 replication in macrophages (Akridge, et al, "TL- 10 is Induced During HTV-1 Infection and is Capable of Decreasing Viral Replication in Human Macrophage", J.
  • Cytokines also are unlikely to be secreted or act individually, but rather as a "cascade" involving other cytokines, and regulated, local, i.e., paracrine, release appears to be critical to optimal activity. Finally, the experience to date, including that with TL-2 therapy, suggests that systemic cytokine therapy will unlikely be of benefit in patients with advanced TflV-1 infection.
  • HTV- 1 -specific CD8+ CTL have been demonstrated to cause immunopathology, including dementia and lymphocytic alveolitis, and may contribute to the immunodeficiency by lysing HTV- 1 -infected CDC4 cells (Riddell, et al, supra; Grant, et al, "Lysis of CDr+ Lymphocytes by Non-HLA- Restricted Cytotoxid T Lymphocytes from HTV-infected Individuals", Clin.
  • autoimmune diseases e.g., rheumatoid arthritis
  • viral induced and result from persistent and acute infections including latent infection (e.g., human herpes virus), chronic infections (e.g., "old dog encephalitis” following canine distemper virus (CDV) infection or lymphocyteic choriomeningitis in mice), and slow infections (both lentiviruses including HIV, feline immunodeficiency virus (FIV), and simian immunodeficiency virus (STV); and a group of unclassified agents which cause subacute spongioform encephalopathies including Cruetzfeld- Jakob disease, Kuru, and Mad Cow Disease).
  • latent infection e.g., human herpes virus
  • chronic infections e.g., "old dog encephalitis” following canine distemper virus (CDV) infection or lymphocyteic choriomeningitis in mice
  • slow infections both lentiviruses including HIV, feline immunodeficiency
  • Such immunosuppressive or chronic diseases that lead to an immunosuppressed state in the host should be treatable in accordance with the precepts of the present invention including, for example, HTV, tuberculosis, measles, dengue fever, malaria, hepatitis (chronic), leprosy, rheumatoid arthritis, multiple sclerosis, canine distemper virus, and the like.
  • RNA viruses are exemplified by, for example, picornaviruses, togaviruses, paramyxoviruses, orthomyxoviruses, rhandoviruses, reoviruses, retroviruses, bunyaviruses, coronaviruses, and arenaviruses.
  • DNA viruses are typified by panoviruses, papoviruses, adenoviruses, herpesviruses, and poxviruses.
  • lymph nodes samples of lymph nodes not needed for diagnosis were obtained from resected specimens. The major part of each lymph node was used for histopathology; approximately 20% to 50% was used for the immunologic studies. Lymph nodes with histologic evidence of malignancy or mycobacterial infection were excluded. Dissociation of tissue was carried out under sterile conditions in a laminar flow hood. Tissue was rinsed in a centrifuge tube with a serum-free medium, Macrophage SFM (Gibco/BRL, Grand Island, NY), and was transferred to a Petri dish. Extraneous tissue was excised with a scalpel, and the tissue was minced into pieces approximately 2 to 3 mm in diameter.
  • Macrophage SFM Gibrophage SFM
  • Tissue fragments were placed in centrifugation tubes with collagenase pre-warmed to 37 * C. Tubes were vortexed at a speed that caused tumbling, but not foaming. Free cells were decanted through three layers of sterile medium-wet nylon mesh into centrifuge tubes. The cells were centrifuged (250 X g) in a refrigerated centrifuge for ten minutes. The supernatant fluid was poured off, and the procedure was repeated until sufficient cells were obtained. Cells from the different digests then were pooled and counted, and the cell viability was assessed by the Trypan blue exclusion test. Cells not needed immediately were cryopreserved in dimethyl- sulfoxide with 5% autologous serum.
  • Lymph node lymphocytes (lOfyml) were suspended in Macrophage SFM, 600 IU/ml human recombinant TL-2 (Proleukin, Cetus Oncology, Emeryville, CA), and MAb anti-CD3 (Orthoclone, OKT3, Ortho Pharmaceutical Corporation, Raritan, NY) at 37 * C for four days. Cells were counted and resuspended every three to four days depending on growth in fresh media with additions.
  • TL-2 Human recombinant TL-2
  • MAb anti-CD3 Orthoclone, OKT3, Ortho Pharmaceutical Corporation, Raritan, NY
  • Target cells were labeled with Na chromate ( 51 Cr) using 100 ⁇ Ci/5 X 10 6 cells/0.5 ml for one hour at 37X.
  • Target cells (lOVlOO ⁇ l) were added to triplicate 6-mm round-bottomed plates. Effector cells generated, as described above, were added in the same media (in 100 ⁇ l) to achieve effector-to-target (E:T) ratios of 40:1, 20:1, 5:1, and 1.5:1. Also included were three maximum release wells containing only labeled target cells plus TRITON X-100 surface active agent and three spontaneous release wells. The plate was centrifuged (200 X g for 5 minutes) and incubated at 37 * C for four hours.
  • PCR cytokine mRNA expression was assessed.
  • Total RNA was extracted by quanidium thiocyanate phenol chloroform using RNA-zol (Cinna/Biotec, Friendswood, TX).
  • 10 X PCR buffer 500 mM KCl, 200 mM Tris HCl, pH 8.3, MgCt ⁇ concentration optimized for each oligo set
  • 2 ⁇ l of 20 pM of the relevant oligonucleotide 0.5 ⁇ l of 10 mM dNTP, and 1.2 U Taq polymerase (Ampli Taq, Perkin-Elmer Cetus) to a final volume of 100 ⁇ l.
  • Amplification was performed with a Perkin-Elmer Cetus thermocycler for 30 cycles (1 min. 94°C melting, 2 min. 55°C annealing, and 2 min. 72 * C extension).
  • cDNA product were amplified with G3PDH primers. Amplified products were separated on a 2% agarose gel and blotted onto nylon filters. The filters were incubated in a pre-hybridization solution for four hours at 65 * C and then hybridized to a digoxigen-labeled internal Cb probe (Boehringer Mannheim, Indianapolis, IN).
  • the filters were washed twice with 2 X SSC 0.5 SDS, then in 2 X SSC 0.1 SDS before being developed with dioxigenin-specific alkaline phosphatase-coupled antibody. mRNA expression was scored as: - (negative); + (positive); and ++ (strongly positive).
  • ELISA enzyme-linked immunoabsorbent assay
  • HTV-1 Co-Culture Quantitative HIV-1 co-cultures were performed per the ACTG Virology Manual.
  • H ⁇ V-1 mRNA H ⁇ V-1 mRNA.
  • PCR was also used to quantify HTV-1 mRNA expression. mRNA was obtained and PCR performed as described above. To verify the presence of HTV- 1 in a sample, oligomer SK38 and SK39 were used (Varas, et al, "Influence of PCR Parameters on Amplifications of HTV-1 DNA: Establishment of Limiting Sensitivity", Biotechniques 11: 384-391, 1991). These oligomers ampkfy a 115-bp fragment of the HIV-1 gag gene. PCR was optimized using the SK38/SK39 primer set at the 0.8 mM dNTPS and 2.8 mM Mg+-
  • Proliferation Assay Proliferation assays were performed by incubation 10 5 lymphocytes, in both the presence and absence of IL-2, with synthetic oligopeptides corresponding to HTV-1 envelope sequence (The Ohio State University Comprehensive Cancer Center Peptide Laboratory). Cells were pulse-labeled with 1 ⁇ Ci [ 3 H] thymidine for one hour at 37"C and harvested using a Ska ron cell harvester.
  • HTV-1 Suppression Assay The effects of culture supernatants on HIV-1 production were assessed using previously described methods (Mackewicz, et al, 1992, supra).
  • Peripheral blood CD4+ T cells were isolated from a normal volunteer using negative selection (Human T cell CD4 Subset Column Kit, R&D Systems, Inc., Minneapolis, MN). These purified CD4 + cells were activated with 10 ng/ml OKT3 and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco/BRL, Grand Island, NY) and 100 TU/ml IL-2. Cells were maintained between 0.5 and 2 x lOfyml by adding fresh complete medium weekly.
  • CD4 + T cells (5 X lOfyml/well) were added to a 24-well plate containing either 20% or 80% of supernatants from lymph node cell expansion cultures from HIV- 1 -infected donors in which 5, 20, or 100 ng/ml OKT3 was used.
  • Control wells were estabkshed from cells similarly expanded from the lymph node of a cancer patient.
  • AD wells, except the "no virus" control wells, were infected with HIV-1 culture supernatant known to contain sufficient HTV-1 to infect lymphocyte cultures at the proportions used.
  • Supernatants were collected from the 24-well plate at twice weekly intervals. At each collection, the same proportion of supernatants from the original expansion cultures was added.
  • Day- 4 and day- 12 supernatants from the 24-well plate were analyzed by quantitative ELISA for HIV-1 p24 antigen (Coulter, Hiyalea, FL) as were the supernatants from the original expansion cultures.
  • the amount of p24 produced was calculated by subtracting the p24 present in the 20% or 80% of the original expansion culture supernatant from the p24 detected in the 24-well plate.
  • the p24 produced in control wells with fresh media alone was compared to the p24 produced in well with 20% or 80% supernatant from the original HTV- 1 lymph node expansion culture or from the control cancer patient expansion culture.
  • Lymph nodes were obtained from 12 HIV- 1 -infected donors. The number of cells obtained per lymph node specimen for immunologic studies varied from 0.3 x 10 8 to 30 x 10 8 . In general, the CD4/CD8 ratio of lymph nodes paralleled that of the peripheral blood. Expansion of lymph node cells was examined in the presence of 600 TU/ml of TL-2 and a range of concentrations of anti-CD3 MAb. Initial exposure to lower concentrations of anti-CD3 MAb, namely 5 ng/ml, resulted in higher cell numbers and also favored CD4 + cell expansion at 10 days (Fig. 1).
  • lymph node cells initially at lOfyml in serum- free media with 5 ng ml of anti-CD3 MAb and 600 TU/ml IL-2 for the initial four days, resuspending cells at 0.25 x 10 6 ceks/ml in media with 20 TU/ml TL-2 for three days, and then resuspending cells at 0.35 x 10 6 cells/ml with media and 20 TU/ml 1-2 for three more days (total of 10 days) appeared to be optimal.
  • lymph node cells expanded 6.0-fold to 48.5-fold in 10-days; expansion rates declining after 10 days. The rate of cell expansion appeared to be positively related to the number of CD4+ cells present initially.
  • Fig. 2 displays expansion of lymph node cells to peripheral blood for two representative patients.
  • CD8+ 80 90 71 75 50 66 29 47 29 73
  • CD8 + ceks The majority of expanded ceks were CD8 + . Importantly, >90% of ak of the CD8 + ceks were CDl lb", the phenotype of CTL and also CD8 + cells which inhibit HTV-1 repkcation non-lyticaky (Mackewicz, et al, 1992, supra).
  • CDllb the CR3 receptor
  • CD8 + ceks has been associated with T- suppressor activity (Landay, et al, "Characterization of a Phenotypicaky Distinct Subpopulation of Leu-2 + Ceks which Suppress T Cell Prokferative Responses", J. Immunol. 131:2757- 2761, 1983).
  • CD45RA + Approximately 3.0% - 16.9% of the CD8 + cells were CD45RA + , 34.0% - 65% were CD30 + , and 42.0% - 78.7% were CDw60 + , the phenotype of CD8 + T H ceks (Manetti, et al, supra; Rieber, et al, "CDw60: A Marker for Human CD8 + T Helper Cells", /. Exp. Med. 170:1385-1390, 1994). Most of the CD4+ ceks that resulted expressed the activation and "memory" marker CD45RO. Very few CD56 + NK ceks ( ⁇ 5%) and no B cells ( ⁇ 1%) expanded. Less than 6% of the expanded expressed the monocyte/macrophage marker, CD 14.
  • Cytokine Production was evaluated by assessing cytokine mRNA expression using standard semi-quantitative PCR, measuring cytokine levels of the culture supernatants using commercially avakable ELISA kits, and by measuring cytokine production (ELISA) of the expanded ceks exposed to immobilized anti-CD3 MAb. Expression of cytokine mRNA over the 10-day culture period is displayed in Table 2 below.
  • TL-2/anti-CD3-expanded ceks consistently expressed mRNA for interferon (TFN) g, TL-1, TL-4, TL-8, tumor necrosis factor (TNF) a, and transforming growth factor (TGF) b-cytokines that have been shown to suppress HIV-1.
  • TGF tumor necrosis factor
  • TGF tumor necrosis factor
  • High concentrations of GM-CSF were present in the culture supernatants (> 500 pg/ml).
  • TL- 5, TL-6, IL-10, and TNF were also present in the cek culture supernatants; secretion of these cytokines increased in response to immobilized anti-CD MAb (Fig. 3).
  • HIV-1 inhibition was greater when using the supernatant form lymph node cell expanded with the highest amount, namely 100 ng/ml, of OKT3.
  • proliferative Responses To examine the spectrum of HTV-reactivity, the proliferative response of expanded cell to oligopeptides corresponding to HIV-1 envelope sequence was assessed in the presence of autologous EBV-transformed B- cells as APCs. Significant proliferative response to all epitopes was observed (Fig. 7).
  • Cvtolvtic Activity The cytotoxic activity of the expanded cells was assessed using 51 Cr-release assays with a variety of target cells including autologous EBV- transformed B-cells pulsed with HTV-associated oligopeptides.
  • Four-day expansion demonstrated significant cytotoxicity (% specific lysis greater than 40% at effector to target ratios of 10:1) versus NK-resistant Daudi cells, i.e., lymphokine activated kiker (LAK) cek activity.
  • LAK lymphokine activated kiker
  • Ten-day expanded ceks were weakly cytolytic versus NK-cek sensitive Raji and NK-resistant Daudi cells (% specific lysis ⁇ 10% at effector to target ratio of 10:1). They did not specificaky lyse autologous EBV-transformed B-ceks that were pulsed with oligopeptides corresponding to the HIV-1 envelope sequence (% specific lysis ⁇ 10% at effector to target ratio of 10:1).
  • Exclusion criteria included: any active ATDS-related opportunistic infection, encephalopathy, or makgnancy except mild Kaposi's sarcoma; significant autoimmune disease or non- AIDS -associated makgnancy; prior splenectomy; prior immunotherapy; or concurrent experimental HIV-1 therapy. Patients had to be receiving approved antiretroviral treatment and Pneumocystis carinii pneumonia
  • PCP prophylaxis
  • lymph node was examined for malignancy and for opportunistic infection. If histologic and microbiologic examinations were negative, lymph node cells were processed and underwent an ex vivo expansion. Patients received a single infusion of autologous expanded cells into a peripheral vein. They were premedicated only with 650 mg of acetaminophen. Patients were monitored frequently for clinical and laboratory evidence of toxicity. Toxicity was graded using World Health Organization Common Toxicity Criteria. Virologic and immunologic response determinations 1 day, 2 days, 1 week, 2 weeks, and 5 weeks after cek infusion and thereafter every 4 weeks. Peripheral blood immunophenotyping was performed by The OSU Hospital Aids Cknical Trial Unit Cekular Immunology Laboratory. Viral loads were assessed by NASBA
  • lymph nodes were minced into pieces approximately 2 to 3 mm in diameter under sterile conditions in a laminar flow hood.
  • the minced lymph nodes were transferred to a 50 ml conical centrifuge tube and allowed to stand at 1 x g for one to two minutes in a serum-free medium (Macrophage SFM, Gibco BRL, Grand Island, NY) to allow dense capsular material to settle. Cells and supernatant were pipetted into a fresh 50 ml tube. The ceks were then centrifuged (250 x g) at room temperature for six minutes. The supernatant and any fatty accumulation on the surface were carefully aspirated and discarded. The cek peket was resuspended in fresh serum-free medium. An aliquot of cells was counted and viabikty assessed by Trypan blue exclusion.
  • a serum-free medium Macrophage SFM, Gibco BRL, Grand Island, NY
  • Lymph node cells were cultured in serum-free medium at a density of 10 6 /ml with
  • MAb anti-CD-3 monoclonal antibody
  • Ceks were harvested and washed in 1 L of 0.9% NaCl and 1.25% human serum albumin (American Red Cross) using a SteriCell harvester (E.I. DuPont de Nemours and Co., Glenolden, PA) and suspended in 300 ml of 0.9% NaCl, 1.25% normal human serum albumin in a transfer bag.
  • the fokowing were performed to characterize the ceks prior to infusion: cek number and viabUity, endotoxin assay of final product supernatant, morphology of cytocentrifuge preparations stained with Diff Quik (Baxter Healthcare Corp., Miami, FL); gram stain; cultures for bacterial contamination; and HTV-1 mRNA. 7.
  • RT-PCR was used to assess an HTV-1 expression.
  • PCR primers were designed using primer analysis software (Oligo, National Biosciences, Inc., Madison, MN) and obtained from Stratagene (La Jolla, CA).
  • RNA was extracted via guanidine isothiocyanate phenol using TRIzol (Gibco/BRL, Gaithersburg, MD). Equal starting concentrations of total RNA (0.5 ⁇ g) were used as a template for the RT reaction.
  • RT synthesized cDNA (2 ⁇ L) was mixed with 1.8 ⁇ L of 10X PCT buffer (500 mM KCl, 200 mM Tris HCl, pH
  • RNA from HTV- 1 -infected and uninfected cells were used as controls.
  • PCR product 3578.
  • Amplification was performed with a Perkin-Elmer Cetus thermocycler for 40 cycles of template denaturing (30 seconds at 94°C), annealing and extension (2 minute at 60 * C). Amplified products were separated on a 1% agarose gel and visualized using a UV kluminator.
  • Cek surface marker analysis was performed on an EPICS ELITE cytofluorograph using fluoresceinated and phycoerythrinated MAb as previously described (Triozzi, et al, "Identification of tumor-reacting lymph node lymphocytes in vivo using radiolabeled monoclonal antibody", Cancer, 73:580-589, 1994).
  • the fokowing MAbs were used: anti-CD3 (LEU4), -CD4 (LEU3a), -CD8 (Leu2a), - l ib (Leul5), -CD14 (LeuM3), and -CD56 (Leul9) (all from Becton-Dickinson); anti-CD30 (DAKO Corporation, Carpinteria, CA); anti-CDw ⁇ O (PharMingen, San Diego, CA); anti-CD45RA (2H4) and -CD45RO (UCHL1; both from AMAC, Westbrook, ME); and anti-HLA-DR (HB103; ATCC, Rockvike, MD). 9.
  • Expanded ceks were stimulated with irradiated (75 Gy), autologous, Epstein-Barr virus (EBV)-transformed B-cell lines transfected with either empty Vaccinia control vector or the Vaccinia vPE16 vector, which contains the HIV-1 env gene.
  • the EBV-transformed cek lines were generated from peripheral blood lymphocytes using B95-8 marmoset cek line supernatant by standard methods (Blumberg, et al, "Effects of human immunodeficiency virus on the cekular immune response to Epstein-Barr virus in homosexual men: characterization of the cytotoxic response and lymphokine production, J. Infect. Dis., 155:877-890 1987).
  • the vectors were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Health (Vaccinia control, Dr. Bernard Moss; vPE16, from Drs. Patricia Earl and Bernard Moss). Expanded ceks were cultured for four additional days with the stimulator ceks at a ratio of 10:1, and then supernatants were cokected. ELISA kits were used to quantify MTP-la and RANTES levels (R&D Systems Inc., Minneapolis, MN). Assays were conducted in dupkcate according to the recommendations of the manufacturer, and data are presented as mean values of these determinations.
  • Cytolytic activity versus the autologous B-ceks expressing env protein, natural kiker (NK) sensitive Raji cells, and NK-resistant Daudi cells was assessed at an effector-to-target ratio of 10:1 for four hours, as previously described (Triozzi, et al, supra). Also included were three maximum release wells containing only labeled target cells plus TRITON X-100 surfactant and three spontaneous release weks (no effector cells). The percent of lysis was determined by the formula: (experimental cpm - spontaneous cpm)/(total cpm - spontaneous cpm). Each variable was tested in triplicate; data are presented as mean ⁇ SD.
  • Expanded/activated lymph node lymphocytes from HIV-1 infected donors did not exhibit any specific cytolytic activity against autologous B cek targets expressing the HTV-1 envelope protein gpl20 (% specific lysis was less than 10% at an effector to target ratio of 10: 1.
  • DTH delayed type hypersensitivity
  • Multitest CMI skin test reaction file (Connaught Laboratories, Swiftwater, PA). Forty-eight hours after application, the largest perpendicular diameters were measured according to the recommendation of the manufacturers. Data are presented as the sum of the means of these determinations. A re-establishment of sensitivity to such antigens would establish immunorestoration of the patients.
  • Substantiaky fewer cells were obtained from two resections (Patients Nos. 3 and 4). On pathologic examination, these two lymph nodes were characterized by lymphocyte depletion and dense plasma cell and eosinophil infiltration replacing most of the normal lymph-node architecture. The three lymph nodes that yield more than 10 8 ceks were characterized histologically by follicular and interfokicular hyperplasia. All lymph nodes were negative for malignancy and evidence of microorganisms, including acid fast bacilk and fungus.
  • Percent CD4 cek also positive for CD45RO.
  • b Percent CD8 cell also positive for CD 1 lb or CDw60.
  • the expanded cells were predominantly CD8 + (Table 4). Less than 3% of the expanded ceks were CD14, CD19, or CD16 positive.
  • the expanded ceks proliferated and secreted MTP-la and RANTES. Expanded ceks were weakly cytolytic versus NK- cek sensitive Raji (data not shown), NK-resistant Daudi cells, and the autologous EBV- transformed B-cells expressing the env gene (Table 4).
  • Expression of HIV-1 mRNA was negative by cells expanded from Patients Nos. 1, 2, and 5; it was positive by ceks expanded from Patents Nos. 3 and 4. Because of the persistent positivity, Patents Nos.
  • Viral loads were performed by certified clinical laboratories using NASBA (copies/100 ⁇ L) or quantitative PCR methodologies (copies/mL, where indicated). Tables 5A-C displays the changes in viral load after infusion. As expected, viral load was observed to increase the day after cek infusion. Viral load was observed to decrease to undetectable levels ( ⁇ 400 copies/sample being the detection limit) four weeks after cell infusion.
  • Tables 5A-C display the changes in peripheral blood CD4 and CD8 lymphocyte counts. Significant changes attributable to cell infusion were/were not observed. Patient No. 2 manifested sustained increases in CD4 count that may have been due, in part, to better compliance with anti-retroviral therapy. As an assessment of immunocompetence, DTH skin test reactivity to common recak microbial antigens was evaluated. All patients were poorly reactive prior to cell infusion; skin test reactivity increased after cek infusion (see Tables 5 and 6, below).
  • Immunorestoration or “immunorestorative effect”, as described herein, relates to the restoration of immune function (as measured by skin tests to antigens) which results by dint of the therapeutic administration of expanded lymph node lymphocytes as disclosed herein.
  • lymph node lymphocytes from an HIV+ donor were stimulated with three different mitogenic stimuli: (1) anti-CD3 and IL-2 (control); (2) anti-CD3, anti-CD28, and TL-2; and (3) phytohemagglutinin (PHA) and IL-2.
  • Anti-CD3 was used at 10 ng/ml
  • anti-CD28 was used at 10 ng/ml
  • PHA was used at 0.5 ⁇ g/ml.
  • 100 Cetus units/ml instead of the usual 100 Cetus units/ml, only 20 Cetus units/ml was used throughout the culture period.
  • Ak cultures were split 1:5 on day 5 of the culture (approximately 0.25 x 10 6 cells/ml after split), then split to 0.35 x 10 6 cells/ml on day 8 of culture, and then were harvested on day 11 of culture.
  • Phenotypicaky all 3 cultures were similar. Also, all 3 cultures were 98%-99% CD3+ (T ceks) and displayed other surface markers in comparable quantities as demonstrated in Table 8, below.
  • CD4 Class Il-restricted T cells helper cells
  • CD45RO activation marker "memory” T cells
  • CD8 Class I-restricted T cells cytotoxic/suppressor cells
  • CD8+CDl lb cytotoxic T cell
  • LAK Natural Killer cell
  • Each virus was used at a low input of infection so individual viral plaques could be detected for quantitation in regards to plaque number and size.
  • all viral inoculums were removed from the monolayers and a 0.6% agarose overlay was added to each monolayer.
  • Incorporated into the agarose overlay was either experimental material at a concentration of 80% (V/V) or control material at a concentration of 80% (V/V).
  • cultures were incubated for 5 days at the appropriate temperature (36.5° C for HSV-1 and 32° C for HRV-13). After 5 days of incubation, cultures were fixed and stained for the quantitation of plaque numbers and plaque size.
  • the experimental material was tested against 2 different viruses, an enveloped virus (HSV-1) and a nonenveloped virus (HRV-13). Both viruses were tested at the same time. In addition, the same test conditions were used for each virus. Only one virus was inhibited, the nonenveloped RNA virus.
  • HSV-1 Virus Herpes Simplex Virus Type 1 (HS V- 1 )
  • the appropriate cells were seeded at 150 ⁇ l per well, prior to the day of inoculation. On the day of inoculation, the media was aspirated from each well. The stock virus was serially diluted in ten-fold steps in EMEM with 2% FBS. Each dilution of the virus was inoculated into 32 repkcate wells, at 50 ⁇ l/well. The viral inoculum was adsorbed for 70 ⁇ 10 minutes at 36 ⁇ 2 * C. After adsorption, the four replicates of each virus dkution were re-fed with an individual cek supernatant or medium control dkution. The cultures were incubated at 36 ⁇ 2 * C until the final CPE observation on day 14 for HSV-1 and on day 17 for Ad-2. TABLE 9
  • Supernatants and controls titers were within 1 log of the certified titer.
  • the lowest dilution of virus tested in 20% Supernatant C tested had CPE present in 3 of the 4 wells at the lowest dilution for the Ad-2 samples.
  • CPE was observed in 4 of the 4 weks at the lowest dilution of virus tested in control medium.
  • the noted Supernatant C was able to suppress or reduce virus infectivity as exemplified by Ad-2. While these tests were limited by available material, they do show the potential for the lymphocyte proliferation technique disclosed herein to be useful in acute viral infections. Note, that these tests relied on the cell supernatants to achieve virus suppression as a means of screening viruses.

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Abstract

La présente invention concerne, dans son ensemble, une nouvelle façon de considérer la thérapie par cellule d'adoption de l'infection par le VIH, thérapie qui tire profit de la réponse immunitaire cellulaire potentiellement efficace se développant initialement chez les individus infectés par le VIH. L'invention concerne également, de façon plus spécifique, un procédé de préparation de cellules destinées au traitement de patients atteint du VIH. Ce procédé qui consiste à prélever chez des patients infectés par le VIH des ganglions lymphatiques, consiste ensuite à provoquer l'expension de cellules productrices de cytokine issues de ces ganglions en soumettant ces cellules à stimulation mitogène en milieu asérique. L'agent thérapeutique ainsi obtenu permet de traiter des patients atteints du VIH. Cet agent est constitué d'un excipient admis en pharmacie et, comme principe actif, de cellules productrices de cytokine produites lors de l'opération consistant à provoquer l'expansion de ces cellules en soumettant à stimulation mitogène en milieu asérique, des cellules productrices de cytokine issues des ganglions lymphatiques prélevés chez des patients atteints du VIH. L'invention concerne enfin pour le traitement de patients atteints du VIH, une thérapie consistant en l'administration au patient d'une quantité suffisante de l'agent thérapeutique de l'invention. L'invention permet ainsi, non seulement de bloquer la réplication du VIH tel qu'on peut l'apprécier au niveau des réductions de charge virale que présentent les patients traités selon la thérapie de l'invention, mais également d'induire un effet de restauration immunitaire chez les patients atteints du VIH.
PCT/US1997/002309 1996-02-21 1997-02-20 Immunotherapie cellulaire WO1997030590A1 (fr)

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WO2001032841A2 (fr) * 1999-10-29 2001-05-10 The Government Of The United States Of America, Represented By The Secretary, Dept. Of Health And Human Services Methode de differentiation in vitro de lymphocytes t generes a partir de cellules souches cd34?+¿

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US5443983A (en) * 1986-08-08 1995-08-22 Regents Of The University Of Minnesota Method of culturing lymphocytes and method of treatment using such lymphocytes

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US5443983A (en) * 1986-08-08 1995-08-22 Regents Of The University Of Minnesota Method of culturing lymphocytes and method of treatment using such lymphocytes

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WO2001032841A2 (fr) * 1999-10-29 2001-05-10 The Government Of The United States Of America, Represented By The Secretary, Dept. Of Health And Human Services Methode de differentiation in vitro de lymphocytes t generes a partir de cellules souches cd34?+¿
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