WO2001062092A1

WO2001062092A1 - Formulations and methods for using the same to elicit an immune response

Info

Publication number: WO2001062092A1
Application number: PCT/US2001/005990
Authority: WO
Inventors: Takami Sato; Kenichiro Hasumi
Original assignee: Thomas Jefferson University
Priority date: 2000-02-25
Filing date: 2001-02-23
Publication date: 2001-08-30

Abstract

Concurrent administration of antigen-pulsed dendritic cells and activated T cells to a patient is disclosed. Such administration results in maturation of dendritic cells, which allows dendritic cells to enter the lymph system. Co-administration of antigen-pulsed dendritic cells and activated T cells induce bidirectional stimulation in each cell population. As a result, cytokine productions such as IFN-η and IL-12 are greatly enhanced, and a Th1 type of immunologic response specific against the antigens is induced. The methods therefore elicit an immune response in a patient, and find application in the treatment of cancer, immune diseases, and viral infections. Formulations comprising the dendritic cells and activated T cells of the present invention are also disclosed.

Description

FORMULATIONS AND METHODS FOR USING THE SAME TO ELICIT AN IMMUNE RESPONSE

FIELD OF THE INVENTION 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.

BACKGROUND INFORMATION

Antigen presenting cells (APCs) play a pivotal role in stimulating an immune response and as such are an important target of cancer vaccines. Dendritic cells (DCs) are potent APCs with the ability to acquire, process, and present antigens to the immune system in the context of major histocompatibility antigens (MHC). 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.

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. 5,849,589; more specifically, this patent discloses methods for differentiating monocytes into DCs by culturing monocytes with granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-α (TNF- ). U.S. Patent No. 5,994,126 discloses methods for producing proliferating cultures of DCs and further producing mature DCs from the proliferating DC precursors; GM-CSF alone or in combination with IL-1, IL-4, IL-13, granulocyte colony stimulating factor (G-CSF) or TNF is used in these methods. 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.

Use of 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. Celluzzi et al., discussed above, teach that MHC Class I presented peptide antigen pulsed onto DCs induces protective immunity to lethal challenge by a tumor transfected with the antigen gene. Celluzzi and Falo, J. Immun. 160:3081- 3085 (1998) report that short-term physical interaction of DCs and tumor cells, with or without cell fusion, results in rapid, efficient and stable DC-tumor cell association; immunization using irradiated DC-tumor cell conjugates induces tumor specific cytotoxic T cells and protects from lethal challenge. None of the above teach or suggest combining pulsed DCs with activated T cells, as claimed herein.

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.

To induce antigen-specific T cell activation and clonal expansion, two signals provided by APCs must be delivered to the surface of resting T lymphocytes. The first signal confers specificity to the immune response, and is mediated by the T cell receptor (TCR) following recognition of specific antigenic peptide presented in the MHC context. The second signal, termed 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.

One method of activating and proliferating T cells is taught, for example, in U.S. Patent No. 5,858,358. There, 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. In contrast, the present invention provides for the administration of both DCs and activated T cells to the patient. In addition, 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.

None of the above teach or suggest simultaneous use of antigen- pulsed DCs and activated T cells for immunotherapy. Accordingly, there remains a need for such methods which promote maturation of DCs and facilitate development of Thl type immune response in regional lymph nodes or injection sites.

SUMMARY OF THE INVENTION 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. 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. 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. As used herein, 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. 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.

There is evidence that immature DCs are not fully capable of migrating into the regional lymph nodes or stimulating antigen specific T cells. "Mature" DCs, in contrast, are believed to express a receptor that allows them to migrate into lymph nodes. Various maturation stimuli have been studied to make cultured DCs fully matured. CD40L is believed to be one of the most potent molecules to promote this maturation. Interaction of CD40L, expressed by activated T cells, with CD40 molecules on the surface of DCs induces IL-12 production by the DCs. The IL-12 produced by DCs in turn promotes interferon-γ production by T cells. IL-12 production also induces memory of immune response. It is therefore an aspect of the present invention to provide a method for inducing IL-12 and IFN-γ production in a patient.

Another aspect of the invention is to promote maturation of DCs and facilitate migration of DCs into the regional lymph nodes for tumor-specific antigen presentation. Yet another aspect of the invention is to provide a method for eliciting an immune response.

It is therefore another aspect of the invention to provide methods for treatment of cancer, immune diseases, and viral infections.

These and other aspects of the invention will be apparent based upon the following disclosure and dependent claims.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the level of IL-12 production in supernatants, determined according to Example 1.

Figure 2 shows the level of interferon-γ 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.

DETAILED DESCRIPTION OF THE INVENTION 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). Generally, the dendritic cells have been antigen pulsed by co-culture with an antigen source, and 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 (ATs) 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-γ by T cells. IFN-γ stimulates expression of CD40 on the surface of antigen presenting cells. Thus, activated DCs will produce cytokine that stimulates T cells; the T cells, in turn, will produce cytokine that will further stimulate DCs. These bidirectional positive interactions are important in eliciting an immune response and therefore are useful in the treatment of cancers, including but not limited to melanoma and renal cell carcinoma. 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. For example, 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. Furthermore, 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.

Any type of dendritic cell can be used according to the present invention. As noted above, 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. Preferably, 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. 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). For example, 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. Preferably, 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. For example, 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. In the present methods, 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, as those terms are used herein, 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" as the name implies, refers to a source of antigens suitable for use in pulsing DCs with antigen. For example, 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 10⁶ 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 10⁶ tumor cells (C.E.U.) should be used for one vaccine injection. If numbers of obtained tumor cells are less than 40 x 10⁶, then 500 μg 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. According to the present invention, 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

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. Upon stimulation with PHA, T cells will be selectively activated and expanded since PHA is a selective T cell activator. Thus, the same PBMC source can be used to generate both the DCs and T cells of the present invention, utilizing different culture conditions.

The term "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. Alternatively, 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. Calcium ionophore increases cytoplasmic free calcium level and induces gene-activation. PHA exerts its mitogenic activity in part by increasing the cytoplasmic calcium level. The use of 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.

According to the present invention, 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 μg (mcg)/ml for about three days and then with calcium ionophore at 1 μg (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-γ.

As noted above, a ratio of DC to AC between about 1:5 and 1:100 is preferred. Most preferred, particularly for stimulation of cytokines, is a DC: AT ratio of 1:40. The antigen-pulsed DCs and the ATs should be mixed together at the predetermined ratio. In one embodiment, the DC/ AT mixture is administered immediately, that is within less than about five minutes, to a patient. In another embodiment, 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.

Simultaneous use of the DCs and ATs according to the present invention allows for CD40L to be presented to DCs. After the interaction with ATs, DCs will be matured and become capable of migrating into lymph nodes. The CD40-CD40L interaction enables DC to produce IL-12, which, in turn, induces a Thl type immune response in the regional lymph node. As a result, an immune response, such as induction or enhancement of anti-tumor T cell response will be elicited. It is also possible that DCs that remain in the injected sites will stimulate antigen-specific T cells in the AT fraction.

An effective amount of each of the antigen-pulsed dendritic cells and the activated T cells should be used. As used herein, the term "effective amount" refers to that amount of each of the cells which will elicit the desired response in the patient being treated. For example, 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. It will be appreciated that 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. The determination of an effective amount is within the skill of one practicing in the art, and will generally be about 1 x 10⁵ to 5 x 10⁶ of DC and 1 x 10⁶ to 1 x 10⁸ 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 10⁶ (1 - 5 x 10⁶) DCs and 4 x 10⁷ (1 - 10 x 10⁷) ATs four times over a six-week period. Administration can be by any means known in the art; intradermal injection is preferred. In addition, 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 10⁵ and 5 x 10⁶ DCs and 1 x 10⁶ to 1 x 10⁸ ATs. The formulations can be utilized in the present methods.

EXAMPLES The following examples are intended to illustrate the invention, and should not be construed as limiting the invention in any way. The following procedures were used for the preparation of activated T cells and antigen pulsed dendritic cells for all of the examples. Preparation of Activated T Cells PBMC are prepared by rapidly thawing cryotubes containing 50 x 10⁶ PMBC in one ml of 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 10⁶ 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 cm² culture flasks (5 ml each).

To stimulate the PBMC with PHA, 40 ml of AIM-V medium and 500 μl 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 cm² culture flasks, each containing 5 ml of PBMC suspension, to yield a final PHA concentration of 10 μg/ml. The final concentration of PBMC is approximately 2 x lOVml. The flasks are gently swirled and kept in a 5% CO₂ incubator at 37°C. The flasks are cultured for three days. If the medium turns yellow at day 2, ten ml of AIM-V medium with 10 μg/ml of PHA is added to the flasks. To stimulate the PBMC, supernatant from each of the 75 cm² 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 cm² flasks. 25 μl of ionomycin solution is added to each flask to yield a final concentration of 1 μg per ml, and then mixed gently by swirling the flask. The flasks are incubated for three hours at 37°C in the 5% CO₂ 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 cm² 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 μl of AIM-V.

Preparation of DCs The procedure for obtaining PBMC cultures having a live cell member equal to or greater than 80 x 10⁶ is performed as described above. Cells are resuspended in 10 ml of AIM-V medium and added to 25 cm² cultured flasks, which are then incubated in a CO₂ incubator at 37°C for two hours. The flasks are gently swung and the floating cells are removed by pipette. DC culture medium which contains 800 U/ml of GM-CSF and 500 U/ml of IL-4 in AIM-V is added to all of the flasks, which are then returned to the CO₂ incubator at 37°C. Cells are cultured for seven days with 2.5 ml of DC culture medium added on day three and day six.

Tumor lysates are prepared from 40 x 10⁶ tumor cells excised from the patient by the freeze and thaw method. Tumor cells are suspended in AIM-V at 10⁷ 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 10⁶ C.E.U. or 500 μg lysates) at 100 μg/ml. The flasks are then incubated in CO₂ incubators at 37° C for two hours.

Following the two-hour incubation, cells from each flask are collected by vigorous pipetting. The cells are transferred to tubes, where they are washed with AIM-V medium, and eventually resuspended with AIM-V medium. The number of live cells is determined by counting the large cells as dendritic cells under the microscope (X 100); small cells are counted as lymphocytes. Based on the optimal AT: DC ratios obtained in the preparatory tests and the actual number of AT obtained, the number of DC for treatment is determined. In general, 1 - 3 x 10⁶ of DCs will be used for each treatment. The DCs are then ready for use in combination in the proper ratio with the activated T cells prepared as above.

Example 1 4 x 10⁴ of DCs suspended in AIM-V medium were prepared as above and co-cultured at 37°C in a CO₂ 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

96 well plate. Supernatants were collected at various time points as shown in Figure 2, and interferon-γ (IFN-γ) levels in the supernatants were measured by ELISA. As can be seen in Figure 2, there was no plateau in IFN-γ production in the wells that contain DC and AT during the 37-hour incubation period. In contrast, IFN-γ levels in the wells that contained AT without DC did not increase after three hours. This indicates that the DC/ AT combination induced INF-γ production.

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. As shown in Figure 3, IL-12 production by DC was completely blocked (greater than 90% blocking) by the anti-CD40L antibody at one μg/ml. In contrast, 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. This demonstrates that the anti-CD40L antibody will block IL-12 production. That the IL-12 production was almost completely blocked by anti-CD40L antibody indicates that CD40 and CD40L interaction is important in IL-12 production by DCs.

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 10⁵ 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. In different experiments, 5 x 10³ 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.

Before the surgery, the patient will undergo leukapheresis. The leukapheresis product will be processed to obtain PBMC. 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.

More specifically, on day -7, patients will be skin tested (DTH reaction) with: 1) inactivated tumor cells (1 x 10⁶ cells); 2) Plain Hanks (100 μl); 3) PPD (RT23 SSI) 2 T.U. and 4) 5 T.U. On day -5, approximately 48 hours after its placement, DTH reactions will be measured. On day 1, patients will receive a 600 μl 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. They will also have skin tests (DTH) to the following materials: 1) inactivated autologous melanoma cells (1 x 10⁶ cells); 2) DC pulsed with autologous melanoma cell lysate (2 x 10⁵ cells); 3) DC only (2 x 10⁵ cells); 4) AIM-V medium (100 μl); 5) Plain Hanks medium (100 μl); 6) PPD (RT23 SSI) 2 T.U. and 7) 5 T.U. On day 58, DTH reactions will be measured.

Every two months after the initiation of the six-week vaccine program, a complete clinical evaluation, CBC with differential, SMA-12 and CT/MRI will be performed. If progression of a metastasis is detected on the follow- up clinical evaluation, patients will be off the study and receive an alternative treatment. Patients who remain relapse-free or who have stable or regressing metastasis after three months of treatment will be given a second cycle of vaccine.

Whereas particular embodiments of this invention have been described above for purposes of illustration, it will be evident to those skilled in the art that numerous variations of the details of the present invention may be made without departing from the invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for eliciting an immune response in a patient comprising: mixing an effective amount of each of antigen-pulsed dendritic cells and activated T cells; and administering the dendritic cell/activated T cell mixture to a patient; wherein said antigen-pulsed dendritic cells have been prepared by incubating dendritic cell precursors with cytokines and co-culturing with an antigen source; and wherein the ratio of antigen-pulsed dendritic cells to activated T cells is between about 1:5 and.1:100.

2. A method of Claim 1, wherein said method results in stimulation of IL-12 production.

3. The method of Claim 1 , wherein said method results in stimulation of interferon production.

4. The method of Claim 1, wherein said method results in triggering a Thl immune response.

5. The method of Claim 1, wherein dendritic cells are derived from epidermal Langerhans cells, dermal dendritic cells, dendritic cells located in the lymph nodes and spleen, dendritic cells in the mterdigitating reticulum cells in lymphoid organs, dendritic cells derived from bone marrow, dendritic cells derived from peripheral blood, and dendritic cells derived from skin.

6. The method of Claim 5, wherein said dendritic cells are derived from peripheral blood mononuclear cells.

7. The method of Claim 1, wherein said dendritic cells are derived from peripheral blood mononuclear cells by co-culture of said peripheral blood mononuclear cells with GM-CSF and IL-4, and said T cells have been activated by sequential culture with PHA and calcium ionophore.

8. The method of Claim 1, wherein said antigen source is a tumor antigen source selected from the group consisting of whole tumor cells, tumor membranes, mRNA extracted from tumor cells, tumor cell lysates and synthesized tumor-related peptides or proteins.

9. The method of Claim 8, wherein said tumor antigen source is autologous.

10. The method of Claim 8, wherein said tumor antigen source is a tumor cell lysate.

11. The method of Claim 10, wherein said lysate is an autologous melanoma cell lysate.

12. The method of Claim 1, wherein said antigen source is a viral protein or peptide.

13. The method of Claim 1, wherein said T cells are derived from spleen, lymph nodes, and peripheral blood.

14. The method of Claim 13, wherein said T cells are derived from peripheral blood mononuclear cells.

15 The method of Claim 14, wherein said T cells are derived from an autologous source.

16. The method of Claim 1, wherein said ratio of dendritic cells activated T cells is about 1:40.

17. The method of Claim 1 , wherein said administration is local.

18. The method of Claim 17, wherein said patient has cancer and said administration is intratumoral.

19. The method of Claim 1 , wherein said dendritic cells and said activated T cells are administered less than five minutes after being combined.

20. The method of Claim 1, wherein said dendritic cells and said activated T cells are co-cultured prior to administration.

21. The method of Claim 1, wherein said effective amount of dendritic cells is between about 1 x 10⁵ and 5 x 10⁶ and said effective amount of activated T cells is between about 1 x 10⁶ and 1 x 10⁸.

22. A formulation comprising: antigen pulsed dendritic cells, prepared by incubation of dendritic cells precursors with cytokines and co-culture with an antigen source; and activated T cells, prepared by sequential co-culture with PHA and calcium ionophore; wherein the ratio of an antigen pulsed dendritic cells to activated T cells is between about 1:10 and 1:100.

23. The formulation of Claim 22, wherein the ratio of antigen pulsed dendritic cells to activated T cells is about 1:40.