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WO2001062092A1 - Formulations et procedes d'utilisation desdites formulations pour provoquer une reaction immunitaire - Google Patents

Formulations et procedes d'utilisation desdites formulations pour provoquer une reaction immunitaire Download PDF

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Publication number
WO2001062092A1
WO2001062092A1 PCT/US2001/005990 US0105990W WO0162092A1 WO 2001062092 A1 WO2001062092 A1 WO 2001062092A1 US 0105990 W US0105990 W US 0105990W WO 0162092 A1 WO0162092 A1 WO 0162092A1
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cells
dendritic cells
antigen
activated
tumor
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Takami Sato
Kenichiro Hasumi
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Thomas Jefferson University
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Thomas Jefferson University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K40/428—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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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

Definitions

  • the present invention is directed to methods for eliciting an immune response using antigen-pulsed dendritic cells in conjunction with activated T cells. These methods have broad application, for example, in increasing cytokine production, enhancing T cell differentiation, and treating cancer, immune diseases and infectious diseases. Formulations comprising antigen-pulsed dendritic cells and activated T cells are also within the scope of the present invention.
  • Antigen presenting cells play a pivotal role in stimulating an immune response and as such are an important target of cancer vaccines.
  • DCs Dendritic cells
  • MHC major histocompatibility antigens
  • Dendritic cells also have a potent array of costimulatory molecules, cytokines and cell adhesion molecules, and are believed to be critically involved in the initiation of primary immune responses, autoimmune diseases and graft rejection. Dendritic cells also induce a potent anti-tumor response.
  • Dendritic cells are found in all tissues and organs of the body. They have been primarily classified by their tissue location and include interdigitating reticulum cells in lymphoid organs, veiled cells in afferent lymph, blood dendritic cells in the circulation, Langerhans cells in the epidermis, and dermal dendritic cells in the dermis of the skin. Dendritic cells are also found in non-lymphoid organs such as the heart, lung, gut, and synovium. As used herein, the term dendritic cell refers to cells from any of these sources.
  • DCs Use and study of DCs in cancer vaccine technology has increased with the development of techniques to generate large numbers of DCs by culturing bone marrow or peripheral blood cells in the presence of one or more cytokines. Methods for obtaining DCs from bone marrow are taught in Celluzzi et al., J. Exp. Med. 183:283-287 (1996). DC can also be obtained from peripheral blood and skin. In addition, methods for differentiating monocytes into DCs are taught, for example, in U.S. Patent No.
  • 6,004,807 discloses methods for generating DCs by culturing CD34 + hematopoietic progenitor cells in the presence of TNF and IL-3 or with GM-CSF.
  • Other methods include culture with stem cell factor (SCF) or FLT3 ligand.
  • SCF stem cell factor
  • DCs in cancer vaccines have been reported, for example, by Nestle et al. , Nature Medicine 4:328-332 (1998), who used DCs generated in the presence of GM-CSF and IL-4 pulsed with either melanoma peptide or melanoma cell lysate and directly injected the DCs into an uninvolved lymph node.
  • Hsu et al. Nature Medicine 2:52-58 (1996) developed a custom made B-cell lymphoma vaccine using DC pulsed with tumor specific idiotype protein, which was given subcutaneously.
  • Cytotoxic T cells are a critical component in the defense against tumors and viral infections. CTLs specifically recognize peptides presented by MHC Class I molecules on the surface of cells, and kill cells that present the peptide. The T cell receptors on the surface of CTLs cannot recognize antigens directly. In contrast to antibodies, antigens on the MHC Class I must first be presented to the T cell receptors for activation to occur.
  • T cell receptor TCR
  • co-stimulation induces T cells to proliferate and become functional.
  • Co-stimulation is neither antigen-specific nor MHC restricted, and is thought to be provided by one or more distinct cell surface molecules expressed by APCs. Delivery to a T cell of an antigen-specific signal with a co-stimulatory signal leads to T cell activation, which can include both T cell proliferation and cytokine secretion. In contrast, delivery to a T cell of an antigen-specific signal in the absence of a co-stimulatory signal is thought to induce a state of unresponsiveness in the T cell, thereby inducing antigen- specific tolerance in the T cell.
  • T cells are activated with a first agent that stimulates TCR/CD3 complex-associated complex, the CD2 surface protein, or by directly stimulating receptor-coupled signaling pathways. Then an accessory molecule on the surface of the T cells, such as CD28 and CD9 is stimulated for proliferation of T cells.
  • U.S. Patent No. 5,846,827 also teaches a method for activating cytotoxic T cells in vitro.
  • the method comprises the steps of dissociating bound peptides from MHC Class I molecules on APCs; associating desired immunogenic peptides with the MHC Class I molecules; and incubating the APCs with the cytotoxic T cells in the presence of a growth factor.
  • Methods of specifically killing target cells in a human patient are also disclosed, the methods comprising administering a pharmaceutical composition comprising autologous T cells activated according to the disclosed method.
  • U.S. Patent No. 5,788,963 discloses incubating or culturing dendritic cells that have been exposed to prostate cancer antigen or specific antigenic peptide in vitro with primed or unprimed T cells to activate the relevant T cell responses in vitro; the activated T cells are then administered to a prostate cancer patient.
  • the present invention provides for the administration of both DCs and activated T cells to the patient.
  • T cells are activated according to the present methods through the receptor-couple pathway, and will stimulate DC cells more efficiently than the art-reported method.
  • the present invention is directed to methods of using dendritic cells (DCs) in combination with activated T cells to elicit an immune response in a patient.
  • DCs dendritic cells
  • the DCs are differentiated from monocytes, preferably in a GM-CSF/IL-4 solution, and then exposed to an antigen source.
  • the cells are washed repeatedly prior to use, so that only antigen that has become associated with the cell remains.
  • T cells are activated (ATs), preferably through sequential exposure to PHA and calcium ionophore.
  • DCs and ATs Prior to use, DCs and ATs are combined in a ratio of between 1:5 and 1:100; the combination is then injected into a patient.
  • the term "patient” refers to members of the animal kingdom, including but not limited to humans.
  • the DC/ AT combination can be injected locally, such as intradermally, subcutaneously or directly into a tumor, or systemically. Formulations comprising these DCs and ATs are also within the scope of the present invention.
  • the present invention utilizes the interaction between DCs and ATs, which is important not only to DC maturation, but also to develop immunologic memory in T lymphocytes.
  • One component of this interaction occurs through the CD40 receptor expressed on the DC, and CD40 ligand (CD40L) expressed by the AT.
  • CD40L CD40 ligand
  • the interaction between CD40 and CD40L results in the production of IL-12 by the DC, and the stimulation of a Thl type immunologic response by the AT specific against the antigens expressed by the DC.
  • the present methods are designed to facilitate and stimulate the DC/ AT interaction to elicit an immune response against a target tumor. These methods also have application against immune diseases and viral infections.
  • FIGURES Figure 1 shows the level of IL-12 production in supernatants, determined according to Example 1.
  • Figure 2 shows the level of interferon- ⁇ production in supernatants, determined according to Example.
  • Figure 3 shows that IL-12 production is blocked by anti-CD40L antibody, determined according to Example 3.
  • Figure 4 shows the CD83 expression on DCs after co-culture of DCs with ATs, determined according to Example 4.
  • Figure 5 shows the IL-12 production at various DC: AT ratios, determined according to Example 5.
  • the present invention is directed to methods for concurrently administering to a patient an effective amount of each of antigen-pulsed dendritic cells (DCs) and activated T cells (ATs).
  • DCs antigen-pulsed dendritic cells
  • ATs activated T cells
  • the dendritic cells have been antigen pulsed by co-culture with an antigen source
  • the T cells have been activated by co-culture with PHA followed by co-culture with calcium ionophore.
  • the ratio of DC: AT is preferably between about 1:5 and 1:100.
  • the present methods for using antigen-pulsed DCs and activated T cells will have numerous applications. In their broadest sense, the present methods elicit an immune response in a patient. It will be understood, therefore, that these methods are useful in treating a patient capable of mounting at least a minimal immune response.
  • the present methods result in maturation of antigen- pulsed DCs. Once mature, these DCs will upregulate costimulatory molecules on their surface and more efficiently stimulate T cells. They also express specific chemokine receptors such as CCR7 and readily migrate into regional lymph nodes; this provides for tumor-specific antigen presentation.
  • Using combinations of DCs and ATs according to the present invention promotes production of various cytokines. For example, the present methods result in enhanced production of IL- 12.
  • IL-12 triggers the development of Thl responses and induces production of IFN- ⁇ by T cells.
  • IFN- ⁇ stimulates expression of CD40 on the surface of antigen presenting cells.
  • activated DCs will produce cytokine that stimulates T cells; the T cells, in turn, will produce cytokine that will further stimulate DCs.
  • the phrase “elicit” or “eliciting an immune response” therefore includes but is not limited to such responses as stimulating cytokine production, including but not limited to interleukin and interferon, maturation of DCs, upregulation of costimulatory molecules by DCs, promoting an anti-tumor T cell response and triggering a Thl response.
  • Tumor antigen-pulsed DCs will become mature and express CD83 after they encounter CD40L-expressing activated T cells. The matured DCs will then stimulate T cells more efficiently, through various costimulatory molecules such as CD86. Production of IL-12 by DCs will shift the immune response from a Th2 or ThO type response to a Thl type response. As a result, development of tumor-specific T cell immune response are developed.
  • Such immune responses are also relevant in the treatment of infections and immune diseases.
  • patients with lepromatous type leprosy have a Th2 type immune response against Mycobacterium leprae.
  • DC and AT treatment with bacterial products according to the present methods would be useful in shifting the Th2 type response against the bacteria to a Thl type response.
  • the present methods could be applicable to immunization of AIDS patients. Since the number of CD4 cells in these patients significantly decreases, induction of anti-viral response by vaccination is usually difficult. Since the current methods supplement the helper effect of CD4 cells, DCs pulsed with HIV-related antigens injected with autologous or allogeneic activated T cells would promote a CTL immune response or antibody production against HIV virus.
  • dendritic cell can be used according to the present invention.
  • DCs are found throughout the body including epidermal Langerhans cells, dermal dendritic cells, dendritic cells located in the lymph nodes and spleen, dendritic cells in the interdigitating reticulum cells in lymphoid organs, dendritic cells obtained from bone marrow, and dendritic cells obtained from peripheral blood and skin.
  • Dendritic cells derived from peripheral blood mononucleate cells (PBMC) are preferred for the present invention.
  • PBMC peripheral blood mononucleate cells
  • autologous DCs are used in the present methods. Using a patient's own DCs provides several advantages. First of all, autologous DCs will present autologous antigens to autologous T cells.
  • autologous DCs The chance of having unknown blood-borne infections can be avoided by using autologous DCs. Furthermore, no foreign DNA will be injected when using autologous cells. Although foreign antigens from allogeneic tumor cells could be presented on MHC of autologous DCs to induce an anti-tumor response against autologous tumors, MHC-matched allogeneic DCs can also be used in the current methods.
  • DCs can be harvested from a patient, or a dendritic cell source, through any means known in the art; preferred is leukapherasis.
  • Leukapherasis involves continuously extracorporealizing blood from a donor using laminar flow properties to separate mononuclear cells from red cells and plasma. The unneeded red cells and plasma are returned to the patient during the leukapherasis procedure. With this technique, the mononuclear cells are selectively removed from many liters of a donor's blood over a several hour period without harming the donor.
  • DC sources are cultured to differentiate precursor cells into DCs with combinations of cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4).
  • cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4).
  • GM-CSF granulocyte/macrophage colony-stimulating factor
  • IL-4 interleukin 4
  • DCs can be obtained from peripheral blood mononuclear cells by culturing them with GM-CSF and IL-4. Induction and expansion of DCs should take place for approximately seven days with GM-CSF and IL-4. After seven days of culture, the DCs become positive for CD1 lc, CD54, CD86, CD40, MHC Class I and II surface antigens, but are low in CD83 and CD14.
  • GM-CSF has been found to promote the proliferation in vitro of precursor DCs and modulates the maturation and function of the DCs.
  • Cells are cultured in the presence of GM-CSF at a concentration sufficient to promote the survival and proliferation of DC precursors.
  • the dose depends on the amount of competition from other cells (especially macrophages and granulocytes) for the GM- CSF, or to the presence of GM-CSF inactivators in the cell population.
  • the cells are cultured in the presence of between about 1 and 1,000 U/ml of GM- CSF. The preferred amount will vary within this range depending on the source of the DCs.
  • GM-CSF at a concentration of between 400 to 800 U/ml has been found to be optimal for culturing proliferating human dendritic cells from blood.
  • a concentration of about 800 U/ml is preferably used with DCs obtained from PBMC.
  • Cells from bone marrow require higher concentrations of GM-CSF because of the presence of large numbers of proliferating granulocytes which compete for the available GM-CSF; doses between about 500 and 1,000 U/ml are therefore preferred for cultures of cells obtained from marrow.
  • IL-4 is used in co-culture with the GM-CSF to inhibit macrophage proliferation and/or maturation.
  • the IL-4 should be provided in an amount sufficient to promote the proliferation of dendritic cells while inhibiting the proliferation and/or maturation of macrophage precursor cells or macrophages.
  • a preferred range is 1 to 1000 U/ml, with about 500 U/ml being most preferred.
  • Antigen-pulsed or “antigen-activated” dendritic cells refer to DCs that have been pulsed with antigen and that express modified antigens for presentation to and activation of T cells.
  • Antigen- pulsed DCs are generally produced by methods standard in the art; basically these methods involve exposing the DCs to or co-culturing the DCs with an antigen source in vitro.
  • Antigen source refers to a source of antigens suitable for use in pulsing DCs with antigen.
  • the antigen source can supply tumor antigens ("tumor antigen source”); viral protein or peptides can also be used as antigen sources for DC co-culturing.
  • the antigen source can be autologous or allogenic. Autologous sources are preferred; this eliminates the need to identify and isolate particular antigens that may or may not work with a given patent.
  • Tumor antigen sources include but are not limited to whole tumor cells, tumor membranes, RNA extracted from tumor cells, tumor peptides obtained from the surface of tumor cells and tumor cell lysates.
  • Tumor-related protein or peptides, naked RNA or DNA that encode tumor-related antigens and vectors that carry tumor antigens can also be used. Any type of tumor cells can be used and should correspond with the type of cancer being treated. When using tumor cells they should first be inactivated, such as by irradiation or other means known in the art.
  • Lysates are prepared by repeatedly freezing and thawing tumor cells, or by hypotonic shock, so as to break up cell membranes and release cytoplasm and cell components; when using lysate, the cell nuclei should be removed by methods known in the art, since nuclei are immunosuppressive. Use of tumor cell lysate is preferred in the present methods; use of autologous tumor cells in the preparation of the lysate is even more preferred. Lysates can be made from 40 x 10 6 cells for one cycle (4 injections) of the DC/ AT treatment, if adequate numbers of tumor cells are available. Tumor lysates obtained from 10 x 10 6 tumor cells (C.E.U.) should be used for one vaccine injection. If numbers of obtained tumor cells are less than 40 x 10 6 , then 500 ⁇ g of lysates can be used for each treatment.
  • Dendritic cells in culture can be exposed to an antigen source in a sufficient amount and for a sufficient period of time to allow the antigen to be processed or bound to the DCs. Preferred methods are described in the Example section.
  • the amount of antigen source and the time necessary to achieve processing or binding of the antigen by the DC may be determined according to any conventional technique, such as an immunoassay. Typically, 2 hours will be sufficient, although both shorter and longer periods of co-culture may also be employed.
  • the DCs are then removed from the culture with the antigen source. DCs are washed repeatedly in a medium such as AIM-V solution, so that none of the antigen source remains. That is, only antigen which has been taken up by DCs will remain. The DCs are then ready for use in the present methods and formulations.
  • T cells include, but are not limited to, CD8 + and CD4 + T cells; any combination of T cells;
  • T cell capable of lysing target cells or providing effector or helper functions that can result in target cell death or enhancement of anti-target effector activity is within the scope of the present invention.
  • T cells can be obtained from any suitable source such as various lymphoid tissues including, but not limited to spleen, lymph nodes, peripheral blood, tumors, ascetic fluid, dermal biopsies and CNS fluids.
  • PBMC are again the preferred source. More preferably, autologous PBMC are used to generate T cells.
  • PHA is a selective T cell activator.
  • the same PBMC source can be used to generate both the DCs and T cells of the present invention, utilizing different culture conditions.
  • T cell activation is used herein to refer to a state in which a T cell response has been initiated or activated by a primary signal, which may or may not be due to interaction with a protein antigen. "Activated T cells” therefore refers to T cells in this state. A T cell is activated if it has received a primary signaling event that initiates an immune response by the T cell.
  • T cells can be activated in numerous ways known in the art. For example, T cell activation can be accomplished by stimulating the T cell TCR/CD3 complex or by stimulating of the CD2 surface protein; an anti-CD3 monoclonal antibody can be used to activate T cells through the TCR/CD3 complex.
  • a primary activation signal can be delivered to a T cell through use of a combination of a protein kinase C (PKC) activator, such as a phorbol ester.
  • PLC protein kinase C
  • Calcium ionophore increases cytoplasmic free calcium level and induces gene-activation.
  • PHA exerts its mitogenic activity in part by increasing the cytoplasmic calcium level.
  • These agents bypasses the TCR/CD3 complex but delivers a stimulatory signal to T cells.
  • These agents are also known to exert a synergistic effect on T cells to promote T cell activation, and can be used in the absence of antigen to deliver a primary activation signal.
  • T cells are preferably activated sequentially by co-culture first with PHA and then with calcium ionophore.
  • Calcium ionophore refers to agents that raise cytoplasmic calcium concentrations, and include, for example, ionomycin and ionophore A23187.
  • Preferred methods for preparing ATs are discussed in the examples, and generally include co-culturing of PBMC with PHA at 10 ⁇ g (mcg)/ml for about three days and then with calcium ionophore at 1 ⁇ g (mcg)/ml for about three hours. This process results in ATs that express CD40L and that are CD69 positive. The ATs are then ready for use in the present methods and formulations. These cells will produce large amounts of . IFN- ⁇ .
  • a ratio of DC to AC between about 1:5 and 1:100 is preferred.
  • the antigen-pulsed DCs and the ATs should be mixed together at the predetermined ratio.
  • the DC/ AT mixture is administered immediately, that is within less than about five minutes, to a patient.
  • the DCs and ATs are co-cultured prior to administration to the patient. Co-culture can be for any length of time in which at least some level of interaction between DCs and ATs is observed.
  • a preferred co-culture time is overnight, at least about six hours, more preferably about twelve hours. The mixture will then be administered to a patient.
  • an effective amount of each of the antigen-pulsed dendritic cells and the activated T cells should be used.
  • the term "effective amount" refers to that amount of each of the cells which will elicit the desired response in the patient being treated.
  • that response may be the amount needed to produce the desired cytokine concentrations, the amount needed to allow sufficient maturation and migration of DCs into regional lymph nodes, or the amount needed to establish T cell memory against a patient's own tumor.
  • the effective amount will vary based on numerous factors, including, but not limited to, the desired result, the size of the patient being treated, the illness being treated, the severity of the illness, the condition of the patient's immune system and other similar factors.
  • an effective amount is within the skill of one practicing in the art, and will generally be about 1 x 10 5 to 5 x 10 6 of DC and 1 x 10 6 to 1 x 10 8 of AT. Higher or lower doses can be used depending on the patient being treated. The number of treatments will also depend on the patient being treated, the illness being treated, the patient's response to treatments, and similar factors. Determination of these parameters are also within the skill of one practicing in the art.
  • a preferred embodiment contemplates administration of 1 x 10 6 (1 - 5 x 10 6 ) DCs and 4 x 10 7 (1 - 10 x 10 7 ) ATs four times over a six-week period. Administration can be by any means known in the art; intradermal injection is preferred.
  • injection can be local, such as directly into a tumor, or into the regional lymph node. It can be systemic, such as into the blood system or lymph system. Administration can be repeated as needed. It is contemplated that the present formulation will be used as a vaccine and that administration will be repeated every two weeks over a period of eight weeks.
  • Cryopreserved dendritic cells and/or activated T cells can be used by any means known in the art.
  • the present invention is also directed to a formulation comprising the antigen-pulsed dendritic cells and activated T cells.
  • the antigen-pulsed DCs and ATs are prepared as generally described above.
  • the two cell types can then be mixed together, preferably in a ratio of DC: AT of between about 1:10 and 1:100.
  • Preferred formulations comprise between about 1 x 10 5 and 5 x 10 6 DCs and 1 x 10 6 to 1 x 10 8 ATs.
  • the formulations can be utilized in the present methods.
  • PBMC Activated T Cells
  • PBMC Activated T Cells
  • Banking medium 2.5% human albumin, 10% DMSO, 2 mM L-glutamine in RPM11640, adjusted pH to 7.2 with NaOH.
  • the cells are thawed in a water bath with the tube being removed before the last ice crystal melts.
  • the DMSO is diluted with Thawing solution containing 0.1 % human albumin, 1 % EDTA in Plain Hanks (Gibco BRL) without phenol red, adjusted pH to 7.2 with NaOH.
  • the procedures are as follows: Add 0.05 ml and swirl for 30 seconds; add 0.1 ml and swirl for 30 seconds; add 0.2 ml and swirl for 30 seconds; and add 0.4 ml and swirl for 30 seconds.
  • the cell suspension is then transferred to a 15 ml conical tube. About 0.8 ml Thawing solution is added to the conical tube which is then swirled for 30 seconds.
  • the cells are allowed to sit at room temperature for five minutes after which time 10 ml of Thawing solution is added to each tube.
  • the tubes are then centrifuged at 250 g for seven minutes. The supernatants are aspirated and the cell pellets are resuspended with 40 ml Plain Hanks solution without phenol red. Cell suspensions are collected into one tube. Live cells are counted by the trypan blue exclusion method. In general, live PBMC equal to or greater than 200 x 10 6 are used for AT culture. The cells are then resuspended in 20 ml of AIM-V medium (Gibco BRL), and the suspension added to four 75 cm 2 culture flasks (5 ml each).
  • AIM-V medium Gibco BRL
  • AIM-V medium To stimulate the PBMC with PHA, 40 ml of AIM-V medium and 500 ⁇ l of PHA solution are mixed.
  • the PHA solution is made by dissolving 5 mg of PHA (Sigma) in 5 ml of AIM-V (1 mg/ml).
  • 20 ml of AIM-V medium with PHA is added to each of the 75 cm 2 culture flasks, each containing 5 ml of PBMC suspension, to yield a final PHA concentration of 10 ⁇ g/ml.
  • the final concentration of PBMC is approximately 2 x lOVml.
  • the flasks are gently swirled and kept in a 5% CO 2 incubator at 37°C. The flasks are cultured for three days.
  • the medium turns yellow at day 2
  • ten ml of AIM-V medium with 10 ⁇ g/ml of PHA is added to the flasks.
  • supernatant from each of the 75 cm 2 flasks is transferred to a 50 ml tube.
  • 10 ml of AIM-V is added to each flask.
  • the tubes are centrifuged at 250 g for seven minutes and the supernatants are aspirated.
  • the pellet in each tube is resuspended with 15 ml of AIM-V medium, and the suspension returned to the original 75 cm 2 flasks.
  • ionomycin solution 25 ⁇ l is added to each flask to yield a final concentration of 1 ⁇ g per ml, and then mixed gently by swirling the flask. The flasks are incubated for three hours at 37°C in the 5% CO 2 incubator.
  • Ionomycin solution is prepared by dissolving 1 mg of ionomycin (Sigma) and 1 ml of DMSO.
  • the AT product is prepared by transferring supernatant from each of the 75 cm 2 flasks to a 50 ml tube, and adding 10 ml of AIM-V medium to each flask. The attached cells from the surface of the flasks are harvested by frequent pipetting. The 50 ml tubes are then centrifuged at 250 g for seven minutes.
  • the supernatants are aspirated and the pellets are resuspended with 10 ml of AIM-V.
  • 30 ml AIM-V is added to the 50 ml tube and the tube centrifuged again at 250 g for seven minutes.
  • the supernatant is aspirated and 10 ml of AIM-V is added to all tubes.
  • the suspensions are then collected into one tube and centrifuged again at 250 g for seven minutes.
  • the cell pellets are resuspended in 40 ml of AIM-V medium and count.
  • the appropriate number of AT can then be transferred to be utilized in the appropriate ratio with dendritic cells.
  • the suspension for treatment is then centrifuged again at 250 g for seven minutes and the cell pellet is resuspended in 400 ⁇ l of AIM-V.
  • Tumor lysates are prepared from 40 x 10 6 tumor cells excised from the patient by the freeze and thaw method. Tumor cells are suspended in AIM-V at 10 7 cells/ml. One ml of tumor cell suspension is put into a cryotube. The cryotubes are frozen at -80° C for 30 minutes and then thawed in a 37° C water bath. The above procedure is repeated twice and then suspensions are centrifuged at 2500g for 10 minutes. The supernatants will be collected into a 15 ml conical tube and protein concentration is measured. On day 7, culture medium is collected from all flasks and centrifuged at 250g for three minutes.
  • Pellets of DC are resuspended in DC culture medium containing tumor lysates (10 x 10 6 C.E.U. or 500 ⁇ g lysates) at 100 ⁇ g/ml. The flasks are then incubated in CO 2 incubators at 37° C for two hours.
  • Example 1 4 x 10 4 of DCs suspended in AIM-V medium were prepared as above and co-cultured at 37°C in a CO 2 incubator with activated, CD40 ligand (CD40L) expressing T cells (AT), also prepared as described above, at a 1:40 ratio in a 96 well plate.
  • Supernatants were collected at various time points as indicated in Figure 1 and IL-12 levels were measured in the supernatants by ELISA. There was no plateau in IL-12 production in the wells that contained DC and AT during the 37- hour incubation. In contrast, IL-12 production by LPS-stimulated DC reached its plateau at 18 hours. No significant IL-12 production was observed in the wells which contained DC without AT. This indicates that the DC/ AT combination stimulated IL-12 production.
  • Example 2 DCs and ATs as prepared above were co-cultured at a 1 :40 ratio in a
  • Example 3 AT were preincubated with anti-CD40L antibody (CD 154) for thirty minutes prior to co-culture with DC.
  • IL-12 production by DC was measured after 18 hours of co-culture.
  • IL-12 production by DC was completely blocked (greater than 90% blocking) by the anti-CD40L antibody at one ⁇ g/ml.
  • isotype-matched control mouse Ig-G at two different concentrations did not suppress IL-2 production by DC.
  • Control DCs had no Ig-G, i.e. , no blocking antibody.
  • Example 4 DCs and ATs were co-cultured for 18 hours; Figure 4 demonstrates that CD83 (one of the maturation markers of DC) was upregulated after this time. This indicates that DCs were maturing in the presence of the AT. In contrast, DCs without co-culture with AT (Immature DCs) do not express CD83 on their surface.
  • Example 5 2 x 10 5 of ATs were co-cultured with various numbers of DCs in a 96 well plate in DC: AT ratios varying between 1:5 and 1:100. Supernatants were collected after 18 hours of incubation and IL-12 levels were measured in the supernatants by ELISA. Significant IL-12 production was observed at DC: AT ratios between 1:5 and 1:100; IL-12 production was maximum of a ratio of 1:40.
  • 5 x 10 3 of DCs were co-cultured with various numbers of AT in a 96 well plate in ratios varying between 1 : 1 and 1 : 80. The production of IL-12 was also maximum at DC: AT ratio of 1:40 (results not shown).
  • Example 6 A proposed human protocol for the methods of the present invention is as follows: Patients with metastatic melanoma will undergo surgical resection of one or more metastasus. Tumor tissue will be processed to obtain tumor cells; tumor cells will be lysed by the freeze and thaw method and tumor lysates will be made.
  • PBMC will be cryopreserved and stored in liquid nitrogen.
  • DC and AT will be obtained from cryopreserved PBMC prior to treatment.
  • Patients will receive a four-course of vaccines every two weeks (day 1, day 15, day 29 and day 43) over a period of six weeks.
  • the vaccine program will be started after the patient has recovered from surgery, but no more than twelve weeks after surgery.
  • Patients will be tested for DTH response against tumor cells pre- and post-vaccination. Toxicity from the treatment will be assessed every two weeks while receiving the vaccine and three months thereafter.
  • DTH reaction patients will be skin tested (DTH reaction) with: 1) inactivated tumor cells (1 x 10 6 cells); 2) Plain Hanks (100 ⁇ l); 3) PPD (RT23 SSI) 2 T.U. and 4) 5 T.U.
  • DTH reactions will be measured.
  • patients will receive a 600 ⁇ l DC and AT vaccine divided in six interdermal injections. Vaccinations will be repeated every two weeks as noted above, for a total of four doses during a six- week period.
  • Each vaccine will consist of DC pulsed with mixed autologous melanoma cell lysates and AT;DC will be stimulated with lysates before mixing with ATs.
  • All vaccines will be injected into a proximal extremity (arms and thighs). If an axiliary or inguinal lymph node dissection has been performed, that extremity will not be used as a site of vaccination. On day 57, exactly two weeks after the fourth vaccine, patients will undergo a complete clinical evaluation, CBC with differential, SMA-12 and CT/MRI.
  • DTH skin tests

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Abstract

L'invention se rapporte à l'administration simultanée à un patient de cellules dendritiques à impulsion antigénique et de lymphocytes T activés. Cette administration génère une maturation des cellules dendritiques, qui permet aux cellules dendritiques de pénétrer dans le système lymphatique. L'administration simultanée des cellules dendritiques à impulsion antigénique et des lymphocytes T activés induit une stimulation bidirectionnelle dans chaque population cellulaire. En conséquence, la production de cytokines telles que IFN-η et IL-12 est considérablement accrue, et une réponse immunologique de type Th1 dirigée spécifiquement contre les antigènes est induite. Lesdits procédés permettent ainsi de provoquer une réponse immunitaire chez un patient, et trouvent leur application dans le traitement du cancer, des maladies immunitaires et des infections virales. L'invention se rapporte également à des formulations comportant les cellules dendritiques et les lymphocytes T activés décrits ci-dessus.
PCT/US2001/005990 2000-02-25 2001-02-23 Formulations et procedes d'utilisation desdites formulations pour provoquer une reaction immunitaire Ceased WO2001062092A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004056397A1 (fr) * 2002-05-29 2004-07-08 Demao Yang Stimulation de l'hematopoiese au moyen de cellules immunitaires activees ex vivo
US7332158B2 (en) 2002-05-29 2008-02-19 Demao Yang Compositions and treatments for myelosuppression by ex vivo activated immune cells
KR20140041517A (ko) * 2011-05-03 2014-04-04 이뮤노베이티브 테라피스, 엘티디. 면역치료를 사용하는 il-12의 유도
US12458249B2 (en) 2018-03-29 2025-11-04 Demao Yang Cell therapy for patients

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US5871728A (en) * 1995-03-31 1999-02-16 University Of Pittsburgh Method of regulating dendritic cell maturation
US5994126A (en) * 1992-04-01 1999-11-30 The Rockefeller University Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens
US6017527A (en) * 1996-07-10 2000-01-25 Immunex Corporation Activated dendritic cells and methods for their activation
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules

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US5994126A (en) * 1992-04-01 1999-11-30 The Rockefeller University Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US5871728A (en) * 1995-03-31 1999-02-16 University Of Pittsburgh Method of regulating dendritic cell maturation
US6017527A (en) * 1996-07-10 2000-01-25 Immunex Corporation Activated dendritic cells and methods for their activation

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004056397A1 (fr) * 2002-05-29 2004-07-08 Demao Yang Stimulation de l'hematopoiese au moyen de cellules immunitaires activees ex vivo
US7048922B2 (en) 2002-05-29 2006-05-23 Demao Yang Stimulation of hematopoiesis by ex vivo activated immune cells
US7332158B2 (en) 2002-05-29 2008-02-19 Demao Yang Compositions and treatments for myelosuppression by ex vivo activated immune cells
EP2011863A1 (fr) * 2002-05-29 2009-01-07 Demao Yang Stimulation de l'hématopoïèse par des cellules immunitaires activées ex vivo
US7758857B2 (en) 2002-05-29 2010-07-20 Demao Yang Stimulation of hematopoiesis by ex vivo activated immune cells
KR20140041517A (ko) * 2011-05-03 2014-04-04 이뮤노베이티브 테라피스, 엘티디. 면역치료를 사용하는 il-12의 유도
EP2704732A4 (fr) * 2011-05-03 2015-02-25 Immunovative Therapies Ltd Induction d'il-12 par immunothérapie
US9233156B2 (en) 2011-05-03 2016-01-12 Immunovative Therapies Ltd. Induction of IL-12 using immunotherapy
KR102121492B1 (ko) * 2011-05-03 2020-06-11 이뮤노베이티브 테라피스, 엘티디. 면역치료를 사용하는 il-12의 유도
US11045541B2 (en) 2011-05-03 2021-06-29 Mirror Biologics, Inc. Allogeneic T-cell compositions for induction of IL-12
US11883490B2 (en) 2011-05-03 2024-01-30 Mirror Biologics, Inc. Induction of IL-12 using immunotherapy
US12458249B2 (en) 2018-03-29 2025-11-04 Demao Yang Cell therapy for patients

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