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WO2005034991A1 - Protocole ameliore pour renforcer l'immunisation par transfert adoptif de cellules dendritiques - Google Patents

Protocole ameliore pour renforcer l'immunisation par transfert adoptif de cellules dendritiques Download PDF

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WO2005034991A1
WO2005034991A1 PCT/US2004/033526 US2004033526W WO2005034991A1 WO 2005034991 A1 WO2005034991 A1 WO 2005034991A1 US 2004033526 W US2004033526 W US 2004033526W WO 2005034991 A1 WO2005034991 A1 WO 2005034991A1
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antigen
administering
animal
cfa
lymph
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Gwendalyn J. Randolph
Veronique Angeli
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Mount Sinai School Of Medicine Of New York University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

  • This invention relates generally to methods for enhancing immunization.
  • the invention further relates to methods for improving the migration of dendritic cells to a lymph node. Additionally, the invention relates to methods for conditioning a lymph node to increase the efficiency of dendritic cell migration to the node.
  • BACKGROUND Dendritic cells are known to play an important role in the immune system, both for their potent antigen presenting ability and their ability to initiate T-cell mediated immune responses.
  • DCs dendritic cells
  • Immature DCs endocytose and process antigen efficiently, but express low levels of costimulatory molecules. Immature DCs bearing antigen can induce T-cell anergy or regulatory T cells specific for that antigen.
  • BM-DCs Immature murine bone marrow-derived DCs
  • lymphoid tissue such as a lymph node (LN)
  • LN lymph node
  • DCs After capturing antigens outside lymphoid tissues, DCs must migrate to lymphoid tissues to prime rare na ⁇ ve antigen-specific lymphocytes that constantly recirculate through peripheral lymphoid tissues (Wu, et al., J. Exp. Med. 1999, 190(5):629-638).
  • LT Membrane lymphotoxin
  • the pivotal role played by DCs in antigen presentation and T-cell activation has resulted in considerable interest in the use of DCs in immunotherapy. This is particularly evident in the areas of vaccinology and cancer immunotherapy.
  • the immune response to an antigen loaded onto a mature or activated DC is significantly greater than to the antigen alone. Typically, the maturation/activation process occurs ex vivo.
  • ex vivo DC generation involves obtaining DC precursor cells from a patient and then differentiating the cells in vitro into DCs before introduction back into the patient.
  • the DCs must be terminally differentiated, or they will de- differentiate into monocytes/macrophages and lose much of their immunopotentiating ability.
  • Ex vivo DC maturation has been successfully accomplished with monocyte conditioned medium; recombinant cytokines such as TNF- ⁇ , GM-CSF, IL-1 and IL-6; bacterial products such as LPS and bacterial DNA; and transfection with genes that encode cytokines or costimulatory molecules.
  • antigen-loaded DC immunizations are generally performed as follows.
  • Dendritic cells are activated in vitro from a patient's white blood cells or, alternatively, from skin cells or mucosal tissue. These DCs are loaded with antigens (in the case of cancer vaccination, the antigens are proteins unique to the cancer). The antigen-loaded DCs are injected subcutaneously/intradermally into the patient to immunize the patient against the tumor. Partial success has been observed and some patients have had complete remissions of their cancer (Banchereau et al., Cell 2001, 106:271-274). This treatment can also be used for the treatment of non-cancerous diseases that are amenable to vaccination. DCs from different sources can be used clinically.
  • Circulatory DCs can be directly isolated from peripheral blood but the numbers are small and, even after Flt3-L pretreatment, the equivalent of a complete leukapheresis is needed to prepare a single DC vaccine (Berger, et al., J Immunol Methods 2002, 268:131-140).
  • DCs can also be generated ex vivo from rare, proliferating CD34+ precursors in blood, or from frequent, nonproliferating blood CD14+ monocytes.
  • the isolation of CD34+ cells requires pretreatment of patients with cytokines. Generation of DCs from CD34+ cells requires a prolonged culture in a complex set of cytokines.
  • DCs can be generated from monocytes acquired from apheresis products, which are subjected to GM-CSF and IL-4 (or IL-13) to convert the monocytes to immature DCs. These immature DCs are differentiated into mature DCs by using autologous monocyte- conditioned medium or a maturation cocktail of ILl ⁇ +IL-6+TNF- ⁇ +PGE 2 . See id. Cultured bone marrow-derived stem cells are another source of DCs (Banchereau, et al., Cancer Res 2001, 61:6451-58). Several problems with dendritic cell immunotherapy have been identified.
  • DC skin injection sites have been pre-treated with adjuvants to improve DC maturation and migration (Nair, et al., "Injection of immature dendritic cells into adjuvant treated skin obviates the need for ex vivo maturation", Dendritic Cells (J7) Keystone Conference Poster Abstracts, March 5, 2003, p. 86). It has now surprisingly been discovered that conditioning the LN that drains the region where the DC is to be administered results in a significant increase in DC migration. This is a significant discovery for the treatment of cancer and other conditions amenable to vaccination because an increase in DC migration can result in an improved response to the vaccine and reduce the number of expensive and difficult-to-produce antigen -loaded DCs required to treat a patient.
  • the present invention provides a method for immunizing an animal to an antigen by detecting an enlarged LN and administering an antigen-loaded dendritic cell to the animal having the enlarged LN. Detecting an enlarged LN can include detecting a further increase in size of an already enlarged LN. ' In one aspect of the invention, a method is provided for immunizing an animal to an antigen by inducing a LN to enlarge, and then administering antigen-loaded DCs to the animal after the LN has enlarged.
  • a method for immunizing an animal to an antigen by administering a lymph-conditioning agent and transplanting antigen-loaded DCs to the animal after a LN is conditioned.
  • the step of administering a lymph- conditioning agent comprises administering an adjuvant in a mixture with an irrelevant antigen.
  • Especially preferred adjuvants include, for example, bacille Calmette-Guerin (BCG) and complete Freund's adjuvant (CFA).
  • the step of administering a lymph-conditioning agent comprises administering a cytokine.
  • Especially useful cytokines are lymphotoxin- ⁇ (LT ⁇ ) and lymphotoxin- ⁇ (LTB).
  • the step of administering a lymph-conditioning agent comprises administering an irrelevant vaccine.
  • antigen-loaded DCs are •administered following routine, prophylactic vaccination that conditions a LN.
  • antigen- loaded DCs are administered to a patient with conditioned LNs resulting from natural exposure to a pathogen, such as S. typhinirium or an environmental stimulus.
  • Figure la depicts the number of cells per draining LN in mice four days following: no injection, CFA injection, KLH injection, or CFA/KLH injection; lb depicts the number of cells per non-draining LN in mice four days following: CFA injection, KLH injection, or CFA/KLH inj ection; 1 c depicts the percentage of CD 11 c + FITC + DCs in un-inj ected and CFA/KLH-injected mice; and Id depicts the number of CD1 lc + FITC + DCs in un-inj ected and CFA/KLH-injected mice.
  • Figure 2a depicts the number of cells per LN in C3HeJ mice and C3HeJ gld/gld mice; 2b depicts the percentage of CDllc + FITC + DCs in C3HeJ mice and C3HeJ gld/gld mice; and 2c depicts the number of CD 11 c + FITC + DCs in C3HeJ mice and C3HeJ gld/gld mice.
  • Figure 3 a depicts migrated FITC-latex labeled DCs in un-inj ected mice; 3b depicts migrated FITC-latex labeled DCs in CFA/KLH injected mice; and 3c depicts the number of migrated FITC-latex labeled DCs in un-inj ected and CFA/KLH injected mice.
  • Figure 4a shows flow cytometry results depicting T cell activation in un-inj ected mice; 4b shows flow cytometry results depicting T cell activation in CFA/KLH-injected mice; and 4c depicts the number of T cells that underwent division in response to OVA- pulsed DCs in un-inj ected and CFA/KLH-injected mice.
  • Figure 5a depicts the number of cells per draining LN in mice four days following: no injection or S. typhinirium injection; 5b depicts the number of migrated FITC+ labeled DCs in un-injected and S. typhinirium injected mice. Results are expressed as means+standard deviation (SD). The values between un-injected and S. typhinirium injected mice were significantly different (p ⁇ 0.05).
  • Figure 5c depicts the number of T and B lymphocytes per draining LN in mice infected with S. typhinirium bacteria.
  • Figure 6a depicts the total number of T and B lymphocytes per draining LN from control and CFA/KLH treated animals; 6b depicts the total number of cells per draining LN in B lymphocyte deficient mice ( ⁇ MT) that were untreated ("None") or CFA/KLH-treated; 6c depicts the number of migrated DCs per draining LN in B lymphocyte deficient mice ( ⁇ MT) that were untreated ("None") or CFA/KLH-treated. Results are expressed as means SD and are combined from 4 experiments. Differences from WT CFA/KLH treated mice were significant, P ⁇ 0.001.
  • Figure 7a depicts the total number of cells per draining LN in WT and B cell deficient mice ( ⁇ MT) following the injection of bacteria into the mice; 7b depicts the number of migrated DCs per draining LN in wild-type and B cell deficient mice following the injection of bacteria into the mice. Results are expressed as means SD and are combined from two experiments. Differences from WT CFA/KLH treated mice were significant, P ⁇ 0.0001.
  • Figure 8a depicts the total number of cells per draining LN in mice that received intraperitoneal injection of L-selectin blocking mAb MEL- 14 or IgG (control) one day before CFA/KLH treatment. Control mice did not receive CFA/KLH treatment.
  • Figure 9a depicts the total number of cells per draining LN in untreated and CFA/KLH-treated mice injected with soluble fusion protein LT ⁇ R-Ig or IgG (control) one day before CFA/KLH treatment; 9b depicts the number of migrated DCs per draining LN in untreated and CFA/KLH-treated mice injected with soluble fusion protein LT ⁇ R-Ig or IgG (control) one day before CFA/KLH treatment. Results are expressed as means SD and are representative of two experiments. Differences from CFA/KLH treated mice receiving control mAb were significant, P ⁇ 0.01.
  • FIG. lOa-d depicts results from experiments in which chimeric mice received bone marrow transplants (Tx) designed to remove expression of LT ⁇ and L-selectin selectively in the B cell compartment.
  • Recipient ⁇ MT mice were lethally irradiated and intravenously reconstituted with a mix of 25% WT BM and 75% ⁇ MT BM (WT/ ⁇ MT), 25% LT ⁇ KO BM and 75% ⁇ MT BM (LT ⁇ KO/ ⁇ MT) or 25% L-selectin BM and 75% ⁇ MT BM (LselKO/ ⁇ MT).
  • Controls included non-transplanted WT and ⁇ MT mice (No Tx). Six weeks after reconstitution, mice were treated with CFA/KLH.
  • FIG. 10a depicts the total number of cells per draining LN cellularity; 10b depicts the number of migrated DCs per draining LN; 10c depicts the number of B lymphocytes per draining LN; and lOd depicts the number of T lymphocytes per draining LN.
  • LN cell counts of WT/ ⁇ MT mice were significantly greater (P ⁇ 0.01) relative to the LT ⁇ KO/ ⁇ MT and LselKO/ ⁇ MT mice.
  • the present invention employs a different strategy: altering lymphoid tissue, especially the lymph node (LN), the desired destination of the DCs.
  • the methods of the present invention can improve the efficiency of DC ( migration by at least about 4-fold.
  • the invention is based, in part, on the following experimental work. Groups of mice were administered injections of keyhole limpet hemocyanin (KLH), complete Freund's adjuvant (CFA), or a mixture of CFA and KLH. A fourth group of mice did not receive any injection.
  • KLH keyhole limpet hemocyanin
  • CFA complete Freund's adjuvant
  • mice from the CFA/KLH-injected group had about a 4.5-fold increase in the number of cells per draining LN compared to the draining LNs of the un-injected group.
  • Mice from the CFA KLH-injected group had about a 4-fold increase in endogenous mature DC migration to the draining LNs relative to the endogenous mature DC migration to the draining LNs in un-injected mice.
  • mice from the CFA/KLH-injected group had about a 4- fold increase in transplanted DC migration to the draining LNs relative to the transplanted DC migration to the draining LNs in un-injected mice.
  • mice were administered injections of S. typhinirium bacteria or were not injected (control).
  • the number of cells per lymph node and the number of migrated DCs per LN were about 3.5 fold greater in the S. typhinirium - injected mice relative to control mice.
  • the experiments described above are significant because an increase in the number of DCs in the LN results in improved immunization. Improved DC migration results in a greater number of DCs in.
  • an increase in DC migration to the LN can result in fewer vaccinations to a patient because a greater number of the transplanted DCs reach their effective site (the LN) and the number of DCs wasted by remaining at ineffective, non-lymphoid sites (e.g., the dermis or subcutaneous tissue) is reduced. Moreover, fewer vaccinations can result in less patient discomfort from repeated injections and a reduction in the risk of adverse effects of vaccination.
  • the invention involves administering antigen-loaded DCs to animals that have undergone conditioning of one or more of the draining LNs (i.e., a LN that will receive an antigen) prior to the appearance of the antigen in the LN, or in animals that are experiencing an immune condition that results in LN enlargement.
  • the draining LNs i.e., a LN that will receive an antigen
  • Antigen presenting cells are a class of cells capable of presenting one or more antigens in the form of antigen-MHC complexes recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented.
  • DCs cytotoxic T lymphocytes
  • DC migration as used herein is understood to mean migration of a DC to lymphoid tissue, especially a LN, from an extra-lymphoid site (e.g., a peripheral skin injection site in the case of transplanted DCs).
  • Lymph node refers to one or more lymph nodes. Lymph node as used herein also encompasses lymphoid tissue such as lymph follicles and Peyer's patches.
  • Lymphadenopathy as used herein generally means one or more LN larger than one centimeter (cm), although this varies by lymphatic region.
  • LN enlargement is one result of lymph-conditioning.
  • "Lymph node enlargement” or "enlarged lymph nodes” means that the LN increases in size relative to its size before conditioning. This includes an increase in LN size over normal size as well as an additional increase in size in an already enlarged LN. Lymphadenopathy is one type of LN enlargement.
  • LN enlargement can be detected by palpation on physical examination, imaging studies (e.g., CT scan, MR imaging, ultrasonography), and/or direct measurement following LN biopsy or examination of LNs following surgical removal of lymph node-containing tissue.
  • LNadenopathy and enlarged lymph node also encompass hypercellular LNs.
  • "Hypercellular lymph node” as used herein means a lymph node that has a greater than normal number of cells as understood by one of ordinary skill in the art of microscopic examination of LNs, or a LN that has a greater-than-baseline number of cells (e.g., a lymph node known to be "hypocellular", which undergoes an increase in cell number so that it is either less hypocellular or normocellular).
  • Hypocellular means having a fewer than normal number of cells and “normocellular” means having a normal number of cells. LN hypercellularity can be detected by microscopic examination of biopsied (or otherwise removed) LNs. "Hypercellular” LNs re sometimes referred to as “congested” LNs, which have an increased volume of subcapsular space that is filled with cells. "Generalized lymphadenopathy” as used herein is lymphadenopathy found in two or more distinct anatomic regions. "Regional lymphadenopathy” as used herein is lymphadenopathy found in one distinct anatomic region.
  • Lymph-conditioning agent as used herein means an agent that, when administered to a patient, results in LN enlargement, lymphadenopathy and/or LN hypercellularity. Lymph-conditioning agents include any one or combination of: an adjuvant in mixture with an irrelevant antigen, a cytokine, and an irrelevant vaccine.
  • An "adjuvant” is a molecule or composition that potentiates the immune response to an antigen. Preferably, the adjuvant is pharmaceutically acceptable. Exemplary adjuvants are set forth below.
  • patient or “subject” as used herein refer to an animal having an immune system, preferably a mammal (e.g., rodent such as mouse). In particular, the terms refer to humans.
  • “Pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the lymph-conditioning agent may be administered.
  • vaccine refers to a composition (protein or vector) that can be used to elicit protective immunity or a therapeutic immune response (“immunotherapy") in a recipient.
  • immunotherapy refers to the process of administering a vaccine to elicit protective immunity or immunotherapy in a recipient.
  • a vaccine of the invention can elicit immunity in a portion of the population, as some individuals may fail to mount a robust or protective immune response, or, in some cases, any immune response. This inability may stem from the individual's genetic background or because of an immunodeficiency condition (either acquired or congenital) or immunosuppression (e.g., treatment with immunosuppressive drugs to prevent organ rejection or suppress an autoimmune.condition). Efficacy can be established in animal models.
  • immunotherapy refers to a treatment regimen based on activation of a pathogen-specific immune response.
  • a vaccination is one form of immunotherapy (e.g., charging DCs with an antigen, preferably with a stimulatory cytokine such as GM-CSF or Flt3 ligand ex vivo (followed by transplantation into the subject) or in vivo).
  • an antigen preferably with a stimulatory cytokine such as GM-CSF or Flt3 ligand ex vivo (followed by transplantation into the subject) or in vivo).
  • antigen means any substance that can be recognized by the immune system and upon such recognition will result in a specific immune response, generally resulting in the production of specific antibodies. While the term antigen can include whole bacteria (live or killed) or virus, immunogenic compositions often contain subunit antigens such as whole proteins or peptides. Subunit antigens can be derived from tumor cells or any type of infectious agent such as bacteria, viruses, parasites, and fungi.
  • an antigen will mean a single molecular entity, whether a protein or the nucleic acid coding for it.
  • the term "irrelevant antigen” means an antigen other than an antigen specific to the condition under treatment, e.g., a protein unrelated to a condition being treated.
  • KLH keyhole limpet hemocyanin
  • the irrelevant antigen is highly immunogenic, as with KLH.
  • gplOO is not an irrelevant antigen.
  • Irrelevant vaccine is used herein to mean a mixture of an irrelevant antigen with an appropriate adjuvant, wherein the irrelevant antigen/adjuvant mixture can be used to directly provide protective immunity or immunotherapy for a patient's condition other than the condition under treatment in the patient.
  • a given vaccine can be irrelevant or relevant, depending on the condition under treatment.
  • flu vaccine administered to a cancer patient to induce LN enlargement prior to tumor antigen-loaded DC injection is an irrelevant vaccine.
  • a flu vaccine administered to a patient with enlarged LNs and for the purpose of immunizing against influenza is a relevant vaccine.
  • Another example of an irrelevant vaccine for the treatment of cancer is pneumococcal vaccine.
  • KLH in complete Freund's adjuvant is not an irrelevant vaccine because it cannot be used to directly provide protective immunity or immunotherapy for a patient's condition (KLH in CFA is, however, a lymph-conditioning agent).
  • a "therapeutic vaccine” or “relevant vaccine” as used herein means an antigen that is directly related to treatment of the cancer or other condition under treatment (e.g., gplOO and an adjuvant for the treatment of malignant melanoma, flu vaccine for the treatment of flu).
  • antigen-loaded DC means a DC pulsed with an antigen and/or a dendritic cell transfected with a vector encoding an antigen.
  • vector and "expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell (e.g., dendritic cell), so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • Vectors include plasmids, phages, viruses, etc.
  • a "cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame.
  • foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA.
  • a segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct.”
  • a large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods known to those skilled in the relevant art.
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host and one or more expression cassettes. Vectors for expression in DCs are discussed in greater detail below.
  • An “endogenous DC” as used herein is a DC that has not been generated in vitro, ex vivo or transplanted.
  • a “transplanted DC” as used herein means a DC that has been introduced into a patient's body. Transplanted DCs include DCs (or their progenitor cells) from tissue culture lines or from a donor, and DCs (or their progenitor cells) that had been removed from the patient's body and cultured ex vivo for a period of time.
  • the term “treat” is used herein to mean to prevent, delay the onset, or treat a disease, or to relieve or alleviate the symptoms of a disease in a subject.
  • DC Preparation Improving the immune response elicited by antigen-loaded DCs is important to: (1) minimize the difficulty and cost of obtaining DCs, and antigen-loading the DCs; (2) shorten the time required for a satisfactory immune response by decreasing the number of immunizations required to deliver an adequate number of DCs to the LN; and (3) decrease patient discomfort and adverse effects related to repeat injections.
  • Vaccination can be accomplished through the targeting of DCs (Steinman, J. Lab. Clin. Med. 1996, 128:531; Steinman, Exp. Hematol. 1996, 24:859; Taite et al., Leukemia 1999, 13:653; Avigan, Blood Rev. 1999, 13:51; DiNicola et ⁇ /., Cytokines Cell. Mol. Ther. 1998, 4:265).
  • DCs play a crucial role in the activation of T-cell dependent immunity.
  • Proliferating DCs can be used to capture protein antigens in an immunogenic form in situ and then present these antigens in a form that can be recognized by, and that stimulates, T cells (see, e.g., Steinman, Exper. Hematol. 1996, 24:859-862; Inaba, et al., J. Exp. Med. 1998, 188:2163-73 and U.S. Pat. No. 5,851,756).
  • DCs can be plated in culture dishes and exposed to (pulsed with) antigen in a sufficient amount and for a sufficient period of time to allow the antigen to bind to the dendritic cells.
  • DCs may be transfected with DNA using a variety of physical or chemical methods as described (see, e.g., Zhong et al., Eur. J. Immunol. 1999, 29:964-72; Van Tendeloo, et al., Gene Ther. 1998, 5:700-7; Diebold et al, Hum. Gene Ther. 1999, 10:775-86; Francotte and Urbain, Proc. Natl. Acad. Sci. USA 1985, 82:8149; U.S. Pat. No. 5,891,432; and Casares et al, J. Exp. Med. 1997, 186:1481-6).
  • the pulsed cells are then be transplanted back to the subject undergoing treatment, e.g., by intravenous, subcutaneous or intradermal injection.
  • autologous DCs i.e., DCs obtained from the subject undergoing treatment
  • MHC-Class matched DCs which may be obtained from a type-matched donor or by genetic engineering of dendritic cells to express the desired MHC molecules (and preferably suppress expression of undesirable MHC molecules.
  • Various strategies are available for targeting DCs in vivo by taking advantage of receptors that mediate antigen presentation, such as DEC-205 (Swiggard et al, Cell. Immunol. 1995, 165:302-11; Steinman, Exp. Hematol.
  • DCs maybe induced to mature in vitro after infection by a vector, prior to transplantation in vivo.
  • host/expression vector combinations i.e., expression systems
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • coli plasmids col El, pCRl, pBR322, SV40 and pMal-C2, pET, pGEX (Smith, et al, Gene 1988, 67:31-40), pMB9 and their derivatives, plasmids such as RP4; gram positive vectors such as Strep, gardonii; phage DNAS, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2m plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • plasmids such
  • Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist and Chambon, Nature 1981, 290:304- 310,), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and control regions that exhibit hematopoietic tissue specificity, in particular: immunoglobin gene control region, which is active in lymphoid cells (Grosschedl et al, Cell 1984, 38:647; Adames et al, Nature 1985, 318:533; Alexander et al, Mol.
  • beta-globin gene control region which is active in myeloid cells (Mogram, et al, Nature 1985, 315:338-340; Kollias, et al, Cell 1986, 46:89-94), hematopoietic stem cell differentiation factor promoters; erythropoietin receptor promoter (Maouche, et al, Blood 1991, 15:2557); and control regions that exhibit mucosal epithelial cell specificity.
  • Vectors particularly useful for vaccination in vivo or ex vivo, are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vaccinia Ankara (MVA) and other recombinant viruses with desirable cellular tropism.
  • viral vectors such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vaccinia Ankara (MVA) and other recombinant viruses with desirable cellular tropism.
  • a vector encoding an immunogenic polypeptide can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA.
  • Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both.
  • Targeted gene delivery is described in international Patent Publication WO 95/28494, published October 1995.
  • Viral vectors commonly used for in vivo or ex vivo targeting and vaccination procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques 1992, 7:980-990).
  • the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell.
  • the replication defective virus is a minimal virus, i.e., it retains only the sequences of its genome which are necessary for encapsidating the genome to produce viral particles.
  • DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), vaccinia virus, and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • vaccinia virus and the like.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt, et al, Molec. Cell. Neurosci. 1991, 2:320-330; International Patent Publication No.
  • viral vectors commercially, including but by not means limited to Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc.
  • Avigen, Inc. Almeda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc.
  • Adenoviral vectors Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).
  • Adenovirus vectors Adenovirases are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types.
  • adenovirases of animal origin which can be used within the scope of the present invention include adenovirases of canine, bovine, murine (example: Mavl, Beard, et al, Virology 1990, 75:81), ovine, porcine, avian, and simian (example: S AV) origin.
  • the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800), for example).
  • replication defective adenovirus and minihium adenovirus vectors have been described (WO94/26914, WO95/02697, WO94/28938, WO94/28152, WO94/12649, WO95/02697 WO96/22378).
  • the replication defective recombinant adenovirases according to the invention can be prepared by any technique known to the person skilled in the art (Levrero, et al, Gene 1991:101:195; EP 185 573; Graham, EMBO J. 1984, 3:2917; Graham, et al, J. Gen. Virol. 1977,36:59).
  • Recombinant adenovirus is.an efficient and non-perturbing vector for human DCs (Zhong et al, Eur. J. Immunol. 1999, 29:964; DiNicola et al, Cancer Gene Ther. 1998, 5:350-6).
  • Recombinant adenovirases are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.
  • Adeno-associated viruses are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect.
  • the replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus).
  • a human helper virus for example an adenovirus
  • the AAV recombinants which are produced are then purified by standard techniques.
  • These viral vectors are also effective for gene transfer into human dendritic cells (DiNicola et al. , supra). Retrovirus vectors.
  • the gene can be introduced in a retroviral vector, e.g., as described in Anderson, et al, U.S. Patent No. 5,399,346; Maim, et al, Cell 1983, 33:153; U.S. Patent No. 4,650,764; U.S. Patent No. 4,980,289; Markowitz, et al, J. Virol. 1988, 62:1120; U.S. Patent No. 5,124,263; EP 453242, EP178220; Bernstein, et al Genet. Eng. 1985, 7:235; McCormick, BioTechnology 1985, 3:689; International Patent Publication No.
  • the retrovirases are integrating viruses which infect dividing cells.
  • the retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env).
  • the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest.
  • vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus” MSV ("murine Moloney sarcoma virus”), HaSV ("Harvey sarcoma virus”); SNV ("spleen necrosis virus”); RSV ("Rous sarcoma virus”) and Friend virus.
  • Suitable packaging cell lines have been described in the prior art, in particular the cell line PA317 (US 4,861,719); the PsiCRLP cell line (WO 90/02806) and the
  • the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender, et al, J. Virol. 1987, 61 : 1639).
  • Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.
  • Retrovirus vectors can also be introduced by DNA viruses, which permits one cycle of retroviral replication and amplifies tranfection efficiency (see WO 95/22617, WO 95/26411, WO 96/39036, WO 97/19182). Lentivirus vectors.
  • lentiviral vectors are can be used as agents for the direct delivery and sustained expression of a transgene in several tissue types, including brain, retina, muscle, liver and blood.
  • the vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the gene of interest.
  • Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lenti virus vectors for gene therapy.
  • An example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate viras particles at titers greater than 106 IU/ml for at least 3 to 4 days (Kafri, et al, J. Virol. 1999, 73: 576-584).
  • the vector produced by the inducible cell line can be concentrated as needed for efficiently transducing nondividing cells in vitro and in vivo.
  • Vaccinia viras is a member of the pox virus family and is characterized by its large size and complexity.
  • Vaccinia viras DNA is double-stranded and terminally crosslinked so that a single stranded circle is formed upon denaturation of the DNA.
  • the viras has been used for approximately 200 years in a vaccine against smallpox and the properties of the viras when used in a vaccine are known (Paoletti, Proc. Natl. Acad. Sci. U.S.A. 1996, 93:11349-53; and Elmer, Infection 1998, 26:263-9).
  • the risks of vaccination with vaccinia viras are well known and well defined and the viras is considered relatively benign.
  • Vaccinia virus vectors can be used for the insertion and expression of foreign genes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al., Proc. Natl. Acad. Sci. U.S.A. 1987, 84:7413-7417; Feigner and Ringold, Science 1989, 337:387-388; see Mackey, et al, Proc. Natl. Acad. Sci. U.S.A. 1988, 85:8027-8031; Ulmer, et al, Science 1993, 259:1745-1748).
  • Useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et al, supra).
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO96/25508), or a cationic polymer (e.g., international Patent Publication WO95/21931).
  • non- viral DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection; see, e.g., U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,853,663, U.S. Pat. No. 5,885,795, and U.S. Pat. No. 5,702,384 and see Sanford, TLB-TECH 1988, 6:299-302; Fynan et al, Proc. Natl. Acad. Sci. U.S.A.
  • the efficiency of antigen-loaded DC immunization is improved by administering a lymph-conditioning agent and transplanting the antigen-loaded DCs to the patient with conditioned lymph nodes.
  • Immunization according to this method is improved because DC migration to a conditioned LN is increased as compared to an un-conditioned LN.
  • the greater number of DCs in the LN result in increased antigen presentation to T-cells and, consequently, a more robust immune response to the antigen.
  • a patient undergoing treatment for a condition is administered a lymph node-conditioning agent
  • a lymph node-conditioning agent e.g., a gplOO-loaded DC
  • an antigen-loaded DC e.g., a gplOO-loaded DC
  • the lymph-conditioning agent is preferably administered by a health professional via cutaneous injection (e.g., subcutaneous or intrade ⁇ nal) in an extremity (an arm or a leg).
  • LN enlargement can be detected by physical examination (e.g., palpation of inguinal LNs), imaging study (e.g., CT scan, MR scan) and/or examination of an excised LN.
  • LN enlargement in the form of hypercellularity can be detected by microscopic examination of an excised LN.
  • An excised LN can be obtained from an excisional biopsy of one or more LNs or from part of a larger specimen (e.g., a patient is administered a lymph-conditioning agent prior to a surgical procedure for melanoma that includes removal of the draining LNs ("lymph node dissection") along with the melanotic skin lesion).
  • LN enlargement indicates that the LN is conditioned.
  • Injection sites for inducing LN enlargement and for administering antigen-loaded DCs are preferably different sites.
  • a patient undergoing treatment for a condition is administered a lymph node-conditioning agent, a time previously determined to be sufficient for lymph conditioning with that agent is allowed to pass, and an antigen-loaded DC is administered to the patient after the LN is conditioned.
  • the lymph-conditioning agent is preferably administered by a health professional via cutaneous injection (e.g., subcutaneous or intradermal) in an extremity (an arm or a leg). Determination of a time sufficient for lymph-conditioning is within the skill of a health professional in the practice of immunotherapy. This time period can vary, to a degree, according to the state of the patient's immune system (e.g., an immunocompromised patient can require more time for lymph node conditioning).
  • a time sufficient for LN conditioning following administration of a lymphnode conditioning agent is typically half-a-day (12 hours) to 7 days, preferably half-a-day to 5 days, and most preferably 2 to 5 days.
  • vaccination with antigen-loaded DCs into a site that drains to the conditioned LN will result in an improved immunization because the LN is conditioned and, therefore, a greater proportion of the antigen-loaded DCs will migrate to the lymph node.
  • vaccination should not occur greater than 10 days following the end of the time sufficient for LN conditioning. In a preferred embodiment, vaccination occurs not greater than two days following the end of the time sufficient for LN conditioning. More preferably, vaccination occurs not greater than one day following the end of the time sufficient for LN conditioning.
  • the timing for administration of anti-tumor immunotherapy with DCs is coordinated with routine prophylactic vaccination protocols.
  • cancer patients and other immunosuppressed patients routinely .receive vaccinations against infectious diseases such as influenza virus and pneumococcus because they are at particular risk from infection.
  • antigen-loaded DCs are administered during the period of lymph node conditioning.
  • the antigen-loaded DCs are administered after LN conditioning has been detected or after a period of time sufficient for the irrelevant vaccine to condition the LN.
  • immunosuppressed patients routinely receive annual flu vaccinations. Such a patient, who is also undergoing cancer treatment, receives a flu vaccination and 2-4 days later (flu vaccine can induce LN enlargement in 2-4 days) tumor-antigen-loaded DCs are administered to the patient.
  • the invention permits administration of antigen-loaded DCs after detecting lymph node enlargement in a patient from natural exposure to pathogens or other environmental stimuli.
  • lymphadenopathy e.g., pharyngitis, tuberculosis, cytomegalovirus (CMV)
  • a cancer patient awaiting antigen-loaded DC immunization can acquire such an illness with its attendant lymphadenopathy.
  • a physician would await resolution of the illness-related lymphadenopathy before administering a vaccine.
  • the antigen-loaded DCs are administered to the patient following detection of the illness-related lymphadenopathy, but not before resolution of the lymphadenopathy.
  • antigen-loaded DCs are administered to a patient following exposure to a bacterial pathogen. Such an exposure can occur, for example, naturally (e.g., strep throat, a bacterial urinary tract infection), iatrogenically (e.g., bacteremia during and/or following a dental procedure), or traumatically (e.g., bacteria in an open wound).
  • antigen-loaded DCs are administered to a patient following lymph node enlargement subsequent to bacterial exposure or after a time sufficient for a bacteria to condition a LN.
  • the patient is known to have palpable or enlarged lymph nodes (e.g., detected by physical exam or an imaging study such as CT scan) and a further increase in LN size is detected.
  • antigen-loaded dendritic cells are administered to the patient in whom LN size is increased.
  • Lymphadenopathy can be induced in more than one distinct anatomic region (e.g., right axillary lymph nodes and left cervical LNs), which constitutes generalized lymphadenopathy.
  • generalized lymphadenopathy is induced, one can administer the antigen-loaded DC vaccine in an anatomic region distinct from the injection site of the lymphadenopathy inducing agent.
  • an adjuvant in mixture with an irrelevant antigen injected into the skin of a patient's right arm causes generalized lymphadenopathy throughout the body.
  • the antigen-loaded DC can be injected into, for example, the skin of the patient's left leg because this region will drain into an enlarged LN.
  • Lymphadenopathy in one distinct anatomic region e.g., left inguinal LNs
  • the antigen-loaded DC vaccine is administered in the anatomic region that drains to the enlarged LN in that region.
  • an adjuvant in mixture with an irrelevant antigen is injected into the skin of a patient's right arm that causes regional lymphadenopathy in the right axilla.
  • a method for immunizing an animal to an antigen comprises conditioning a LN by administering an adjuvant in mixture with an irrelevant antigen.
  • the adjuvant is preferably pharmaceutically acceptable.
  • Preferred adjuvants are complete Freund's adjuvant (CFA) and bacille Calmette-Guerin (BCG).
  • Adjuvants for this purpose also include, among others, polyargmine, imiquimod, FTY720, attenuated Shigella (see Science 1995, 270:299), attenuated Salmonella (see Cell 1997, 91:765), attenuated Listeria monocytogenes, emulsion adjuvants (e.g., Ribi, MF59, SAF-1, saponin, polyphosphazine, PLGA), and MPLTM (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4 ⁇ 912,094. See generally, Liu, M.A., Nature Med. Vaccine Suppl. 1998, 4:515.
  • AGP aminoalkyl glucosamine phosphate compounds
  • Corixa Hamilton, MT
  • AGP aminoalkyl glucosamine phosphate compounds
  • One such AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy- 4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3- tetradecanoyoxytetradecanoylamino]-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529).
  • This 529 adjuvant is formulated as an aqueous form or as a stable emulsion.
  • Useful adjuvants also include oil and water emulsions, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, etc., Amphigen, Avridine, L121/squalene, D- lactide-polylactide/glycoside, muramyl dipeptide, killed Bordetella, saponins, such as Quil A or Stimulon® and QS-21 (Antigenics, New York, NY), described in U.S. Patent No.
  • coli heat-labile toxin particularly LT-K63, LT-R72, CT-S109, PT- K9/G129; see, e.g., PCT Publication Nos. WO 93/13302 and WO 92/19265.
  • TLRs Toll-like receptors
  • Immature bone marrow derived dendritic cells express a full set of toll- like receptors, which, upon recognition of their cognate ligand molecules (also known as Pathogen Associated Molecular Patterns (PAMP)), induce dendritic cell maturation and migration (Medzhitov R, Toll-Like Receptors And Innate Immunity, Nature Reviews Immunology 1, 135 -145 (2001)).
  • PAMP Pathogen Associated Molecular Patterns
  • Toll-Like Receptor binding molecules that may be used as adjuvants are: lipopolysaccharides (LPS), peptidoglycans, lipoteichoic acids, phosphatidyl cholines and phosphorylchone on microbial membranes, hpoproteins, lipopeptides, bacterial DNAs, viral single and double-stranded RNAs, unmethylated CpG- DNAs, mannans and terminal mannose residues, a variety of other bacterial and fungal cell wall components such as outer membrane proteins (OMPs) and outer surface proteins (OSPs) and plant alkaloids.
  • LPS lipopolysaccharides
  • peptidoglycans peptidoglycans
  • lipoteichoic acids phosphatidyl cholines and phosphorylchone on microbial membranes
  • hpoproteins lipopeptides
  • bacterial DNAs viral single and double-stranded RNAs
  • Examples disclosed elsewhere in this application include the 65kDa mannoprotein (MP65) from Candida albicans, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif (U.S. Patent No. 6,207,646), and anionic polymers.
  • CFA activates the TLR9 class of TLRs via CpG.
  • Suitable adjuvants include, but are not limited to: surface active substances (e.g., hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl- dioctadecylammonium bromide), methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g., aluminum phosphate, etc.
  • surface active substances e.g., hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl- diocta
  • a method for immunizing an animal to an antigen comprises conditioning a lymph node by administering a cytokine.
  • cytokine Especially preferred cytokines are lymphotoxin- ⁇ (LT ⁇ ) and lymphotoxiri- ⁇ (LT ⁇ ).
  • GM- CSF granulocyte-macrophage colony stimulating factor
  • IL- 12 cytokine interleukin- 12
  • Other cytokines include, but are not limited to, the interleukins 1-alpha, 1-beta, 2, 4, 5, 6, 7, 8, 13, 14, 15, 16, 17 and 18, the interferons-alpha, beta and gamma, granulocyte colony stimulating factor, and the tumor necrosis factors alpha and beta.
  • Antigens DCs can be loaded with a variety of antigens such as tumor-specific antigens, viral antigens, bacterial antigens, protozoan antigens, and fungal antigens.
  • antigens for use in the present invention include tumor-specific antigens (TSAs).
  • TSA tumor-specific antigens
  • a TSA is an antigen expressed exclusively or to a much greater degree by a tumor cell.
  • TSAs include ErbB receptors, Melan A (MARTI), gplOO, tyrosinase, TRP-l/gp 75, and TRP-2 (in melanoma; for additional examples, see also a list of antigens provided in Storkus and Zarour, Forum (Genova) 2000 Jul-Sep, 10(3):256-270); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV E6 and E7 proteins (in cervical cancer); HER3, HER4, Mucin (MUC-1) (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen (PSA) (in prostate cancer); carcinoembryonic antigen (CEA) (in colon, breast, and gastrointestinal cancers); CA125 (in ovarian cancer); CD20 (in B cell lymphoma); HER2/neu/c-erbB-2 (in breast cancer); PI A tumor antigen (e.g., as disclosed in MARTI),
  • Additional antigens for the invention include viral protein or peptide antigens such as, for example, those derived from influenza virus (e.g., surface glycoproteins hemagluttinin (HA) and neuraminidase (NA) or the nucleoprotein (NP) as described in Bodmer et al, Cell 1988, 52:253 and Tsuji et al, J. Virol. 1998, 72: 6907-6910 or NP CTL epitopes as described in Gould et al, J. Virol. 1991, 65:5401; Murata et al, Cell Immunol. 1996, 173:96-107 and PCT Application No.
  • influenza virus e.g., surface glycoproteins hemagluttinin (HA) and neuraminidase (NA) or the nucleoprotein (NP) as described in Bodmer et al, Cell 1988, 52:253 and Tsuji et al, J. Virol. 1998, 72:
  • immunodeficiency virus e.g., a simian immunodeficiency viras (SIV) antigen (e.g., SIV-env CTL epitope as disclosed in PCT Application No. WO 98/5.6919), or a human immunodeficiency virus antigen (HIV-1) such as gpl20 CTL epitopes as disclosed, e.g., in PCT Application No. WO 98/56919, gpl60, pl8 antigen (e.g., CD8+ T cell epitopes and gp41 CTL epitopes as disclosed in PCT Application No.
  • SIV simian immunodeficiency viras
  • HAV-1 human immunodeficiency virus antigen
  • herpes viras e.g., a glycoprotein, for instance, from feline herpes viras, equine herpes virus, bovine herpes virus, pseudorabies viras, canine herpes viras, herpes simplex viras (HSV, e.g., HSV tk, gB, gD), herpes zoster viras, Marek's Disease Virus, herpes viras of turkeys (HVT), cytomegalo viras (CMV), or Epstein-Barr virus); hepatitis C viras; human papilloma virus (HPV); human T cell leukemia viras (HTLV-1); bovine leukemia viras (e.g., gp51,30 envelope antigen); feline leukemia virus (FeLV) (e.g., FeLV envelope protein, a Newcastle Disease Virus (ND)
  • HSV herpe
  • Mycobacterium tuberculosis-specific e.g., Bacillus Calmette-Guerin (BCG) - 38kD protein; antigen 85 complex (see Klein et al, J. Infect. Dis. 2001, 183:928-34, see also a list of antigens in Klein and McAdam, Arch. Immunol. Ther. Exp. (Warsz.) 1999, 47:313-320), Listeria monocytogenes-specific (e.g., as disclosed in Finelli et al, Immunol. Res.
  • BCG Bacillus Calmette-Guerin
  • typhoid vaccine includes the entire bacterium, killed to make it harmless to the recipient of the vaccine. Attenuated organisms are nonpathogenic because they have either been genetically engineered or cultured to reduce their pathogenicity (i.e. BCG tuberculosis vaccine).
  • antigens for use in the invention include but are not limited to protozoan antigens such as those derived from Plasmodium sp., Toxoplasma sp., Pneumocystis carinii, Leishmania sp., Trypanosoma sp., and malaria-specific antigens, e.g., synthetic peptide antigens comprising at least one CD8+ T cell epitope of the malarial circumsporozoite (CS) protein.
  • Fungal antigens of the invention include, for example, those isolated from Candida (e.g., 65kDa mannoprotein (MP65) from Candida albicans), trichophyton, or ptyrosporum.
  • antigens can be derived from any animal or human pathogen or tumor.
  • DNA encoding pathogen-derived antigens of interest attention is directed to, e.g., U.S. Patent Nos. 4,722,848; 5,174,993; 5,338,683; 5,494,807; 5,503,834; 5,505,941; 5,514,375; 5,529,780; U.K. Patent No. GB 2 269 820 B; and PCT Publication Nos. WO 92/22641; WO 93/03145; WO 94/16716; WO 96/3941; PCT/US94/06652.
  • antigens derived from tumor viruses reference is also made to Molecular Biology of Tumor Viruses, RNA Tumor Viruses, Second Edition, Edited by Weiss et al, Cold Spring Harbor Laboratory Press, 1982.
  • additional antigens useful in the compositions of the invention see also Stedman's Medical Dictionary (24th edition, 1982).
  • mice C57BL/6, C3H/HeJ, and C3H/HeJ -gld/gld mice were obtained from Jackson Laboratory (Bar Harbor, Maine). The C57/BL6 mice received forward footpad injections of (1) 25 ⁇ l CFA; (2) 25 ⁇ l of KLH (2.5 mg/ml); or (3) 25 ⁇ l of KLH emulsified in CFA (CFA/KLH) (1:1, vohvol). A fourth group of C57/BL6 mice did not receive an injection. Migration analysis was performed four days after injection (this time was selected based on experiments showing maximum expansion of the draining LNs (brachial and axillary) after CFA/KLH injection at four days post-injection).
  • FITC sensitization and migration assay A solution of 8 mg/ml fluorescein (FITC) in acetone/dibutylphtalate (1:1, volume:volume) was prepared. Three days following the footpad injections, each mouse received two 25 ⁇ l skin injections (one on each side of the mouse) in areas that drain to axillary and brachial LNs. After 18 hours, when the maximum accumulation of FITC CD1 lc DCs (CD1 lc is a cell surface marker on mature DCs) was reached, LN suspensions were prepared as described in Robbiani, et al., Cell 2000, 103:757- 68.
  • the total number of cells was counted and the samples were analyzed by two-color flow cytometry by staining with PE-conjugated anti-CD l ie mAb.
  • the number of migrated DCs (FITC + CD1 lc + cells) per LN was calculated by multiplying the percentage of FITC + CD1 lc + cells by the total number of LN cells.
  • Bone marrow-derived DCs were prepared as described by Inaba, et al., J. Exp. Med. 1992, 176:1693-1702; except that subpopulations of bone marrow cells were not immunodepleted at the initiation of culture.
  • the DCs (5xl0 6 ) were pulsed with 0.002% (wt/vol) FITC-conjugated latex particles of 1 ⁇ m diameter (obtained from Polysciences) at day 6 of the culture and again at day 8.
  • FITC-conjugated latex particles 1 ⁇ m diameter (obtained from Polysciences)
  • each mouse received four skin injections (two in each side of the mouse, at dorsal sites that drain to brachial LNs) of lO ⁇ l of DCs resuspended in PBS at 20x10 6 cells/ml.
  • Migration of I-A b + cells carrying latex to draining LNs was assessed two days after injection. The mice were sacrificed and the draining LNs were collected.
  • TCR T-II T-cell receptor
  • OVA ovalbumin
  • the cells were labeled with 5 ⁇ M carboxyfluorescein-succinimydil-ester (CFSE) for 10 minutes at 37°C (Molecular Probes). The reaction was stopped with an excess of RPMI 10% FCS and the cells were washed twice in the same medium.
  • CFSE carboxyfluorescein-succinimydil-ester
  • C57/BL6 recipient mice that had been treated four days earlier with CFA/KLH or CFA alone or KLH alone received intravenous injections with lOxlO 6 CFSE labeled T cells.
  • 2x10 5 bone marrow-derived DCs that had been pulsed with 20 ⁇ g of OVA overnight were injected on both sides of each mouse at four sites of the dorsal skin that drain to the brachial LNs.
  • the draining brachial LNs were collected and cell suspensions were stained with anti-CD4-PE for assessment by flow cytometry.
  • OVA-specific CD4 + T cell proliferation was traced by following dilution of CFSE labeling.
  • CFA/KLH injection increased endogenous DC migration.
  • the mobilization of endogenous DCs was studied using an assay based on topical administration of FITC that results in the appearance CDllc + FITC + DCs in the draining LNs (Robbiani, et al., Cell 2000,103:757-68).
  • the migration of CDl lc + FITC + DCs in the draining LNs was determined in control mice and in mice injected with CFA/KLH.
  • the percentage of CDl lc + FITC + DCs was similar in both groups. Fig. lc.
  • the data also suggested that the cause of increased migration originated in the LN because no stimulus was administered to the periphery (e.g., no peripheral injection of an immune system stimulant) and the activated T cells that fail to apoptosize are concentrated in the LN.
  • Migration of transplanted DCs increased in CFA/KLH-injected mice. Bone marrow-derived DCs were labeled in vitro with FITC-conjugated particles, and then transplanted into un-injected mice or CFA/KLH-injected mice. The DCs were injected in a site distal (further removed form the draining LN) to the site of CFA/KLH injection.
  • CFSE-labeled TCR transgenic OT-II cells were transferred into syngenic mice that had been injected with CFA/KLH or that had not been injected. Bone marrow-derived DCs pulsed with OVA were injected into the bone marrow of the mice. Six days later, flow cytometry (CFSE + , CD4 + ) of draining LNs showed that T cells were efficiently activated (as determined by CFSE dilution) and had undergone up to seven divisions in both CFA/KLH-injected and un-injected mice. Figs. 4 a and b.
  • EXAMPLE 2 Bacteria Increase DC Migration 10 S. typhinirium bacteria were injected into the forward footpads of C57/BL6 mice three days prior to FITC injection. Control mice did not receive an injection. FITC sensitization and migration assays were performed as in Example 1. The number of cells per lymph node ( Figure 5a) and the number of migrated cells per LN ( Figure 5b) were about 3.5 fold greater in the S. typhinirium -injected mice relative to control mice. The differences between control and S. typhmirium-injected mice were statistically significant (p ⁇ 0.05). The number of T and B lymphocytes per draining LN in mice injected with S. typhinirium bacteria (Figure 5c) was significantly greater relative to mice that were not injected with bacteria. These experiments showed that S. typhinirium inoculation increases draining LN cellularity and endogenous DC migration to draining LNs.
  • EXAMPLE 3 B Lymphocytes Have a Significant Role in DC Migration
  • C57BL/6 and B lymphocyte deficient mice ( ⁇ MT) were treated with CFA/KLH or untreated in accordance with the protocol described in Example 1 above.
  • the total number of cells (T and B lymphocytes) per draining LN from control and CFA/KLH treated C57BL/6 mice was determined ( Figure 6a).
  • the total number of cells per draining LN in ⁇ MT mice that were untreated (“None") or CFA/KLH-treated ( Figure 6b), and the number of migrated DCs per draining LN in ⁇ MT mice that were untreated or CFA/KLH-treated ( Figure 6c) was determined. Results were combined from four experiments and expressed as means SD.
  • L-selectin is an adhesion molecule expressed on lymphocytes, which is essential for lymphocyte homing to lymphoid tissues such as LNs.
  • Wild-type mice that were treated with CFA/KLH or untreated received intra-peritoneal injection of L-selectin blocking mAb MEL- 14 or IgG (control) one day before CFA/KLH treatment. Cellularity was determined 4 days after CFA/KLH treatment.
  • the total number of cells per draining LN ( Figure 8a) and the number of migrated DCs per draining LN ( Figure 8b) were determined. The results were representative of two experiments and were expressed as means SD.
  • EXAMPLE 6 The Role of the LT ⁇ R Signaling Pathway in DC Migration Soluble fusion protein LT ⁇ R-Ig or IgG (control) was injected into the forward footpad of wild-type mice that were CFA/KLH treated or untreated one day prior to CFA/KLH treatment in order to investigate the effect of blocking the LT ⁇ R signaling pathway on CFA/KLH-induced LN hypertrophy and DC migration. LN cellularity determinations were made four days after CFA/KLH treatment. The total number of cells per draining LN (Figure 9a) and the number of migrated DCs per draining LN ( Figure 9b) were determined. Results were representative of two experiments and expressed as means SD.
  • EXAMPLE 7 The Role of LT ⁇ and L-Selectin Expression on B Lymphocytes for CFA/KLH-induced LN Hypertrophy and DC Migration Chimeric mice received bone marrow transplants (Tx) designed to remove LT ⁇ and L-selectin expression on B cells.
  • Recipient ⁇ MT mice were lethally irradiated and intravenously reconstituted with a mix of (1) 25% WT BM and 75% ⁇ MT BM (WT/ ⁇ MT); (2) 25% LT ⁇ KO BM and 75% ⁇ MT BM (LT ⁇ KO/ ⁇ MT) or (3) 25% L-selectin BM and 75% ⁇ MT BM (LselKO/ ⁇ MT).
  • the WT/ ⁇ MT mice Upon reconstitution, the WT/ ⁇ MT mice expressed both LT ⁇ and L-selectin; the LT ⁇ KO/ ⁇ MT mice expressed L-selectin but not LT ⁇ , and the LselKO/ ⁇ MT mice expressed LT ⁇ but not L-selectin.
  • Controls included non-transplanted WT and ⁇ MT mice (No Tx).
  • mice Six weeks after reconstitution, mice were treated with CFA/KLH. A control group of wild-type mice was untreated with CFA/KLH. Three days later, the mice received FITC epicutaneously. Eighteen hours after FITC application, cell counts were determined. Five animals were independently analyzed in each group.
  • Figure 10a depicts the total number of cells per draining LN
  • 10b depicts ' the number of migrated DCs per draining LN
  • 10c depicts the number of B lymphocytes per draining LN
  • lOd depicts the number of T lymphocytes per draining LN.
  • the LN cell counts of WT/ ⁇ MT mice were significantly greater (P ⁇ 0.01) relative to the LT ⁇ KO/ ⁇ MT and LselKO/ ⁇ MT mice.

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Abstract

L'invention concerne de manière générale des procédés permettant de renforcer l'immunisation. L'invention concerne en outre des procédés pour améliorer la migration de cellules dendritiques vers un ganglion lymphatique. L'invention concerne par ailleurs des procédés de conditionnement d'un ganglion lymphatique destinés à accroître l'efficacité de la migration de cellules dendritiques vers ce ganglion.
PCT/US2004/033526 2003-10-07 2004-10-07 Protocole ameliore pour renforcer l'immunisation par transfert adoptif de cellules dendritiques WO2005034991A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104984004A (zh) * 2015-06-25 2015-10-21 四川金堂海纳生物医药技术研究所 一种治疗全身淋巴结肿大的内服药物及其制备方法

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