EP0348413A1 - Procede de production de lignees de lymphocytes t a specificite antigenique et utilisation therapeutique - Google Patents

Procede de production de lignees de lymphocytes t a specificite antigenique et utilisation therapeutique

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Publication number
EP0348413A1
EP0348413A1 EP88902046A EP88902046A EP0348413A1 EP 0348413 A1 EP0348413 A1 EP 0348413A1 EP 88902046 A EP88902046 A EP 88902046A EP 88902046 A EP88902046 A EP 88902046A EP 0348413 A1 EP0348413 A1 EP 0348413A1
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EP
European Patent Office
Prior art keywords
antigen
cells
hcmv
specific
population
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP88902046A
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German (de)
English (en)
Inventor
Yung-Nan Liu
Richard C. Gehrz
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Childrens Hospital Inc
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Childrens Hospital Inc
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Publication of EP0348413A1 publication Critical patent/EP0348413A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • 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
    • C12N2501/20—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • T lymphocytes One population, called T lymphocytes, is the effector agent in cell-mediated immunity, while the other (B lymphocytes) is the primary effector of antibody-controlled, or humoral immunity.
  • the stimulus for B cell antibody production is the attachment of an antigen (Ag) to B-cell surface immunoglobulin.
  • Ag antigen
  • B cell populations are largely responsible for specific antibody (Ab) production in the host.
  • B cells require the cooperation of T cells for effective Ab production.
  • T helper (T h ) cells are antigen-specific cells that are involved in primary immune recognition and host defense reactions against bacterial, viral, fungal and other antigens.
  • the T cytotoxic (T c ) cells are antigen-specific effector cells which can kill target cells following their infection by pathological agents.
  • T helper (T h ) cells are antigen-specific, they cannot recognize free antigen.
  • the antigen For recognition and subsequent T h , cell activation and proliferation to occur, the antigen must be presented to receptors or a receptor complex on the T h cell togetherwith major histocompatibility complex (MHC) class II products, or "leukocyte antigens".
  • MHC major histocompatibility complex
  • T h cell recognition of pathogenic antigens is MHC class II "restricted" in that a given population of T h cells must be either autologous or share one or more of the restricting leukocyte antigen (LA) specificities expressed by the MHC of the host.
  • LA leukocyte antigen
  • T c cells recognize Ag in association with class I MHC LAs.
  • T h cells In the case of T h cells, this function is performed by a limited number of specialized cells termed "antigen-presenting cells” (APC).
  • APC antigen-presenting cells
  • T helper (T h ) cells recognize processed soluble antigen in association with class II MHC LA, expressed on the surface of macrophages.
  • T helper (T h ) cells recognize processed soluble antigen in association with class II MHC LA, expressed on the surface of macrophages.
  • other cell types such as resting and activated B cells, dendritic cells, epidermal Langerhan's cells and human dermal fibroblasts, have also been shown to present antigen to T cells.
  • Epstein-Barr virus transformed human lymphoblastoid B cells (LCL) have been shown to present tetanus toxoid, M . Leprae and Candida albicans to autologous antigen-specific T cells.
  • T h cell If a given T h cell possesses receptors or a receptor complex which enable it to recognize the MHC-class II LA-antigen complex, it becomes activated, proliferates and generates lymphokines such as interleukin 2 (IL-2).
  • lymphokines such as interleukin 2 (IL-2).
  • the lymphokines in turn cause the proliferation of several types of "killer cells", including T c cells and macrophages, which can exhibit antimicrobial and tumoricidal activity. After stimulation subsides, survivors of the expanded T h cells remain as memory cells in the body, and can expand rapidly again when the same antigen is presented.
  • T cells in the recovery from acute viral infections has been well established for some viruses, in particular the myxovirus, (influenza), the poxviruses (ectromslia and vaccinia), and the arenavirus, (lymphocytic choriomeningitis virus).
  • myxovirus influenza
  • poxviruses ectromslia and vaccinia
  • arenavirus lymphocytic choriomeningitis virus
  • T c or T h cells have been made to isolate and maintain homogeneous populations of T c or T h cells and to characterize them in terms of their antigen specificity and MHC restriction. These attempts usually involve the stimulation of mononuclear cells from a seropositive human or murine host with bacterial or viral preparations in combination with non-prolifsrative APCs, such as irradiated autologous mononuclear cells (MNCs).
  • MNCs irradiated autologous mononuclear cells
  • Proliferating polyclonal populations of T h cells or T c cells are cloned by limiting dilution to obtain homogeneous populations and then further proliferated and characterized by a variety of techniques.
  • Rosenberg et al. in the case of cloned LAK cells, one of the major obstacles in cloning T lymohocytes is the limited availability of autologous, or alternatively, allogeneic MHC LA-matched MNC
  • APCs other than autologous MNCs have been employed as APCs.
  • T. Issekutz et al., J. Immunol., 129, 1446 (1982) first disclosed that autologous Epstein-Barr virus (EBV)-transformed LCL lines can present antigens associated with tetanus toxoid to tetanus-reactive polyclonal T cells and T cell clones.
  • EBV Epstein-Barr virus
  • One of the clones proliferated in the presence of irradiated, virus-infected, autologous MNCs or in the presence of irradiated, infected Epstein-Barr virus transformed allogeneic lymphoblastoid cells (LCL).
  • LCL Epstein-Barr virus transformed allogeneic lymphoblastoid cells
  • EBV-transformed LCL lines can present antigens associated with M. leorae bacili to M. leorae-reactive cloned T cell lines.
  • a further paper by this group described the isolation of a T cell clone which may recognize only M. leprae antigens.
  • the cloning method used autologous EBV-transformed LCLs as APCs. The advantage of using EBV-transformed LCLs is that they may provide a continuous and unlimited source of APCs. [J.B.A.G. Haanen et al., Scand. J. Immunol., 23, 101 (1986).]
  • HCMV Human cytomegalovirus
  • DNA 1.5X10 8 Da Human cytomegalovirus
  • HCMV Human cytomegalovirus
  • HCMV is ubiquitous (30-70% of adults are seropositive) but the principal site(s) of latency of HCMV are uncertain, as are the molecular events involved in its reactivation.
  • HCMV infection and reactivation can be asymptomatic, but are associated with appreciable morbidity and mortality in immunosuppressed subjects.
  • HCMV is the most common cause of opportunistic infection in bone marrow transplant patients. More than 80% of those who develop HCMV pneumonia die despite current treatment modalities.
  • Peripheral blood lymphocytes are also not likely to be useful as therapeutic agents, even if they could be obtained in sufficient quantities.
  • the numbers of T helper cells or T cytotoxic cells reactive with a particular antigen are extremely low and thus, selective expansion of antigen-specific T cells in vitro will be required to obtain sufficient numbers for therapeutic purooses.
  • therapeutic administration of allogeneic PBLs would activate T cells recognizing "foreign" leukocyte antigens, resulting in a mixed leukocyte culture reaction. This mixed leukocyte culture reaction may activate undesirable non-specific immune responses, or alternatively, induce suppressor cells which might inhibit desired antigen-specific immune responses.
  • T h cells and T c cells which are specific to antigens associated with viruses such as HCMV ana other pathogenic agents.
  • the present invention is directed to methods for the efficient production of a homogeneous population of T helper (T h ) cells which are specific for a viral antigen, such as an HCMV antigen. It is believed that the present methods provide the basis for the efficient production of cloned T h cells while using a minimum amount of antigen and lymphokine support, while maintaining both the antigenic specificity and functional activity of the clones.
  • the present method comprises:
  • MNC mononuclear cells
  • APC autologous, non-proliferative, antigen-presenting cells
  • clonally expancing tne antigen-specific T helper ceil in the presence of an amount of non-proliferative antigen-presenting cells comprising a mixture of (i) autologous MNCs, allogeneic MNCs or mixtures thereof; and (ii) autologous iymphoblastoid cells (LCLs), allogeneic LCLs or mixtures thereof; and an amount of said viral antigen effective to proliferate said cloned antigen-specific T helper cell, to yield said homogeneous population, and wherein said LCLs are present in an amount effective to increase the proliferation rate of the T helper cell over that caused by the MNCs.
  • non-proliferative antigen-presenting cells comprising a mixture of (i) autologous MNCs, allogeneic MNCs or mixtures thereof; and (ii) autologous iymphoblastoid cells (LCLs), allogeneic LCLs or mixtures thereof; and an amount of said viral anti
  • step (d) antigen-specific monoclonal T h cells may be derived by limiting dilution, or by single cell isolation by micromanipulation or flow cytometry. Antigen-specific polyclonal T cell lines, as well as monoclonal antigen-specific T cells, have potential therapeutic benefit.
  • T cell line may appropriately refer to an expanded population of polyclonal T cells which is uniquely reactive with a given antigen, or to a homogeneous population of monoclonal T cells which has been derived by the expansion from a single progenitor T cell.
  • the present invention comprises the proliferation of antigen-specific T h cells, it also provides a method for proliferating a homogeneous population of T helper cells which are specific for a viral antigen comprising combining said homogeneous population of T helper cells with an amount of the antigen and an amount of a mixture of non-proliferative, antigen-presenting cells (APC) effective to cause the proliferation of the population of T helper cells, wherein said mixture of APCs comprise (i) autologous or allogeneic lymphoblastoid cells (LCL) and (ii) autologous or allogeneic mononuclear cells (MNC), wherein the LCLs are present in an amount effective to increase the proliferation rate of the T helper cells over that caused by the MNCs.
  • APC non-proliferative, antigen-presenting cells
  • the LCLs are derived from a continuous lymphoblastoid cell line, such as can be produced by viral transformation, hybridoma technology and the like.
  • the APCs are rendered non-proliferative by techniques known to the art, e.g., by x-irradiation, treatment with chemical agents such as mitomycin C and the like.
  • MNCs and LCLs have been found to cause the proliferation of T h cells when they are derived from the same host as the T h cells or are allogeneic MNCs or LCLs which share one or more class II restricting antigens (abbreviation: "MHC
  • LA-matched LA-matched
  • this effect is observed in the case of a viral antigen such as an HCMV antigen, when a mixture of EBV-transformed autologous LCLs ana autologous MNCs are employed as the APCs in a ratio of at least about 1:10.
  • a viral antigen such as an HCMV antigen
  • the ratio of T h cells to LCLs added is about 1:1-3 and the ratio of T h cells to MNCs is about 1:3-10.
  • HCMV-specific T h clones could be expanded from 1-2 ⁇ 10 6 cells to 10 9 cells in 2 weeks by using 1-2 ⁇ 10 7 MNC and 1x10 6 LCL as APC.
  • a further aspect of the present invention comprises increasing the proliferation rate of the viral antigen-specific T h cells by conducting the initial proliferation step (steps a-c, hereinabove) or the subsequent expansion step (step (d), hereinabove) in the presence of an amount of monoclonal antibody which is specific for said viral antigen.
  • the proliferation rate of a T h cell specific for an HCMV viral antigen is increased by combining MNCs isolated from the blood of an HCMV-seropositive donor mammal with a monoclonal antibody specific for an antigen present on an HCMV gene product such as a structural protein.
  • the monoclonal antibody is employed in combination with (a) an effective proliferation-stimulating amount of said antigen, and (b) an amount of autologous AFCs selected from the group consisting of (i) non-proliferating MNCs, (ii) a non-proliferating LCL derived from a continuous LCL, and (iii) mixtures thereof, e.g., mixtures which can cooperatively interact to further increase the proliferation rate of the T h cells.
  • the autologous, non-proliferating antigen-presenting cells are selectad from the group consisting of x-irradiated autologous MNCs, an x-irradiated Epstein-Barr Virus (EBV)-transformed lymphoblastoid cell line (LCL) and, mixtures thereof.
  • x-irradiated autologous MNCs an x-irradiated Epstein-Barr Virus (EBV)-transformed lymphoblastoid cell line (LCL) and, mixtures thereof.
  • EBV Epstein-Barr Virus
  • LCL lymphoblastoid cell line
  • the present method can also be employed to produce a homogeneous population of T cytotoxic (T c ) cells which are specific for an HCMV antigen.
  • This aspect of the present invention comprises the steps of: (a 1 ) isolating a population of mononuclear cells (MNC) from the blood of a donor mammal, preferably a human, wherein the MNC population comprises T c cells specific for said viral antigen;
  • HCMV antigen HCMV antigen
  • an amount of interleukin-2 (IL-2), effective to cause the proliferation of a T c cell which is specific for said antigen IL-2
  • HCMV antigen effective to proliferate said cloned antigen-specifIc T c cell, to yield said homogeneous population.
  • Steps (b 1 ), (c 1 ) or (c 1 ) are preferably carried out in the presence of a monoclonal antibody which is specific for an HCMV antigen, and which acts to increase the proliferation rate of said T c cell.
  • polyclonal T c lines reactive with a particular viral antigen can be expanded by continuous stimulation of T c blasts exhibiting cytotoxic activity against target cells expressing the desired antigen.
  • Homogeneous populations of virus-specific T c clones derived from a single progeny T cell may be obtained by limiting dilution, or by single cell isolation by micromanipulation or by flow cytometry, followed by expansion according to step (d 1 ).
  • T c cell proliferation method examples include (1) the use of whole HCMV-viral antigen or the use of HCMV-infected autologous or allogeneic fibroblasts as a source of cell-associated viral antigen, e.g., an HCMV immediate-early protein; (2) the addition of additional amounts of the viral antigen, IL-2 and the autologous, non-proliferative APCs during the course of step (c 1 ) to cause further proliferation of said T c cell, and (3) the use, in step (b 1 ) or in step (d 1 ), of antigen-presenting cells which further comprise non-proliferative MNCs derived from a continuous autologous MNC line in an amount effective to increase the proliferation rate of the T c cells over that caused by the autologous mononuclear antigen-presenting cells.
  • This continuous, autologous MNC line preferably comprises a virus-transformed LCL, i.e., an EBV-transformed LCL.
  • the present invention is also directed to a method for proliferating a homogeneous population of T c cells which are specific for an antigen, such as a viral antigen, i.e., an HCMV antigen.
  • the method comprises combining said homogeneous population of T cytotoxic cells with an amount of said antigen, an amount of interleukin-2 (IL-2) and an amount of a mixture of non-proliferative, antigen-presenting cells (APC) effective to cause the proliferation of said population of T c cells, wherein said mixture of APCs comprise allogeneic virus-transformed lymphoblastoid cells (LCL) and allogeneic mononuclear cells (MNC).
  • the method further comprises combining said population of T c cells with an amount of monoclonal antibody specific for said antigen, which monoclonal antibody acts to increase the proliferation rate of said population of cells.
  • the term "antigen" refers to a compound such as a polypeptide, polypeptide complex, glycoprotein, nucleic acid or the like, which elicits an immune response.
  • a pathological target antigen may be a portion of the pathological target itself, e.g., a viral envelooe glycoprotein, or it may be an antigen expressec by a diseased tissue such as neoplastic tissue or a virus-infacted cell.
  • An "antigen-specific" T lymphocyte becomes activated in the presence of a single antigen when it is oresented by an antigen-oresenting cell (APC), in association with a specific leukocyte antigen (LA) expressed by the antigen-presenting cell.
  • An antigen-soecifIc T h cell will proliferate in a suitable medium in the presence of the antigen for which it has specificity when said antigen is presented to the T h cell by an APC which also expresses the leukocyte antigen for which the T h cell has specificity.
  • the specific leukocyte antigen will be a human MHC class II antigen, whereas human T cytotoxic cells are specific for MHC class I antigens.
  • Preferred embodiments of the present invention comprise homogeneous populations of T c or T h cells, and methods for the production thereof, wherein a given cell population is specific for an HCMV antigen.
  • a T cell or a T cell clone is said to be antigen-specific, although the composition comprising the antigen may be specified, the epitope, or specific antigenic site, on the antigenic composition, is not necessarily known.
  • three different homogeneous populations of T helper lymphocytes may be specific for one particular HCMV structural protein or glycoprotein. While they all respond to the same antigen, and thus, have the same antigen specificity, they may have different specificities at the submolecular level, meaning they may respond to differert regions of the antigen, or epitopes.
  • HCMV-specific T lymphocytes have the same submolecular-specificity only if they recognize the same epitope on the same HCMV-associated antigen. They may still be specific for the same antigen, however, if the antigen has a plurality of epitopes as presented by APCs in different instances.
  • the HCMV DNA genome is transcribed in sequential order, beginning with the restricted transcription of immediate-early genes encoding regulatory proteins required for subsequent expression of early genes.
  • the major immediate-early gene (I-El) encodes a 68 kD regulatory protein which is expressed on the membrane of infected cells 6-24 hours after onset of infection.
  • HCMV-specific T cytotoxic cells are thought to primarily recognize this immediate-early protein as part of their role in immune surveillance to prevent reactivation of latent HCMV.
  • Transcription of early genes precedes the onset of DNA synthesis. Included among the early gene products are virus-specific polymsrases and kinases necessary for DNA replication. These enzymes do not appear to play an important role in immune responses.
  • the late HCMV genes encode a variety of immunogenic structural proteins and glycoproteins. Included among these proteins are disulfide-bridged envelope glycopeptide complexes, non-glycosylated envelope proteins, tegument glycoproteins bridging the viral nucleocapsid with the outer envelope; and matrix proteins forming the internal capsid structure of the virus.
  • Peak* (MoAb Reacprecipitated After Reduction tivity) (mol wt., kD) (mol wt., kD)
  • the immun ⁇ genic glycopeptide complexes and glycopeptides listed on Table 1 are useful to selectively stimulate the proliferation of T h cells derived from the blood of an HCMV-seropositive donor.
  • monoclonal antibodies of known binding specificity such as 9E10, 41C2 and 9B7 can be used to purify viral antigens. These monoclonal antibodies can also be used to augment antigen recognition by T h cell clones, thereoy facilitating their expansion in the presence o f limited amounts of viral antigen.
  • T cell recognition of different HCMV proteins is important in planning the proper course of immunotherapy with the T cell lines.
  • the T h cells exemplified herein primarily recognize structural proteins of HCMV. They are, therefore, likely to be important in the recognition of cell-free virus (i.e., in cases of acute viremia).
  • Data in mice and humans show that certain T cytotoxic cells recognize Immediate-early proteins, which are expressed on the surface of infected cells. Since these proteins are only produced after the viral genes are expressed by the host cells, such T c cells are important in preventing reactivation of latent HCMV and their administration may be indicated for both prophylaxis and specific HCMV treatment.
  • the preferred T cell lines recognize viral antigens in association with human LA products.
  • therapeutic T cells must be either autologous or share one or more of the restricting specificities expressed by the patient.
  • allogeneic cells are cells from a donor individual of the same species as the recipient which are major histocompatibility complex matched for an appropriate human leukocyte antigen (HLA) class of antigens expressed by the recipient (abbreviation "MHC LA-matched"). Therefore, when dealing with a patient with a pathological infection such as chronic HCMV disease, one can expand autologous antigen-specific T cell lines in vitro for subsequent T cell therapy via re-administration.
  • HLA human leukocyte antigen
  • a further aspect of the present invention is directed to a method of using T lymphocytes (T cells) for a therapeutic treatment of a mammal, such as a human patient, having a viral infection, such as an HCMV infection.
  • T lymphocytes T cells
  • the method comprises treating said infected mammal with an amount of a homogeneous, clonally-expanded, viral antigen-specific T cell population effective to elicit an increased immune reaction to the viral infection, wherein said T cell population is specific (a) for at least one leukocyte antigen (LA) present on the surface of an antigen-presenting cell (APC) of said mammal, and (b) for an antigen of said virus.
  • the clonally-expanded T cell population will consist essentially of T h cells or T c cells.
  • a plurality or "bank" of homogeneous, clonally-expanded, viral antigen-specific T cell populations in order to (a) promote immune system recognition of antigens expressed during different stages of infection and/or (b) to guarantee that an effective number of the total T cell populations will be MHC-matched to the recipient.
  • the present method of T cell immunotherapy comprises the administration of a plurality of T cell populations which comprise T cells having specificity (a) for a plurality of different antigens of said virus, and (b) for at least one LA present on the surface of an APC of said mammal.
  • the present method can also comprise the administration of a plurality of T cell populations, each comprising T cells having specificity (a) for at least one antigen of said virus, and (b) for a plurality of different LAs present on the surfaces of APCs of a plurality of allotypes of a single species of said mammal, wherein at least one LA is LA-restriction-matched with said mammal.
  • Polyclonal lines as well as T cell clones may have significant therapeutic potential.
  • antigen-specific polyclonal T cell lines include T h and/or T c cells which can recognize the target viral antigen in association with all restricting MHC LAs expressed by the T cell donor.
  • virus-specific T h and T c clones derived from a single progenitor T cell is that they represent a homogeneous population with cells of known antigen specificity, functional activity, and MHC LA- restriction specificity. Therefore, they are likely to exhibit maximal therapeutic effect since all cells in the population exhibit the same functional activity. From a practical point of view, however, a much larger "bank" derived from discrete T cell populations will be required if monoclonal T cells are to be used.
  • the methods of the present invention permit the administration of a therapeutically-effective amount of T h cells and/or T c cells
  • the afflicted mammal may, but need not be, concurrently treated with exogenous lymphokines such as IL-2 (TCGF).
  • exogenous lymphokines such as IL-2 (TCGF). Since the administration of large doses of IL-2 has been associated with adverse reactions in some patients, the ability to enhance the immune response in the absence of IL-2 can provide a substantial improvement in the efficacy of T cell therapy.
  • the present invention has been described primarily in terms of the production of T h ana T c cells which are specific for viral antigens, anc tne treatment of viral infections with homogeneous populations of these T cells.
  • homogeneous populations of T h and T c cells which are specific for antigens on a wide variety of pathological targets can be prepared using the present method, and that these populations will be effective to augment or stimulate the immune response to a wide variety of pathogens or pathological targets .
  • a pathological target is an entity within a mammalian body which is The cause of, or a result of, a disease which threatens the health and well-being of the mammalian host.
  • the target may be an entity which is foreign to the host such as a virus, bacterium, fungus, or the like, or it may be a product of a disease or pathological condition, such as a virus-infected cell, a neoplastic cell, and the like.
  • the pathological target of the treatment is occasionally a secondary infection which is more threatening to the intended recipient because a primary illness has weakened the recipient's ability to respond immunologically to the secondary infection.
  • Many aspects of the primary illness may weaken the intended recipient's immune response to the secondary infection, including therapeutic treatments which act to suppress aspects of the recipient's immune system. Such treatments include, but are not limited to, radiation therapy, chemotherapy and the like.
  • the present therapeutic method can employ allogeneic T cells from individuals of the same species who are MHC LA-matched. This will enable physicians and pharmacists to simply use pre-formed dosage forms of allogeneic T cells to treat a patient. Since T cells which are not matchad are simply ineffective in aiding the patient, the dose may contain many cells, some of which are matched and will aid the patient and some of which are not matched and will not aid the patient.
  • a single dosage form may be used to treat a number of patients sharing one or more LAs expressed by the allogeneic therapeutic cells.
  • the dose may contain both T c and T h cells which have different MHC class restrictions. Obviously, such a therapeutic treatment does not require the preparation of doses on a case-by-case basis.
  • the T cells can be expanded in large culture mediums, allowing for efficiencies of scale.
  • the cells can be made immediately available without requiring individualized collection, leukapheresis and culture.
  • a dosage form may be provided which contains allogeneic T h and T c cells which are specific for a variety of antigens which are all associated with a particular pathogen.
  • a unit dosage may contain a plurality of T h and T c cell clones which have been individually cultured and then mixed.
  • the dosage form may contain T h and T c cells with a pre-selected number of LA restrictions, and also a pre-selected number of antigen specificities. If a particular pathogen is known to express 10 or 20 known epitopes on a variety of antigens, structural or otherwise, the dose may contain appropriate T cells having specificity for each.
  • Treatment methodologies will normally involve the parenteral administration of a theraoeutically effective amount of autologous or allogeneic T cells in a suitable liquid vehicle, such as a physiological salt solution.
  • a suitable liquid vehicle such as a physiological salt solution.
  • One such protocol is that provided by S. A. Rosenberg et al., New England J . Med., 313, 1485 (1985), but the number of cells delivered per dose, and the number of doses administered will vary widely, depending upon factors determined by the clinician, including the pathology under treatment, the physique and the physical condition of the patient and other factors.
  • the whole HCMV antigen preparation was resuspended in 2-4 ml TN buffer (Tris-NaCl buffer (50 mM Tris hydrochloride, pH 7.5, 10 mM NaCl, 2 mM phenylmethylsulfonyl fluoride)) containing 1% Triton-X 100 (TX-100). After incubation at ambient temperature for 60 minutes, this solution was layered onto a linear 20-60% sucrose gradient for rate zonal centrifugation at 120,000 g for 60 minutes. The TX-100 solubilized material remained at the top of the gradient, while the HCMV nucleocapsids were banded by velocity sedimentation.
  • Tris-NaCl buffer 50 mM Tris hydrochloride, pH 7.5, 10 mM NaCl, 2 mM phenylmethylsulfonyl fluoride
  • TX-100 Triton-X 100
  • detergent extracts of Towne strain HCMV were prepared using 1.0% Nonidet P-40 (NP-40, Sigma Chemical Company).
  • 10-15 mg of the purified whoie HCMV antigen were suspended in 3-5 ml of TN buffer containing 1% NP-40.
  • Detergent-virus suspensions were stirred for one hour at room temoerature.
  • the extracted proteins were separated frdm insoluble proteins by nighspeed centrifugation for 1 hour.
  • the extract obtained by either of these methods was designated "HCMV detergent extract”.
  • HCMV-specific T helper cells appear to recognize HCMV proteins and glycoproteins contained within the whole HCMV antigen preparations
  • HCMV-specific cytotoxic T cells recognize primarily HCMV-encoded proteins expressed on the surface of infected cells.
  • I-E1 protein which is expressed on the cell membrane within 6-24 hours of infection, appears to be important in expansion of HCMV-specific cytotoxic T cells. Therefore, autologous fibroblasts or allogeneic fibroblasts sharing one or more class I leukocyte antigens recognized by the T cell donor were infected with Towne strain HCMV at a multiplicity of infection of 5-10 for 3 hours.
  • the cells were infected in the presence of cyclohexamide (50 ⁇ g/ml). After removal of cyclohexamide, Actinomycin D (5 ⁇ g/ml) was added to prevent further DNA transcription, thereby allowing for selective expression of I-E genes of HCMV.
  • HSV-I Herpes simplex virus type I
  • HCMV-specific T cell blasts were prepared in 25 cm 2 upright tissue culture flasks by stimulating 10 ⁇ 10 6 mononuclear cells (MNC) at a concentration of 1 ⁇ 10 6 MNC/ml with 10 ⁇ g of heat-inactivated whole HCMV antigen (56°C, 1 hr) suspended in RPMI 1640 medium supplemented with 15% heat-inactivated HCMV- seronegative human serum (PHS).
  • MNC mononuclear cells
  • PHS heat-inactivated whole HCMV antigen
  • the HCMV-specific blasts were either further expanded in bulk culture, or cloned by limiting dilution in 96 well U-bottomed tissue culture plates at 0.3 cell/well in the presence of x-irradiated (5,000 R) autologous MNC (MNC IRR ) as feeder cells, whole HCMV antigen at a concentration of 1 ⁇ g/ml (for T h cells) or cell- associated antigen at a fibroblast:MNC ratio of 1:20 (for T c cells), and 10-20% interleukin-2 (IL-2) (TCGF, Biotest, Frankfurt, Germany). Thereafter, cells were refed every 3-4 days with fresh IL-2-containing media. Whole HCMV antigen and autologous x-irradiated feeder cells were added to the media every 7-14 days.
  • MNC IRR autologous MNC
  • T h and T c lines and clones employed in the Examples hereinbelow were characterized as to phenotype, proliferative responses, IL-2 production and cytotoxic activity as follows:
  • T cell lines were analyzed for expression of CD phenotypic determinants using the monoclonal antibodies OKT3 (total T cells), OKT4 (helper/inducer T cells), and OKT8 (cytotoxic/ suppressor T cells) (Ortho Pharmaceuticals Inc., Raritan, NJ) using an indirect immunofluorescence assay. Fluorescence was detected either by fluorescence microscopy using a Zeiss flourescent microscope or flow cytometry using the EPICS sp cell 541 (Coulter Corp., Hialeah, FL). Polyclonal T h lines were predominantly CD3+4+8-; all T h clones expressed the CD3+4+8- phenotype. T c lines were predominantly CD3+4-8+; T c clones were CD3+4-8+.
  • T cell lines and clones were rested in tissue culture media in the abse ⁇ ce of TCGF overnight, and then restimulated for 72 hrs with either whole HCMV antigen or HSV antigen to determine the specificity of proliferative response of T h clones. All T h lines and clones demonstrated positive proliferative responses to HCMV, whereas responses to HSV were similar to tissue culture media background control. Thus, all T h lines and clones were HCMV-specific. T c cell clones proliferated poorly to HCMV antigen in the absence of IL-2.
  • T h clones were stimulated with whole HCMV antigen, and the supernatants harvested at 24 hrs and assayed for IL-2 activity using the murine CTLL-20 (IL-2 dependent) cell line. All T h clones were shown to produce IL-2, as demonstrated by the survival and growth of CTLL-20. T c clones were not tested for IL-2 production.
  • T h clones were tested for cytotoxic activity against the NK target cell line, K562; autologous uninfected and CMV- and HSV- infected fibroblasts as target cells. No NK or virus-specific cytctoxic activity was observed with any of the T h clones.
  • T c clones described herein exhibit HCMV-specific cytotoxic activity restricted by MHC class I antigens, anc therefore exhibit the characteristics of virus-specific cytotoxic T cells.
  • HCMV-T h clones obtained from donor WRC were characterized extensively as to phenotype, proliferative responses, IL-2 production and cytotoxic activity. All clones were CD3+4+8-; proliferative to HCMV but not HSV (as described in Example II (2) above), all produced IL-2 when stimulated with HCMV but not HSV, and none exhibited cytotoxic activity. Thus, all clones were considered to be T helper cells.
  • All clones also exhibited class II MHC restriction specificity, as determined by blocking with anti-class II monoclonal antibodies and reactivity to whole HCMV antigen presented by autologous or allogeneic antigen presenting cells sharing the appropriate Dw restriction determinant expressed in association with DR, DQ or DP.
  • HPLC-2UR contains primarily gpB, reactive with MoAb 9E10; 2UR also contains an immunologically distinct gpC, which is non-reactive with MoAb's reactive with either gpA or gpB.
  • HPLC-3UR contains primarily gpA reactive with MoAb 41C2.
  • T h clones All clones also reacted significantly to Triton X-100 extracts of Towne HCMV, although to a lesser extent than that to whole Towne viral particles. Thus, it appears that the majority of T h clones either recognize primarily proteins or glycoproteins included within the envelope of the virus, or proteins that associate sufficiently with the viral envelope to be recovered in the detergent extract. The lower responses compared to whole Towne antigen may reflect inhibitory effects of residual detergent, or a loss of immunogenicity due to structural alterations resulting from the extraction procedure. Of interest, all T h clones also showed a low but significant proliferative response to precipitates of viral antigen following detergent extraction.
  • nucleocapsid proteins which presumably comprise the dominant protein in the precipitate, may also express antigenic determinants recognized by T helper cells.
  • T cell clones were then stimulated with purified glycoprotein complexes obtained by anion exchange HPLC fractionation of unreduced detergent extract of whole HCMV antigen (see Example I (1)).
  • glycoprotein complexes contained within peaks 2UR and 4UR include a glycoprotein complex recognized by a monoclonal antibody (9E10). This antibody is cross-reactive with several other viruses, and is capable of neutralizing HCMV in the absence of complement.
  • a separate and distinct group of glycoprotein complexes are isolated to a high degree of purity in peak 3UR, and are recognized by a separate class of monoclonal antibodies which uniquely react with HCMV, and neutralize HCMV efficiently only in the presence of complement.
  • glycoproteins derived from the 3UR complex have molecular weights of 130,000; 90,000; and 50,000-52,000 kD; the two glycoproteins derived from the 2UR/4UR complex recognized by the 9E10 monoclonal antioody have molecular weights of 93,000 and 50,000-52,000 kD.
  • a third glycoprotein has been demonstrated in peax 2UR which is distinct from those glycoproteins recognized by the 9E10 monoclonal antibody. Therefore, there are at least thrae major glycoprotein complexes contained within the envelope of HCMV that have been i ⁇ entified as incorporating in excess of 95% of tne total glycoproteins within the HCMV viral envelope. Since these HPLC methods appear to yield glycoprotein complexes of >95% purity, it was of interest to demonstrate the pattern of T h reactivity to these individual glycoprotein complexes.
  • T h clones As shown in Table 3, of the seventeen T h clones tested for reactivity with HPLC-purified glycoprotein complexes, two responded to peaks 3UR and 4UR but not to peak 2UR. A third T h clone responded to all three HPLC peaks. It is likely that these clones are primarily responding to the major glycoprotein complex which is also recognized by monoclonal antibodies which detect the glycoprotein complex principally isolated in peak 3UR. It is likely that tailing of peak 3UR into peak 4UR accounts for the apparent cross-reactivity.
  • the immunodominant HCMV antigenic determinant recognized by these cloned T helper cells may reside either in a nonglycosylated membrane protein or in an internal protein. If so, this may be similar to influenza virus, in which antibody recognition involves primarily the surface glycoproteins (i.e., hemagglutinin), whereas cytotoxic T lympnocytes are known to recognize primarily internal nucleocapsid proteins.
  • Table 4 summarizes data regarding the specificity of HCMV-specific T h clones reactive with individual HCMV (glyco)proteins. Clones were generated by initial stimulation of MNCs from seropositive ⁇ onor WRC with whole HCMV antigen to select for T h reactive with structural proteins and glycoproteins of HCMV.
  • Envelope glycoprotein gpA peak 3UR purified by anion exchange HPLC of detergent extract of Towne CMV.
  • HSV whole virus purified from supernatants of infected cell cultures
  • WRC T h clones obtained by initial stimulation with whole Towne virus; 18% of T h clones were gpA specific; 33% of T h clones were specific for HPLC-purified 64 kD protein.
  • MNC* Irradiated autologous MNC as APC
  • N.D. Not determined
  • T h clones tested were specific for gpA as described in detail in Example I.1.
  • Representative examples include WRCT3#3 and WRCT2#41.
  • Thirty-three percent of the HCMV-specific T h clones were found to be reactive with whole virus as well as a partially-purified matrix protein of molecular weight 64,000 daltons. This protein was obtained by reverse phase HPLC and gel filtration, according to a modification of the method of B. R. Clark, J . Virol., 49, 279 (1984).
  • HCMV Whole Towne HCMV obtained by centrifugation of the supernatant of the HCMV-infected fibroblasts was solubilized in 6 M guanidinium chloride and run on reverse phase HPLC with a C-18 column. Proteins adsorbed to the column were eluted with acetonitrile and detected by monitoring the column effluent at 214 nanometers. Peaks collected were pooled and the 64 kD protein was identified by SDS-PAGE.
  • the 64 kD protein was further purified from co-eluted proteins by size-exclusion chromatography using TSK 4000 and TSK 3000 SEC columns linked in series. An aggregate form of the 64 kD protein was isolated as determined by SDS-PAGE using this method.
  • Clones WRC-T2/88 and WRC-T2/131 are representative of HCMV-specific T h clones which proliferated in response to the 64 kD matrix protein.
  • many of the clones i.e., WRC-L33 and WRC-L42
  • WRC-L33 and WRC-L42 were non-reactive with either the major e ⁇ velope glycoprotein, gpA, or the abundant 64 kD internal matrix protein.
  • HCMV-specific T helper cells exist to a variety of structural HCMV proteins.
  • HCMV-Infected Human Skin Fibroblasts Human diploid skin fibroblasts (SF) were obtained from skin biopsies of adult donors and newborn foreskins. Cells were passaged at least 6 times, and frozen at 1°C/minute in
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS fetal calf serum
  • DMSO dimethylsulfoxide
  • HSV- and Influenza-infected Skin Fibroblasts An SF monolayer in a 25 cm 2 flask was infected with HSV or influenza at a multiplicity of infection of 5-10 and incubated overnight. The cells were labelled the same way as HCMV-infected SF. 3. K562
  • the erythroleukemia cell line K562 was used as a source of target cells to measure NK activity.
  • K562 cells (1 ⁇ 10 6 ) were suspended in 1 ml medium and 375 ⁇ Ci of Na 2 CrO 4 in a 25 cm 2 flask overnight at 37°C in an atmosphere of 5%
  • the cells were resuspended in RPMI supplemented with 10% FCS after three washings.
  • SFs were infected with HCMV at a multiplicity of infection of 5-10 for three hours in the presence of cyclohexamide (15 ⁇ g/ml). After removal of cyclohexamide, Actinomycin-D (5 ⁇ g/ml) and Na 2 CrO 4 (375 ⁇ ci) were added to the culture. The cells were then washed three times and resuspended in RPMI in 10% FCS as target cells. The cytotoxicity as.say was performed under Actinomycin-D to prevent DNA transcription. Uninfected SFs treated with cyclohexamide and Actinomycin D in parallel were used as control.
  • SR (% 51 Cr release HCMV-infected targets) - (% 51 Cr release uninfected targets). Results were expressed as the mean from triplicate or quadruplicate wells. Spontaneous release from both infected and uninfected SF target cells was less than 40%.
  • Mononuclear cells from seropositive donor SP-CN were stimulated for 7-10 days in bulk culture with autologous skin fibroblasts (SF) infected for 20 hours with Towne HCMV. T cell blasts were restimulated weekly with HCMV-infected fibroblasts, autologous irradiated MNC as feeder cells and IL-2. Resulting polyclonal CD8+ T cell lines were evaluated for cytotoxic activity against uninfected and HCMV-infected autologous fibroblasts and the NK target cell line K562. The results of this study are presented on Table 5, below. TABLE 5 Cytotoxic activity of SP-CN cell lines stimulated with Towne HCMV-infected autologous skin fibroblasts (SF) Lysis of target cells b
  • SP-CN CA line 1 50:1 9.1 57.5 11.2 28.9 56.9 25:1 5.1 44.4 9.7 25.3 46.0 12:1 5.2 40.8 4.8 23.1 36.4
  • CD8+ T c cell lines and clones were obtained from seropositive donor SP-RK by initial stimulation of MNC with sucrose gradient-purified Towne HCMV viral particles, followed by repeated stimulation with HCMV viral particles, autologous irradiated MNC as feeder cells, and IL-2.
  • SP-RK line 4 50:1 3.0 21.3 -2.9 7.0 6.3 2.9 1 8.4
  • T cell lines and clone are characteristic of HCMV-specific, MNC class I LA-restricted T cytotoxic cells which recognize an antigen(s) expressed by whole viral antigen (i.e., structural proteins).
  • HCMV-T h clone WRC-T3#3 was used at a concentration of 1.5 ⁇ 10 4 cells per well as a source of responder cells.
  • Autologous x-irradiated MNC (10 5 ) were added as a source of antigen-presenting cells.
  • An optimal dilution of whole HCMV antigen or 0.1 ug/ml of HPLC-purified 3UR was added as a source of HCMV-specific antigenic stimulation.
  • MoAb MoAb 41C2 (anti-gpA) MoAb 35F10 (anti-pD) Concentration Whole HCMV Whole HCMV ( ⁇ g protein/well) 3UR Ag b 3UR Ag b 10 19,6673 5,674 17,577 2,349
  • monoclonal antioody 41C2 directed against gpA, augmented the proliferative response of all six T h clones to whole HCMV antigen, despite the fact that only 3 of the 6 clones respond to HPLC-purified gpA in the absence of antibody (see Table 7).
  • the monoclonal antibody directed against protein D did not augment the response of any of the clones tested.
  • Whole serum from the seropositive donor augmented the response of all clones, whereas that from the seronegative donor did not.
  • EXAMPLE VI Presentation of HCMV Antioen to T Helper Cell Clones by MNC, LCL or Mixtures Thereof 1. Epstein-Barr Virus (EBV)-transformed Lymphoblastoid Cell Lines (LCL).
  • EBV Epstein-Barr Virus
  • SP-CN LCL An autologous LCL from serooositive donor SP-CN (SP-CN LCL) was established by the method of Sugden and Mark, J . Virology, 23, 503 (1977) and grown in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS).
  • FCS heat-inactivated fetal calf serum
  • AMG LCL (DR 2,2/Dw 2,2) and NHS LCL (DR 3,8) were gifts from Dr. F. Bach (IRC, University of Minnesota, Minneapolis, MN).
  • HCMV-specific T cell clones were generated from a seropositive donor (SP-CN). Briefly, mononuclear cells (MNC) were cultured at 10 5 cells/ml in RPMI 1640 medium supplemented with 15% heat-inactivated HCMV-seronegative human serum (PHS) and 1 ⁇ g/ml whole HCMV antigen.
  • SP-CN seropositive donor
  • MNC mononuclear cells
  • T cell blasts were isolated and cloned by limiting dilution at 0.3 cells/well in round-bottom wells containing 0.2 ml RPMI-15% PHS medium with 10,000 autologous x- irradiated (5,000 R) MNC, 1 ⁇ g/ml whole HCMV antigen (Towne strain) and 15% IL-2 (TCGF, Biotest, Frankfurt, West Germany). Plating efficiencies of 2% to 12% were observed. The clones were expanded in culture medium containing 10-20% IL-2, and restimulated with autologous x-irradiated MNC/LCL mixture and 1 ⁇ g/ml whole HCMV antigen every 1-2 weeks.
  • HCMV-specific cloned T h cells were assayed 7-14 days after restimulation.
  • the microcultures were set up in round-bottom plates (Flow Lab., Inc., VA) with a total volume of 0.2 ml/well containing 10 4 HCMV-specific T h cells, various concentrations of x-irradiated autologous MNC and/or LCL and whole HCMV antigen (Towne strain) or HSV antigen.
  • the cultures were incubated for 3 days, and 1 ⁇ Ci/well 3H-thymidine (New England Nuclear, Boston, MA) was added to each well for the final 16-24 hours of culture.
  • Wells were harvested with an automatic cell harvester, cells collected on glass fiber filter paper, and radioactivity measured in a Beckman 5801 liquid scintillation spectrometer.
  • HCMV-specific T h clones generated from a seropositive donor were tested for HCMV-specific proliferative activity using either x-irradiated autologous MNC (10 5 /well) or x- irradiated autologous LCL (10 4 - 3 ⁇ 10 4 /well) as APC.
  • HSV antigen was included as an antigen specificity control.
  • Table 9 shows the results obtained using seven clones. TABLE 9: PROLIFERATIVE ACT IVITY OF HCMV-SPECIFIC T h CELL CLONES USING EITHER IRRADIATED AUTOLOGOUS MNC OR LCL
  • a x-irradiated autologous MNC (5,000 rads) were added at 10 5 MNC/well.
  • b x-irradiated autologous MNC (8,000 rads) were added at 10 4 3 ⁇ 10 4 /well.
  • the data is expressed as (mean + 1 S.D.) CPM from triplicate wells.
  • T h clones listed on Table 9 proliferated well in response to HCMV antigen but not HSV antigen when MNC were added to the cultures as APC.
  • the magnitude of proliferation ranged from 8114 to 68,307 cpm. Most of these clones also exhibited
  • T helper (T h ) cells recognize antigen associated with class II MHC products on the surface of APC (P. Erb et al., J. Exper. Med., 142, 460 (1975)).
  • T h clones recognize antigen associated with class II MHC products on the surface of APC.
  • LCL LCL as APC
  • LCL from other MHC class II-matched donors also presented HCMV antigen well to the two high-responding clones (SP-CN/T3-16 and SP-CN/T3-9), but poorly to the five low-responding clones.
  • the proliferation assays were set up by using 10 4 T cells/well, 10 4 -10 5 MNC IRR and/or LCL IRR /well and 2 ⁇ g/well whole Towne HCMV antigen.
  • 3 H-thymidine incorporation of clone SP-CN/T5-43 plus SP-CN LCL IRR (10 4 -10 5 /well) was in the range of 59-92.
  • 3 H-thymidine incorporation of clone SP-CN/T5-43 plus SP-CN LCL IRR at LCL concentrations of 10 4 /well, 3 ⁇ 10 4 /well and 10 5 /well were 629; 1,279; and 2,466; respectively.
  • LCL alone as APC at a range of 10 4 LCL/well to 10 5 LCL/well did not stimulate any significant T cell proliferative response to whole HCMV antigen above background.
  • the addition of LCL at 10 4 did not stimulate any significant T cell proliferative response to whole HCMV antigen above background.
  • the addition of LCL at 10 4 did not stimulate any significant T cell proliferative response to whole HCMV antigen above background.
  • T h cells were not expanded even when both LCL and whole HCMV antigen were added to the culture.
  • a combination MNC and whole HCMV antigen led to a 3- to 43-fold increase in T cell numbers during 12 days of culture depending on the amount of MNC that were involved.
  • LCL were added along with MNC and HCMV antigen, a 29- to 620-fold Increase in T cell numbers was observed over the same period of time under the same tissue culture conditions, clearly indicating that LCL were augmenting the expansion of activated T h cells.
  • the cells were taken from cultures shown in Table 7 on day 7 and added to flat-bottomed wells at 0.2 ml/well in triplicate. The cultures were labeled with [ 3 H] -thymidine for 18 hrs. before harvesting.
  • T h cell expansion is proportional to the concentration of MNC IRR in the range of 10 4 to 10 5
  • LCL cloned T h cells

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Abstract

Le procédé décrit sert à la préparation de lignées de lymphocytes T auxiliaires ou cytotoxiques, qui peuvent être utilisées comme agents thérapeutiques pour accroître la réponse immunitaire chez des mammifères à un pathogène, tel qu'un virus, qui contient ou provoque l'expression cellulaire dudit antigène.
EP88902046A 1987-03-11 1988-02-09 Procede de production de lignees de lymphocytes t a specificite antigenique et utilisation therapeutique Withdrawn EP0348413A1 (fr)

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US6207161B1 (en) 1986-07-16 2001-03-27 City Of Hope Induction of cytolytic T-lymphocytes with cytomegalovirus polypeptides
AU2925789A (en) * 1987-12-17 1989-07-19 Browning's Clinical Pathology Services Limited Lymphokine activation of cells for adoptive immunotherapy, e.g. of hiv infection
US6905680B2 (en) 1988-11-23 2005-06-14 Genetics Institute, Inc. Methods of treating HIV infected subjects
US6534055B1 (en) 1988-11-23 2003-03-18 Genetics Institute, Inc. Methods for selectively stimulating proliferation of T cells
US7479269B2 (en) 1988-11-23 2009-01-20 Genetics Institute, Llc Methods for selectively enriching TH1 and TH2 cells
JP2639834B2 (ja) * 1988-11-30 1997-08-13 有限会社ジオリサーチ 免疫記憶細胞懸濁液およびその調製方法
DE3921211C1 (fr) * 1989-06-28 1990-11-29 Idt Ag Fuer In Vivo Diagnostik Und Therapie, Zuerich, Ch
WO1992020783A1 (fr) * 1991-05-16 1992-11-26 Idt Ag Für In Vivo Diagnostik Und Therapie Procede de preparation de lymphocytes b et t specifiques d'antigenes et anticorps monoclonaux ainsi obtenus
JPH06510799A (ja) * 1992-06-25 1994-12-01 シティ・オブ・ホープ サイトメガロウイルスのポリペプチドによる細胞溶解性ｔ−リンパ球の誘導
US7175843B2 (en) 1994-06-03 2007-02-13 Genetics Institute, Llc Methods for selectively stimulating proliferation of T cells
US5827642A (en) * 1994-08-31 1998-10-27 Fred Hutchinson Cancer Research Center Rapid expansion method ("REM") for in vitro propagation of T lymphocytes
IL125884A0 (en) * 1996-03-04 1999-04-11 Targeted Genetics Corp Modified rapid expansion methods ("modified-rem") for in vitro propagation of t lymphocytes
EP0904350B1 (fr) 1996-03-04 2010-08-04 Calyx Bio-Ventures Inc. Procedes modifies d'expansion rapide (rem modifie) permettant la propagation in vitro de lymphocytes t
CA2335516A1 (fr) * 1998-06-26 2000-01-06 Keld Kaltoft Methode de developpement et de selection des lymphocytes t associes a une pathologie

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