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WO1997022349A1 - Procedes relatifs a l'activation de lymphocytes t in vivo par des cellules dendritiques a impulsion d'antigene - Google Patents

Procedes relatifs a l'activation de lymphocytes t in vivo par des cellules dendritiques a impulsion d'antigene Download PDF

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Publication number
WO1997022349A1
WO1997022349A1 PCT/US1996/019954 US9619954W WO9722349A1 WO 1997022349 A1 WO1997022349 A1 WO 1997022349A1 US 9619954 W US9619954 W US 9619954W WO 9722349 A1 WO9722349 A1 WO 9722349A1
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cells
antigen
cell
human
antigens
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PCT/US1996/019954
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English (en)
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Edgar G. Engleman
Ronald Levy
Frank Hsu
Claudia Benike
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The Board Of Trustees Of The Leland Stanford Junior University
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Priority to AU11523/97A priority Critical patent/AU1152397A/en
Publication of WO1997022349A1 publication Critical patent/WO1997022349A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to methods of using isolated human dendritic cells to present exogenous antigens for the induction of immune responses m vi vo
  • it relates to the isolation of dendritic cells from human blood, ⁇ _0 exposing the cells to lymphoma-derived immunoglobulins or proteins derived from human immunodeficiency virus (HIV) as antigens, and infusing the antigen-pulsed dendritic cells into patients to induce and/or augment an antigen-specific immune response
  • HIV human immunodeficiency virus
  • the methods of ⁇ _5 the invention described herein have a wide range of applications, including but not limited to, the clinical use of antigen-pulsed dendritic cells as vaccines and/or immunotherapeutICS against cancel and infectious agents such as viruses.
  • T and B cells two functionally distinct populations of lymphocytes known as T and B cells, respectively.
  • the T cells may be further divided into 30 two subsets by function and phenotype A subset of " cells responds to antigen stimulation by producing lymphokines which "help" or activate various other cell types m the immune system.
  • Another T cell subset is capable of developing into antigen-specific 35 cytotoxic effector cells, being able to directly kill antigen-positive target cells
  • th ⁇ ⁇ B cell response is primarily carried out by secretory proteins, antibodies. Antibodies function by neutralizing antigens or m conjunction with other 5 effector cells of the immune system m mediating antibody-dependent cellular cytotoxicity.
  • Helper T cells can be distinguished from classical cytotoxic T lymphocytes (CTL) and B cells by their cell surface expression of a
  • CD4 10 glycoprocem marker termed CD4.
  • type 1 helper T cells THI
  • IL-2 mterleukm-2
  • ⁇ -IFN ⁇ - mterferon
  • TH2 type 2 helper T cells
  • CD4 + T cells like CD8 * CTL
  • a second T cell subpopulation is the classical CTL which express the CD8 surface marker Unlike most TH, these cells display cytolytic activity
  • antibody products play a principal role in the defense against viral infections and cancer.
  • B cells recognize antigens by antibodies, either acting as cell surface receptors or as secreted _o proteins, which bind directly to antigens on a solid surface or in solution, whereas T cells only recognize antigens that have been processed or degraded into small fragments and presented on a solid phase such as the surface of antigen-presenting cells (APC) .
  • APC antigen-presenting cells
  • MHC major histocompatibility complex
  • MHC 20 man, the MHC is known as HLA.
  • MHC class I gene products are found on all somatic cells, and they were originally discovered as targets of major transplantation rejection responses.
  • Class II gene products are mostly expressed on cells of various types
  • MHC-encoded proteins have been shown to function as receptors for processed antigenic fragments on the surface of APC (B orkman et al . ,
  • APC The presentation of antigens to T cells is carried out by specialized cell populations referred 35 to as APC.
  • APC include macrophages/monocytes , B cells, and bone marrow- derived dendritic cells (DC) .
  • DC are sometimes also referred to as "professional" APC.
  • APC are capable of internalizing exogenous antigens, cleaving them into 5 smaller fragments in enzyme-rich vesicles, and coupling the fragments to MHC-encoded class I or class II products for expression on the cell surface (Goldberg and Rock, 1992, Nature 357:375-379) . Since APC express both MHC-encoded class I and class II 0 glycoproteins, they can present antigenic fragments to both CD4 + and CD8* T cells for the initiation of an immune response.
  • APC not only can present antigens to T cells with antigen-specific receptors, 5 but can provide all the signals necessary for T cell activation. Such signals are incompletely defined, but probably involve a variety of cell surface molecules as well as cytokines or growth factors . Further, the factors necessary for the activation of o naive or unprimed T cells may be different from those required for the re-activation of previously primed memory T cells. The ability of APC to both present antigens and deliver signals for T cell activation is commonly referred to as an accessory cell function. 5 Although monocytes and B cells have been shown to be competent APC, their antigen presenting capacities in vi tro appear to be limited to the re-activation of previously sensitized T cells. Hence, they are not capable of directly activating functionally naive or 0 unprimed T cell populations.
  • dendritic cell refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid
  • lymphoid DC of the spleen include lymphoid DC of the spleen, Langerhans cells of the epidermis, and veiled cells in the blood circulation. Although they are collectively classified as a group based on their lymphoid DC of the spleen, Langerhans cells of the epidermis, and veiled cells in the blood circulation. Although they are collectively classified as a group based on their lymphoid DC of the spleen, Langerhans cells of the epidermis, and veiled cells in the blood circulation. Although they are collectively classified as a group based on their
  • mouse epidermal Langerhans cells unlike splenic DC, are not active APC in mixed leucocyte reaction (MLR) , unless cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) (Witmer-Pock et al . , 1987, J. Exp. Med. 166:1484-1498; Heufler et al . , 1988, J. Exp. Med. 167:700-705) .
  • MLR mixed leucocyte reaction
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • Most human Langerhans cells express the CD1 and CD4 markers, while freshly isolated blood DC express CD4 weakly, but not CD1.
  • cultured peripheral blood DC express CDlc, but not CD4. Additionally, it has not been established the extent to which the functional characteristics observed with mouse DC are applicable to human DC, especially the DC obtained from non-splenic tissues; in part, due to inherent differences between the human and murine immune systems.
  • the present invention relates to the use of isolated and antigen-pulsed human DC as APC in vi vo to induce and/or augment antigen-specific immune responses It also relates to pharmaceutical composition containing such cells
  • the invention is based, m part, on the Applicants' discovery that human DC partially purified from human blood by sequential density gradient centrifugation function as potent APC m vi vo in patients with B cell ly phomas
  • Example 6 infra , when isolated autologous DC are pulsed with immunoglobulin "custom made" from four patients' lymphoma cells and re-infused into the patients, ldiotype-specific T cell proliferative responses are detected Most importantly, one patient's tumor undergoes a complete regression over the course of such treatment. This patient also developed a tumor- specific CTL response.
  • Example 7 further demonstrates that HLA-matched donor DC pulsed with a recombinant HIV envelope protein, gpl60, induce HIV-spec fic T cell proliferative and cytotoxic responses, upon their infusion into an HIV- seropositive recipient
  • a wide variety of uses is encompassed by the invention described herein, including but not limited to, the in vi vo administration of antigen-pulsed DC- as 5 vaccines for priming primary immune responses and/or as immunotherapeutics for augmenting secondary immune responses .
  • Such responses may be induced against any antigen of interest, ranging from tumor antigens such as lymphoma idiotypes, p53 tumor suppressor protein, - j _0 human prostate carcinoma antigen known as prostatic acid phosphatase, melanoma antigens MAGE and MART-1, and oncogene products to infectious agents, including viruses such as HIV and its encoded antigenic products .
  • tumor antigens such as lymphoma idiotypes, p53 tumor suppressor protein, - j _0 human prostate carcinoma antigen known as prostatic acid phosphatase, melanoma antigens MAGE and MART-1
  • oncogene products to infectious agents, including viruses such as HIV and its encoded antigenic products .
  • FIG. 1 Peripheral blood lymphocytes were isolated by "FICOLL/HYPAQUE" gradient centrifugation of patient's blood drawn at the indicated
  • IMDM containing 5% FCS and 30 units/ml of IL-2. After a total of 5 days at 37°C in 5% C0 2 atmosphere, the cells were pulsed for 16 hours with 1 ⁇ ci/well 3 H-thymidine . Data are
  • FIG. 1 A first figure.
  • FIG. 2A and B demonstrate the regression of paraaortic adenopathy.
  • FIG. 2C and D demonstrate the regression of a paracardiac mass and paraaortic adenopathy. Prior to treatment, these nodal masses were documented to be slowly progressing.
  • FIG. 3 Peripheral blood mononuclear cells (10 5 per well) from an HIV-infected individual with a CD4 count of 480 CD4 cells per ⁇ l. were cultured with purified HIV-l MN gpl60 (Immuno AG, Vienna, Austria) for 7 days. [ 3 H] -thymidine was added during the last 12 hours of culture and [ 3 H] -thymidine incorporation determined using standard methods. The patient was infused monthly with DC pulsed with gpl60 (arrows) . Peripheral blood mononuclear cells were collected one week after each infusion, and a t one and two weeks before the first infusion. Results are expressed as stimulation index (S.I.) ; counts per minute with antigen/counts per minute without antigen.
  • S.I. stimulation index
  • FIG. 4 Autologous Epstein Barr virus-transformed B cells were pulsed with vaccinia constructs that carried the HIV env gene or the ⁇ galactosidase gene and used as target cells.
  • Peripheral blood mononuclear cells were isolated from the HIV-infected individual and then incubated with the target cells infected with vaccinia virus .
  • Standard chromium release assays were performed one week after each infusion, and at one and two weeks before the first infusion. Patient was treated with gpl60 pulsed-DC at monthly intervals (arrows) . Background lysis of target cells pulsed with control vaccinia 5 virus expressing the ⁇ galactosidase gene was subtracted. Standard error of the mean never exceeded 5% of the mean.
  • Immunoglobulin or antibody molecules are polypeptides composed of heavy and light chains, which possess highly specific variable regions at their amino termini .
  • the variable regions of heavy and light chains collectively form the unique antigen ⁇ ic recognition site of the immunoglobulin protein. These variable regions contain determinants that can themselves be recognized as antigens by the immune system, and they are referred to as idiotypes .
  • B-cell malignancies are products of clonal proliferation of tumor cells synthesizing a single immunoglobulin molecule (or monoclonal :, . ibody) with unique variable regions in the heavy and light chains. Since such tumors are monoclonal in origin, the 25 immunoglobulin expressed by all cells of a given tumor in a patient is identical, and can be distinguished from normal B cells by virtue of its unique idiotype.
  • B-cell lymphomas are tumors of mature lymphocytes, which generally express ir on their cell 30 surface.
  • the idiotypic de .rminants of the surface immunoglobulin of a B-cell /mphoma can thus serve as a tumor-specific marker fo. he malignant clone since it is not expressed by any ⁇ iher tissues in the body.
  • Studies in animals as well as in humans have 35 demonstrated the usefulness of the immunoglobulin idiotype as a tumor-specific antigen and as a target for passive immunotherapy in vivo (Campbell et al . , 1990, J. Immunol. 145:1029; Campbell et al . , 1988, J. Immunol. 141:3227) .
  • KLH keyhole-limpet hemocyanin
  • an immunologic adjuvant was also associated with toxicity. Furthermore, an approved human adjuvant, alum, may not be effective in inducing anti- idiotypic responses.
  • DC may be isolated from any source where they are found using variants of the procedure described herein, pulsed with any antigens or fragments thereof, and remfused nto pat- nts containing cells or tissues that express the antigens for the lmmunologic destruction of such cells and tissues .
  • the present invention relates to an antigen presentation system using DC for the activation of T cells m vivo . Due to their presence m low numbers m most tissues, DC must first be isolated and enriched. Although DC are found m both lymphoid and non-lymphoid tissues, a natural and easily accessible source of DC in man is the peripheral blood, which contains an estimate of fewer than 1 DC per 100 white blood cells. The potency of the accessory cell function of DC in antigen presentation allows for the use of these cells in relatively small numbers when enriched, and absolute purity is not necessary for the generation of a T cell response in vivo . However, it is most preferable that a highly purified DC population (>90%) be used for in vivo administration.
  • DC may be isolated from a normal human or from a patient suffering from a disease. Additionally, such individuals may be treated with colony stimulating factors to increase their number of DC prior to isolation. For example, GM-CSF ("LEUKINE", Immunex Corporation, Seattle, WA) may be infused into an individual at 250 mcg/m 2 /day for several days up to three weeks intravenously prior to obtaining the peripheral blood mononuclear leukocytes (PBML) for the purification of DC. This procedure may increase the yield of DC for antigen pulsing and subsequent infusion.
  • PBML peripheral blood mononuclear leukocytes
  • Human DC may be isolated from any tissues where they reside, using a variety of separation methods.
  • Example 6, infra presents variants of such methods as illustrations for isolating DC from the human peripheral blood. This procedure is principally designed to avoid the exposure of DC to antigens such as fetal calf serum, sheep red blood cells and murine monoclonal antibodies which have been used in the separation of peripheral blood leukocytes. Since DC may be able to present such proteins to T cells, even in the absence of other exogen usly added antigens, conventional methods of DC isolation may lead to activation of T cells not specific for the antigens of interest, thus potentially masking the response sough .
  • antigens such as fetal calf serum, sheep red blood cells and murine monoclonal antibodies which have been used in the separation of peripheral blood leukocytes. Since DC may be able to present such proteins to T cells, even in the absence of other exogen usly added antigens, conventional methods of DC isolation may lead to activation of T cells not specific for the antigen
  • human PBML may be isolated from blood samples, particularly buffy coats or leukocytes prepared by apheresis, by "FICOLL HYPAQUE” gradient centrifugation followed by "PERCOLL” discontinuous centrifugation (Markowicz and Engleman, 1990, J. Clin. Invest.
  • the high buoyant density (HD) fraction contains ⁇ and at ⁇ -T cells, B cells, and NK cells, whereas DC are in the low buoyant density (LD) fraction of the "METRIZAMIDE” or "NYCOPREP 1.068" gradient.
  • the LD fraction can then be subjected to a second "METRIZAMIDE” or "NYCOPREP 1.068" gradient to obtain a further enriched population of DC.
  • DC may also be further enriched using additional protocols, depending on the level of purity required.
  • Isolated DC can be pulsed immediately with any antigen of interest. Alternatively, DC may be isolated by procedures involving repetitive density gradient centrifugation, positive selection, negative selection, or a combination thereof.
  • the above-mentioned density gradient methods are preferred because they do not contain xenogeneic proteins in the form of mouse antibodies or sheep red blood cells which may be internalized and presented by DC prior to the addition of an exogenous antigen of interest.
  • Positive selection methods may utilize affinity chromatography with antibodies directed to DC surface markers. Positive selection does not necessarily require the use of antibodies that recognize DC- specific determinants. For example, B cells and monocytes may be depleted first from the DC-containing fraction after density gradient centrifugation, plastic adhesion, and Fc receptor panning, then an antibody to MHC-Class II antigen can be used to positively select for DC. Negative selection includes modifications of the protocol disclosed herein, supra.
  • a DC-containing cell preparation may be reacted with one or more antibodies directed at cell surface antigens not expressed by DC for the removal of non-DC.
  • Antibodies to any T cell, B cell, " monocyte, and granulocyte markers may be used. Examples of such antibodies include anti-CD3, anti-CD4, anti-CD5, and anti-CD8 specific for T cells; anti-CD12, anti-CD19 and anti-CD20 specific for B cells; anti-CD14 specific for monocytes; and anti-CD16, and anti-CD56 specific for natural killer cells (Becton Dickinson, San Jose, CA and Ortho
  • the cells may be removed by adsorption to a solid surface coated with an anti-mouse antibody, as the majority of monoclonal antibodies directed at cell surface markers are of mouse origin, or if the antibodies are conjugated with biotin, the antibody-bound cells can be removed by an avidin or streptavidin-coated surface; or if the antibodies are conjugated to magnetic beads, the cells expressing antigens recognized by the antibodies can be removed in a magnetic field (Harlow and Lane, 1988, Antibodies: A Laboratory Manual,, Cold Spring Harbor Laboratory Press) . 5.2. USE OF DENDRITIC CELLS AS ANTIGEN PRESENTING CELLS
  • APC APC process complex antigens into smaller fragments by enzymatic degradation, and present them in association with MHC-encoded molecules to T cells.
  • macrophages/monocytes have been studied most extensively as APC, murine DC have been shown to also possess potent accessory cell function.
  • the present invention demonstrates that DC isolated from human blood present antigens for the activation of antigen-specific T cells in vi vo .
  • the potent accessory cell function of DC provides for an antigen presentation system for virtually any antigenic epitopes which T and B cells are capable of recognizing through their specific receptors.
  • Example 6, infra demonstrates that human DC can present immunoglobulin idiotypes as antigens to T cells in vivo . T cell activation is manifested by T cell proliferation in response to antigen.
  • Example 7, infra shows that similarly prepared DC can present an HIV-encoded protein to HLA-matched T cells in a recipient to induce both T cell proliferative and cytotoxic responses against HIV-infected target cells.
  • DC are useful in vi vo in presenting antigens encoded by infectious agents such as viruses, microorganisms and their products, as well as tumor antigens expressed by cancer cells (Urban and Schreiber, 1992, Ann. Rev. Immunol. 10: 637-644) .
  • Infectious agents against which the present invention may be applicable include, but are not limited to, bacteria, parasites, fungi, and viruses.
  • the multitudes of antigens encoded by these agents, which may be processed and presented by DC include but are not limited to, external surface proteins, and structural proteins including internal enzymes .
  • antigens encoded by any genes of the HIV genome including the env, gag, pol , nef , vif , rev, and tat genes may all be presented by DC in vi vo .
  • peptide fragments which are degradative products of proteins within DC may also be directly added to DC during pulsing.
  • hepatitis B virus hepatitis C virus
  • cytomegalovirus herpes simplex virus
  • varicella zoster herpes simplex virus
  • Staphylococcal species hepatitis C virus
  • Mycobacterium species hepatitis C virus
  • tumor-associated antigens In addition to immunoglobulin idiotypes as tumor-specific antigens, a large number of human tumor-associated antigens have been identified by monoclonal antibodies (Reisfeld and Cheresh, 1987, Adv. Immunol. 40: 323-377) . Although these cellular- antigens are selectively expressed in higher quantities by certain tumor cells, it has not been established that they naturally elicit an immune response in cancer patients or can be used effectively to induce such a response. Unlike animal tumor models in which tumor-reactive T and B cells can be induced through hyperimmunization with tumor cells or tumor antigens, intact human tumor cells or oncogenic proteins may not be easily tested in humans without an approved clinical protocol.
  • lymphocytes obtained from cancer patients whose cells presumably have been exposed to antigens expressed by their autologous tumor cells in vivo have been shown in some systems that tumor development is accompanied by a down-regulation of tumor specific immune responsiveness mediated by suppressor cells, and if so, T cells isolated from cancer patients may have already come under the influence of such suppression m vivo so as to not function in a manner similar to that of T cells obtained from tumor-immune hosts.
  • these attempts to activate human tumor-reactive T cells have generally used monocytes as APC, which have been shown to be much less effective than DC, especially if the T cells have not been primed adequately in vi vo against the tumor antigens .
  • the antigen-pulsed DC described herein may be used as a cellular adjuvant for presenting tumor antigens in vivo .
  • the potent accessory cell function of DC can present tumor antigens to T cells of cancer patients m vivo, whose immune response is apparently inadequate to eliminate the tumors m vivo .
  • Whole tumor cells in viable or irradiated form, tumor membrane preparations, and tumor antigens purified from natural sources or expressed as recombinant products or chemically synthesized as peptides may be used to pulse DC m vi tro
  • oncogene products have been shown to be capable of inducing murine T cell activities.
  • oncogenic forms of the ras gene product p21, and the fusion product p210 of the bcr-abl gene induce T cell proliferative responses, when used to immunize mice (Peace et al , 1991, J Immunol. 146: 2059-2065; Chen et al . , 1992, Proc. Natl. Acad. Sci. USA 89 1468-1472) .
  • oncogenic proteins which are different from their normal cellular counterparts as a result of ammo acid substitutions may possess new immunogenic determinants that are recognizable by T cells It is not necessary that such proteins be expressed naturally on the cell surface, as cytoplasmic and nuclear proteins may be processed, attached to MHC-encoded products intracellularly, and translocated to the cell surface in a complex form (Gould et al . , 1989, J. Exp. Med. 170: 1051-1056) . Since oncogene products are expressed in a variety of tumor types including colon cancer, leukemia and lymphoma, antigen-pulsed DC may be used to activate T cells in vi vo against such cancers.
  • HER-2/neu gene product United States Patent No. 4,968,603
  • estrogen receptor milk fat globulin
  • p53 tumor suppressor protein Levine, 1993, Annu. Rev. Biochem. 62:623
  • mucin Tum-Papadimitriou, 1990, International Publication No. W090/05142
  • telo erases nuclear matrix proteins, MART-1, MAGE-1, MAGE-2, MAGE-3 (van der Bruggen et al . , 1991, Science 254:1643; Celis et al . , 1994, Proc. Natl. Acad.
  • Bacterial, parasitic, fungal, viral, and tumor antigens of cellular or viral origin may be introduced to DC by addition to DC cultures followed by incubation, by the osmotic lysis of pinosomes after pinocytotic uptake (Moore et al . , 1988, Cell 54: 777- 785) , or by uptake in antigen-containing liposomes.
  • Antigens may be used as purified naturally occurring whole polypeptides, purified recombinant whole polypeptides, whole organisms or cells in viable or dead forms, protein fragments generated by enzymatic digestion, or synthetic peptides produced by solid phase chemical methods (Creight-on, 1983, Protein Structures and Molecular Principles, W.H. Freeman and Co.
  • the amount of antigens necessary for pulsing DC may vary depending on the nature, size, and purity of the molecules. In general, polypeptides may be used at 1-100 ⁇ g/ml, and small peptides at 1-50 ⁇ g/ml . Introduction by osmotic lysis of pinosomes requires larger amounts of proteins in the range of 200-500 ⁇ g/10 6 APC. Alternatively, exogenous genes encoding specific antigens of interest or expression vectors containing such genes or portions thereof may be incorporated into DC in expression vectors using conventional methods, including transfection, recombinant vaccinia viruses and retroviruses (Sambrook et al .
  • antigen pulsing of DC Any of the aforementioned methods for introducing exogenous antigens into DC as well as any others commonly used by those skilled in the art are hereinafter collectively referred to as antigen pulsing of DC.
  • the present invention relates to a method of activating an immune response in a human patient to an antigen.
  • the method includes the isolation of human DC, preferably from the peripheral blood, pulsing the cells with an antigen in vi tro , and then administering the antigen-pulsed cells into a patient whose cells or tissues express the antigen.
  • DC may be pulsed with antigens according to the various methods described in Section 5.2.1, supra , washed, and administered in vivo as vaccines and/or immunotherapeutics for the elicitation or augmentation of a pre-existing but weak T cell response.
  • the isolated cells may be first expanded in number prior to incubation with antigens. It has been shown that GM-CSF and tumor necrosis factor-of induce the differentiation of human hematopoietic progenitor cells into DC (Caux et al . , 1992, Nature 360:258) .
  • isolated DC may be expanded by treatment with such cytokines in culture.
  • Immunization with antigen-pulsed DC may increase both the magnitude and the specificity of a response. It may be desirable to repeat such immunizations at time intervals of days or weeks.
  • the potency of DC as APC may alleviate the need of using conventional adjuvants to augment the response, although it does not preclude the use of adjuvants to further enhance immune reactivity.
  • the antigen-pulsed DC may be administered in combination with cytokines that can maintain their number and activity for prolonged periods in vi vo .
  • cytokines include colony stimulating factors such as GM-CSF and interleukins such as IL-12.
  • the antigen-pulsed DC may be suspended in any known physiologic saline at a concentration sufficient to induce an immune response as detected by assays which measure T cell proliferation, T cell cytotoxicity, antibody production or reduction of the number of antigen-positive cells or tissues.
  • a patient usually receives the total number of isolated DC pulsed with antigen.
  • a patient may be infused with several million up. to several hundred million DC.
  • the cells may be infused into autologous, HLA-matched allogeneic or even HLA-mismatched allogeneic patients.
  • Human DC were obtained from buffy coats of lymphoma patients after leukophoresis .
  • PBML were isolated by "FICOLL-HYPAQUE" gradient centrifugation (Boyum, 1968, Scand. J. Clin. Lab. Invest: 21:21-29) .
  • a buffy coat was diluted with Dulbecco's PBS without divalent ions such as Ca 2* or Mg 2* (referred to as DPBS) up to 10 ml.
  • 10 ml of "FICOLL” was gently underlaid into each tube and centrifuged at 1000 x g for 35 minutes at room temperature. The interface was collected and washed with DPBS three times.
  • the preparation was further fractionated over a four layer discontinuous "PERCOLL” gradient (30%, 40%, 50.5% and 75%) (Pharmacia, Uppsala, Sweden) (Markowicz and Engleman, 1990, J. Clin. Invest. 85:955) .
  • Original "PERCOLL” density was prepared at 1.130g/ml DPBS and 15 ml of a 50.5% "PERCOLL” solution was made and shaken in a conical polypropylene tube to create a foam on a surface of the "PERCOLL” solution. The tube was gently underlaid with about 6.5 ml of 75% "PERCOLL” .
  • the tube was then slowly overlaid with 3 to 3.5 ml of 40% "PERCOLL” dropwise along the side of the tube which was being slowly rotated, followed by an overlay of 2.5ml of 30% “PERCOLL” in the same manner.
  • the gradients were kept on ice for use within about 4 hours .
  • the collected cell fractions were diluted with DPBS at least 3 volumes and centrifuged at 1000 x g for 12 minutes at 4°C.
  • the cells were washed twice with DPBS supplemented with 5% human serum at 400 x g for 5-6 minutes at 4°C.
  • the HD cells (3-7 x 10 ⁇ /50 ml of RPMI containing 10% pooled human serum) were then cultured overnight in teflon vessels at 37°C.
  • the cultured cells were subjected to gradient centrifugation in "METRIZAMIDE” (15.5%) by overlaying the cells onto 10 ml of 15.5% (wt/vol) "METRIZAMIDE” (Sigma Chemical Co.) followed by centrifugation at 650 xg for 10 min at room temperature. This fraction was further depleted of contaminating monocytes by a solid phase absorption procedure for about 20 min. on human IgG-coated petri dishes. The IgG was commercial preparation tested and approved for intravenous human use . The DC were then enriched over a second "METRIZAMIDE” gradient (14%) .
  • the HD cells from the first "METRIZAMIDE” gradient consisted of a mixture of at ⁇ and ⁇ -T cells, B cells, and NK cells.
  • the purity of the DC obtained using this procedure were 60-90%.
  • the DC could be enriched 5 after overnight culture by centrifugation over a "NYCOPREP 1.068" discontinuous gradient (Nycomed Pharma AS, Oslo, Norway) .
  • About 2.5 x 10 8 cells were suspended in 15-20 ml of a solution made up of 85% DPBS, 10% human serum and 5% EDTA.
  • these cells could be further enriched by another round of "NYCOPREP” centrifugation to obtain a LD fraction of 80-90% DC.
  • the LD cells after the first "NYCOPREP” 5 step could be negatively selected by incubation with antibody-coated petri dishes to remove CD3", CD14 + , CD16 * and CD20 + cells.
  • the non-adherent cell population also contained 80-90% DC.
  • density gradient centrifugation was preferred in order to 0 avoid the use of xenogeneic proteins in the form of antibodies against leukocyte markers. All procedures described herein could produce a yield of 1-2.5 x 10 6 cells from 400-500 ml of whole blood.
  • the purity of DC following each step of DC 5 enrichment was assessed by staining with an anti-HLA-DR (anti-MHC class II) antibody (CA141) conjugated to fluorescein, and phycoerythrin- conjugated anti-CD14 (anti-monocyte) .
  • the base-line studies used to evaluate clinical disease in all patients included complete physical examination, chest radiography, routine blood counts and chemistry tests, abdominal and pelvic CT scanning, with or without bipedal lymphangiography, which were all normally part of routine clinical care of such patients. All underwent re-staging during the course of the study according to the objective procedures used at base line.
  • a cell suspension obtained from the tumor biopsy specimen was washed in phosphate buffered saline (PBS) and mixed with the HAT sensitive heterohybridoma B5/K6H6 (B5) in a ratio of 1:4 or 1:5
  • PBS phosphate buffered saline
  • B5 is a heterohybridoma that was produced by the fusion of the NS-1 mouse myeloma fusion partner with a human lymphoma cell.
  • the B5 clone has lost the ability to spontaneously secrete immunoglobulin.
  • a subclone was identified which had retained the ability to secrete immunoglobulin when fused with human B-cells. This subclone was drug-marked so that it would not survive in HAT medium.
  • Studies by Carroll et al . (1986, J. Immunol. Methods 89:61) show B5 to be a useful fusion partner for rescuing idiotype secretion from non- secreting human B-cell tumors. No mouse immunoglobulin is produced by B5 or hybridomas derived from i .
  • the mixture of tumor cells and fusion partner cells was washed 2 times in Hank's balanced salt solution (HBSS) . Supernatant was aspirated and the cell pellet was exposed to 40% polyethylene glycol (PEG) for 2 minutes. The PEG was diluted slowly by adding HBSS, and the cells were centrifuged. The cells were then resuspended in complete medium (RPMI Medium, 10% fetal bovine serum, glutamine 4 mM) to density of 2xlO G cells per ml, and 0.1 ml was then added to the wells of a microtiter plate.
  • RPMI Medium 10% fetal bovine serum, glutamine 4 mM
  • HAT media Complete media with the addition of hypoxanthine 10 ⁇ 4 M, thymidine 1.6 X 10 ⁇ 3 M and aminopterin 4 x 10 "5 M) .
  • the microtiter plates containing the cells were incubated for 10-20 days at 37°C in a humidified 5% C0 2 in air incubator. The supernatants were removed from the wells and the cells were fed with the medium described above but without the aminopterin (HT medium) .
  • An ELISA assay was used to determine which wells of the microtiter plate contained hybridomas which were secreting the rescued tumor immunoglobulin.
  • the tumor biopsy specimen used for cell fusion contained a large predominance of malignant cells which expressed a single light chain type, either kappa or lambda.
  • Wells secreting immunoglobulin with the heavy and light chain type corresponding to the known immunophenotype of the tumor specimen were selected and expanded.
  • Goat anti-human Ig heavy chain-specific reagents were diluted in 0.05 M sodium bicarbonate buffer (pH 9.5) .
  • the 96-well microtiter plates were coated with this antibody in a volume of 50 microliters per well. The plates were incubated overnight at 4°C. The plates were then washed 5 times with normal saline containing 0.05% Triton X (wash buffer) . Nonspecific binding was blocked by incubating the wells with 5% non-fat milk in PBS for 1 hour and then washing 5 times with wash buffer.
  • Hybridoma supernatants were added (50 microliters per well) and incubated for 1 hour at room temperature. The plates were again washed and developed with 50 microliters per well of horseradish peroxidase conjugated to goat anti-human Ig light chain-specific reagents. The plates were incubated for 60 minutes at room temperature. Antibody reactivity was determined by added ABTS substrate and the optic density was read at 405 nm using a microtiter plate reader. As the hybridomas were grown, the supernatants were tested for immunoglobulin content using the ELISA described above.
  • Dilutions of the supernatants were compared to a standard curve generated by using purified human immunoglobulin of the same isotype as that expressed on the patient's tumor.
  • the idiotype protein was confirmed to be derived from the patient's lymphoma by cloning and comparing the DNA sequences of the variable region genes from both the lymphoma and rescue fusion.
  • Hybridomas that constantly produced in excess of 2 micrograms per ml were expanded to large volumes (2000-5000 ml) . The cell density was allowed to increase until the media was fully spent and the supernatants were collected and pooled. 5
  • the rescued tumor idiotype protein was purified from bulk supernatant using affinity chromatography.
  • IgM class a murine monoclonal anti-human IgM heavy chain-specific antibody was coupled to cyanogen bromide activated sepharose B4 or 0 an equivalent solid phase matrix at a concentration of 2-6 mg/ml of swollen beads (immunoadsorbent) .
  • Rescued tumor idiotypes of the IgA class was purified using an immunoadsorbent column consisting of a goat or rabbit anti-human IgA-specific antibody similar]y coupled to 5 a solid phase matrix.
  • Idiotypes of the IgG class were purified using a column of protein A coupled to a solid phase. The idiotype-containing supernatant was gravity flowed through the immunoadsorbent column.
  • the column was washed with 200 ml of o physiologic saline to remove the unbound protein.
  • the idiotype was eluted with 0.1 M glycine-HCl buffer (pH 2.4) using absorption at 280 nm to monitor the protein effluent.
  • the protein peak was collected and the immunoglobulin level measured using an ELISA technique 5 or absorption at 280 nm.
  • the protein purity was determined by SDS-PAGE.
  • KLH Keyhole Limpet Hemocyanin Megathura 0 crenulata
  • the KLH was supplied in a form containing more than 60% protein in BES buffer and magnesium sulfate. The protein purity, which was determined by the vendor, was greater than 5 90%.
  • the KLH was dialyzed extensively against physiologic saline and then passed through a detoxigel column (Pierce Chemical Company, Rockford, Illinois, Cat. #20339) in PBS pH 7.4 or it was passed over a QAE Zeta Prep 15 disk (LKBm, Broma, Sweden, Cat. #224-202) using 25 mM Tris-HCl pH 8.0.
  • LAL limulus amoebocyte lysate
  • idiotype specific protein and KLH were then dialyzed extensively against sterile physiologic saline. Each product was then concentrated or diluted to achieve a final concentration of 1 mg/ml in sodium chloride. The proteins were then sterile filtered through 0.45 ⁇ m filters. The final concentration was determined by measuring the absorption at 280 nm or performing an ELISA on a small, expendable aliquot of the final product . The final product was aseptically filled into sterile containers under a laminar flow hood. The vials were then frozen and stored at -20 C until needed. Sterility of the final product was confirmed by a standard 5-day bacterial culture assay performed by clinical laboratory.
  • Enriched DC were resuspended in RPMI media at a concentration of approximately one million cells/ml .
  • the DC were split into two independent cultures of five million cells into 24 well plates.
  • Each set of DC was then cultured with either sterile, purified idiotype immunoglobulin protein or KLH at a concentration of fifty micrograms/ml for 4-5 hours or overnight in a humidified 37° C incubator with 5% C02.
  • DC pulsed with either idiotype protein or KLH were washed three times in physiologic, pyrogen- free, injection grade saline and resuspended into a volume of 100 ml in an intravenous injection administration bag.
  • the patients received the total number of DC isolated from their own blood. Since each isolation yielded a different number of DC, each cell infusion contained between 5xl0 6 to lOOxlO 6 antigen-pulsed DC.
  • the patients were premedicated with acetaminophen and diphenhydramine, and received the transfusion of DC through a peripheral intravenous line or a central catheter (if available) over a period of 30-60 minutes. Vital signs were monitored continuously prior to and during the infusion. All therapy was monitored and supervised closely by a physician. Patients received similarly prepared antigen-pulsed DC for up to four times .
  • Microtiter plates were coated either with idiotype protein or with a panel of other immunoglobulins of the same isotype.
  • Patient serum w s serially diluted.
  • Pretreatment samples were used as a negative control.
  • Binding of antibodies in the patient's serum to the test panel of immunoglobulins including the patient's idiotype was detected by goat anti-human light chain-HRP antibodies specific for the light chain opposite to the patient's idiotype or by a mixture of anti-human heavy chain isotype-specific-HRP antibodies that did not react with the patient's idiotype .
  • the patient received 0.5 mg of soluble idiotype protein in sterile physiologic saline subcutaneously as a boost, and a separate 0.5 mg boost of sterile KLH at a separate subcutaneous site.
  • the cell infusions and boosts were well tolerated and two weeks following each infusion fresh lymphocytes isolated from peripheral blood were challenged in vi tro with graded doses of either KLH or idiotype, and their proliferative activity was assayed.
  • the second patient had residual lymphoma in periaortic and parailiac regions prior to DC infusion. Despite a measurable anti-idiotypic proliferative response, this patient subsequently developed progressive disease four months after the primary series of infusions. Following a fourth immunization, the patient has remained stable and has not required further therapy.
  • the third patient had equivocal radiographic findings of tumors, but showed evidence of disease in bone marrow and blood by the use of a sensitive assay involving polymerase-chain reaction for detecting tumor-specific immunoglobulin heavy chain gene rearrangement. The sensitivity of this technique allowed the detection of one tumor cell in 10 7 normal PBML.
  • stage IV A follicular lymphoma with growing disease prior to DC therapy.
  • This patient completed three cell infusions and has shown a minor response with regression of some peripheral lymph nodes.
  • all patients developed tumor idiotype-specific proliferative responses during the course of DC infusion.
  • HIV envelope antigen gpl60
  • HIV envelope antigen gpl60
  • DC were further enriched by "METRIZAMIDE” centrifugation as described in Section 6.1.1, supra .
  • 10-20X10 6 cells/ml were resuspended in RPMI/10% human serum plus 2 ⁇ g/ml of the same gpl60 antigen, and placed into teflon vessels at 37°C for 14-18 hours. Following incubation, the cells were washed by centrifugation in DPBS/5% human serum at room temperature for 10 minutes at 500xg. The pellet was resuspended in 15 ml of the same buffer and cell clusters allowed to disaggregate at room temperature for 15 min. The cells were then underlaid with 15 ml of 14% "METRIZAMIDE"/RMPI/10% human serum, centrifuged and washed three times in RPMI/10% human serum. About
  • 10 6 cells were removed for phenotype analysis. Prior to infusion of antigen-pulsed DC in vivo, 10-20X10 6 cells/ml in RPMI/10% human serum were again incubated with 50 ⁇ g/ml of gpl60 in teflon vessels overnight. In cases where DC were pulsed with HIV peptides, the cells were pulsed at a concentration of 5 ⁇ g of peptides with 10 6 cells/ml. Also, only one pulsing step was used for peptides, which were presumed to not require further processing. Several HLA-A2.1-restricted HIV-l peptides were used. The amino acid sequences, HLA binding affinities and CTL- inducing activities of these peptides are given in Table 1 below.
  • the enriched DC preparation was collected the next morning and centrifuged at room temperature for 10 minutes at 500 xg. The pellet was resuspended in DPBS and cell viability was examined by trypan blue exclusion. After two additional washing steps, the cells were placed into a 100 ml injection grade saline administration bag. Using a 10ml syringe and a 19 gauge needle, 8-10ml of saline was removed from the bag for use in resuspending the cell pellet. Thereafter, the cell suspension was brought up into the syringe and injected into the bag, followed by injection of 5 ml of previously frozen autologous serum.
  • EXAMPLE Asymptomatic HIV-seropositive patients generally exhibit a vigorous T cell response against HIV. However, such a response subsides as clinical symptoms of the disease begin to develop. In order to augment the T cell immunity against HIV, an HIV- seropositive patient was infused with DC prepared from an HLA-matched sibling that had been pulsed with HIV gpl60 in vi tro .
  • Donor DC cells were enriched from peripheral blood and pulsed with HIV-encoded gpl60 and administered into the patient three times at one month intervals.
  • PBML peripheral blood and pulsed with HIV-encoded gpl60 and administered into the patient three times at one month intervals.
  • PBML peripheral blood and pulsed with HIV-encoded gpl60 and administered into the patient three times at one month intervals.
  • PBML peripheral blood and pulsed with HIV-encoded gpl60 and administered into the patient three times at one month intervals.
  • PBML were obtained from the patient and assayed for T cell proliferative and cytotoxic responses against HIV- infected cells.
  • FIG. 3 demonstrates that shortly after the first cell infusion, T cells obtained from the patient proliferated in response to HIV gpl60 in culture. The T cell proliferative activity continued to rise for two weeks and was maintained at the same level for over six weeks after the first infusion.
  • T cells obtained from the same patient also exhibited HIV-specific CTL (FIG. 4) . Therefore, the administration of HIV antigen-pulsed DC induced both HIV-specific CD4 * and CD8 * T cells. Since a delay of clinical symptoms of HIV correlates with a detectable HIV-specific T cell response in an HIV- infected individual, an augmentation of such T cell responses will likely contribute to the control of the spread of HIV, thereby delaying HIV clinical latency, and prolonging of survival of infected individuals.
  • the present invention is not to be limited in scope by the exemplified embodiments, which are intended as illustrations of individual aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims . All publications cited herein are incorporated by reference in their entirety.

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Abstract

L'invention concerne des procédés qui consistent à isoler un certain nombre de cellules dendritiques humaines et à s'appuyer sur elles pour présenter des antigènes exogènes, induisant ainsi les réponses immunes in vivo. En particulier, on isole des cellules dendritiques du sang humain, qui sont ensuite exposées aux immunoglobulines dérivées des lymphomes ou aux protéines dérivées du virus de l'immunodéficience humaine (VIH) tenant lieu d'antigène, et on injecte ensuite au patient les cellules dendritiques qui ont ainsi reçu une impulsion par antigène afin d'induire et/ou d'accroître une réponse immune propre aux antigènes. Les procédés décrits ont un large éventail d'applications, y compris mais pas exclusivement l'utilisation clinique de cellules dendritiques à impulsion d'antigènes comme vaccin et/ou substances d'immunothérapie contre le cancer et des agents infectieux tels que les virus.
PCT/US1996/019954 1995-12-20 1996-12-17 Procedes relatifs a l'activation de lymphocytes t in vivo par des cellules dendritiques a impulsion d'antigene WO1997022349A1 (fr)

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