HK1128161A - Compositions and methods for inducing the activation of immature monocytic dendritic cells - Google Patents

Description

Compositions and methods for inducing activation of dendritic cells of immature monocytes

Related applications

Priority of united states provisional application 60/748,885, filed on 8/12/2005, is hereby incorporated by reference.

Background

Antigen Presenting Cells (APCs) are important in eliciting an effective immune response. They not only present antigen to T cells with antigen-specific T cell receptors, but also provide the signals necessary for T cell activation. These signals have not been fully determined, but involve many cell surface molecules and cytokines or growth factors. The factors necessary for activation and polarization of naive T cells may be different from those required for reactivation of memory T cells. The ability of an APC to provide antigen and deliver a signal to a T cell is commonly referred to as helper cell function (APC). Although monocytes and B cells have been shown to be competent (component) APCs, their in vitro antigen presenting capacity appears to be limited to the reactivation of previously primed T cells. Thus, monocytes and B cells are not able to directly functionally activate naive or unprimed T cell populations. They also fail to deliver a signal that polarizes the immune response induced or when it is induced.

Dendritic Cells (DCs) are professional antigen presenting cells of the immune system that are thought to activate naive and memory T cells. Dendritic cells are increasingly prepared ex vivo for use in immunotherapy, particularly of cancer. The preparation of dendritic cells with optimal immunostimulatory properties requires understanding and exploiting the biological knowledge of these cells for ex vivo culture. Various protocols for these cell cultures have been described, with different advantageous aspects ascribed to each protocol. Recent protocols include the use of serum-free media, the use of maturation conditions that confer desirable immunostimulatory properties to the cultured cells.

Activation of dendritic cells initiates the process of converting immature DCs (which are phenotypically similar to skin langerhans cells) into mature antigen presenting cells that can migrate to lymph nodes. This process results in an asymptotic and progressive loss of the strong antigen uptake capacity that characterizes immature dendritic cells and in an upregulation of the expression of costimulatory cell surface molecules and various cytokines. Different stimuli can initiate maturation of DCs. This process is very complex and can take at least 48 hours to complete in vitro. One other consequence of maturation is the alteration of the migratory properties of the cells in vivo. For example, maturation induces several chemokine receptors, including CCR7, which direct cells to the T cell region of the draining lymph node where mature DCs activate T cells that are resistant to antigens in the context of class I and class II MHC molecules presented on the surface of DCs. The terms "activate" and "mature" and "activated" and "mature" describe the induction and completion of a transition from immature DCs (partially characterized by the ability to take up antigen) to mature DCs (partially characterized by the ability to effectively stimulate a T cell response from the newly formation). The terms are generally used interchangeably in the art.

Known maturation protocols are based on the in vivo environment that DCs are believed to encounter during or after exposure to antigens. The best example of this method is the use of Monocyte Conditioned Medium (MCM) as cell culture medium. MCM is produced in vitro by culturing monocytes and used as a source of maturation factors. (see, e.g., U.S. 2002/0160430, incorporated herein by reference). The major components of MCM responsible for maturation are reported to be the (pro) inflammatory cytokines interleukin 1 β (IL-1 β), interleukin 6(IL-6) and tumor necrosis factor α (TNF α).

Maturation of DCs can therefore be triggered by a number of different factors that are involved in the signal transduction pathway through the host. As a result, there is no single maturation pathway or outcome, but in fact there are many mature DC strategies, each with their own unique functional characteristics. Conceptually this is the reason, since the various threats to the body to which the immune system must react are manifold, and therefore require different attack strategies. For example, when bacterial infection is sufficiently cleared by activated macrophages supplemented with specific antibodies, viral infection is sufficiently attacked by cytotoxic T cells that effectively kill the virus-infected cells. Killing of cancer cells typically involves the binding of cytotoxic T cells, natural killer cells and antibodies.

In vitro maturation of DCs can therefore be designed to induce the immune system to favor one type of immune response over another, i.e., a polarized immune response. Directed maturation of DCs describes the concept that the outcome of the maturation process dictates the type of immune response that ensues from treatment with mature DCs. In its simplest form, directed maturation results in the generation of DC populations that produce cytokines that direct T cell responses that are polarized to either Th 1-type or Th 2-type responses. DCs express up To 9 different To 11-like receptors (TLR1 To TLR9), each of which can be used To trigger maturation. Not surprisingly, the interaction of bacterial products with TLR2 and TLR4 resulted in directed maturation of DCs, leading to a polarized response that was best suited for the treatment of bacterial infections. Conversely, maturation triggered by TLR7 or TLR9 appears to be more likely to result in an antiviral type response. As another example, addition of interferon gamma (IFN- γ) to the most mature protocol results in the production of interleukin 12 by mature DCs, which dictates a Th1 type response. Conversely, the addition of prostaglandin E2 has the opposite effect.

Factors that may be used for directed maturation of activated DCs may thus include, for example, interleukin 1 beta (IL-1 beta), interleukin 6(IL-6), and tumor necrosis factor alpha (TNF α). Other maturation factors include prostaglandin E2(PGE2), poly-dIdC, vasointestinal peptide (VIP), bacterial Lipopolysaccharide (LPS) and mycobacterial or mycobacterial components such as specific cell wall components. Other maturation factors include, for example, imidazoquinoline compounds, such as imidazoquinoline-4-amine compounds, e.g., 4-amino-2-ethoxymethyl- α, α -dimethyl-1H-imidazo [4, 5-c ] quinoline-1-ethanol (designated R848) or 1- (2-methylpropyl) -1H-imidazo [4, 5-c ] quinolin-4-amine, and their derivatives (WO 00/47719, incorporated herein by reference in its entirety), synthetic double stranded polynucleotides such as poly [ I ]: poly [ C (12) U ], etc., agonists of To 11-like receptors (TLRs) such as TLR3, TLR4, TLR7 and/or TLR9, sequences of nucleic acids comprising mature unmethylated CpG motifs known To induce DCs, etc. In addition, any combination of the above agents can be used to induce maturation of dendritic precursor cells.

Fully mature dendritic cells differ qualitatively and quantitatively from immature DCs. Once fully mature, DCs express higher levels of MHC class I and class II antigens, as well as higher levels of T cell costimulatory molecules, i.e., CD80 and CD 86. These changes increase the ability of dendritic cells to activate T cells because they increase the antigen density on the cell surface and the intensity of the T cell activation signal by the counterparts (counterparts) of costimulatory molecules on T cells such as CD28 and the like. In addition, mature DCs produce large amounts of cytokines that stimulate and polarize T cell responses.

Generally, methods for ex vivo DC generation include obtaining a cell population enriched for DC precursor cells from a patient, and then differentiating the DC precursor cells into mature DCs in vitro prior to introduction back to the patient. Some believe that DCs must be fully differentiated, or they will dedifferentiate back into monocytes/macrophages and lose much of their immunopotentiating capacity. Ex vivo maturation of DCs generated from monocytes has been successfully achieved using the methods and reagents listed above.

Generally, to produce immature Dendritic Cells (DCs), one must first purify or enrich monocytic precursors from other contaminating cell types. Such purification or enrichment is typically performed by attaching monocyte precursors to the surface of plastic (polystyrene) because monocytes have a greater propensity for attachment to plastic than other cells found in, for example, peripheral blood, such as lymphocytes and Natural Killer (NK) cells. After substantially removing contaminating cells by vigorous washing, monocytes are cultured with cytokines that convert the monocyte precursors to immature DCs or directly to mature DCs. Methods for differentiating monocyte precursor cells from immature DCs were first described by Sallusto and Lanzavecchia (J.Exp.Med., 179: 1109) -1118, 1994, which is incorporated herein by reference), which use the cytokines GM-CSF and IL-4 to induce the differentiation of monocytes into immature DCs. Although this combination of cytokines is most commonly used, various other combinations have been described to achieve the same goal, such as the replacement of IL-4 with IL-13 or IL-15. The end result of this process is a "veil" cell that expresses T cell costimulatory molecules as well as high levels of molecules of the Major Histocompatibility Complex (MHC), but does not express the dendritic cell maturation marker CD 83. These cells are similar to langerhans cells in the skin, and their primary physiological function is to capture invading microorganisms.

Variations of this method include different methods of purifying monocytes, including, for example, Tangential Flow Filtration (TFF), or by binding antibodies attached to beads to surface molecules on monocytes. The beads with bound cells are then concentrated in a column or on a magnetic surface so that contaminating cells can be washed away after the monocytes are eluted from the beads. In another method of obtaining dendritic cell precursors, cells expressing the stem cell marker CD34 from blood (U.S. patent No. 5,994,126, incorporated herein by reference) or from bone marrow are purified. These cells can be cultured with the essential cytokine GM-CSF to differentiate into mature DCs. These DCs appear to have very similar characteristics and functional properties to immature DCs generated from monocytes.

Immature DCs have a high capacity to take up and process antigens, but have a limited ability to initiate immune responses. The ability to elicit an immune response is obtained by maturation of immature DCs. This maturation is also known as activation of DCs or activation of DCs. The maturation process is initiated by contact with maturation-inducing cytokines, bacterial products, or viral components, etc., as indicated above.

Although these methods are capable of producing mature DCs, there are disadvantages to using recombinant molecules and cell supernatants for DC maturation. These aspects include lot-to-lot quality and yield inconsistencies of these reagents and the introduction of large quantities of foreign proteins that can compete with the antigen of interest for transport into monocytic dendritic cell precursors for processing. The foreign protein may also be toxic or cause autoimmunity if administered to a patient. Such agents can also be expensive to produce, making immunotherapy prohibitively expensive.

Summary of The Invention

The presently described methods and compositions provide for the induction of activation of immature Dendritic Cells (DCs), and the priming of these cells for antigen-specific immune responses. In one aspect, methods are provided for producing a cell population enriched for activated dendritic cells by contacting immature dendritic cells with a tissue culture substrate and a culture medium under culture conditions suitable for dendritic cell attachment to the tissue culture substrate and for in vitro culture of the immature dendritic cells for a time sufficient to form a cell population enriched for activated dendritic cells. In the method, it is not necessary to add a dendritic cell maturation agent to induce activation of the dendritic cell population. The immature dendritic cells can be contacted with a predetermined antigen prior to, during, or after contacting the immature dendritic cells with the tissue culture surface. The predetermined antigen can be, for example, a tumor-specific antigen, a tumor-associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide antigen (e.g., a synthetic peptide antigen), or an isolated antigen. Furthermore, the antigen may be a soluble antigen or a particulate antigen. In particular embodiments, the antigen may be a cell lysate or a membrane preparation derived from a patient's tumor or tumor cell line.

In certain embodiments, the method can also optionally include obtaining a cell population enriched for monocytic dendritic cell precursors; and culturing the precursor in the presence of an agent that induces differentiation of the monocytic dendritic cell precursor cells to form a population of mature dendritic cells. Suitable dendritic cell differentiation agents include, for example, GM-CSF, interleukin 4, a mixture of GM-CSF and interleukin 4, or a mixture of GM-CSF and interleukin 13 or interleukin 15. Monocytic dendritic cell precursors can be obtained as cells isolated from a human subject.

In another aspect, methods are provided for producing a cell population enriched for activated dendritic cells. The methods generally include providing a cell population enriched for immature dendritic cells; and contacting the immature dendritic cells with a tissue culture substrate having a dendritic cell differentiation inducing agent and a culture medium under culture conditions suitable for attachment of the dendritic cells to the tissue culture substrate and for in vitro culture of the immature dendritic cells. The cells are cultured for a time sufficient to form a cell population enriched for activated mature dendritic cells. When administered to a mammal, the resulting activated mature dendritic cell population produces an antigen-specific immune response in the mammal. The immature dendritic cell population can be contacted with a predetermined antigen prior to, during, or after contact with the tissue culture substrate under conditions suitable for attachment. The predetermined antigen can be, for example, a tumor-specific antigen, a tumor-associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide antigen (i.e., a synthetic peptide), or an isolated antigen. The antigen may be a soluble antigen or a particulate antigen. In particular embodiments, the antigen is a cell lysate or membrane preparation derived from a patient's tumor or tumor cell line. Agents that direct the response of the maturation propensity of DCs to respond to Th1 and/or Th2, such as interferon gamma (IFN γ), may also be added.

In certain embodiments, the method may also optionally comprise obtaining monocytic dendritic cell precursors; and culturing the precursor in vitro in the presence of a differentiating agent, thereby forming the immature dendritic cells. Suitable differentiating agents include, for example, GM-CSF, interleukin 4, interleukin 13, interleukin 15, or mixtures thereof. Monocytic dendritic cell precursors can be isolated from a human subject in need of treatment or from a histocompatibility-matched individual.

In another embodiment, a composition for activating T cells is provided. The composition can comprise a dendritic cell population that is activated and matured by contacting the tissue culture substrate and a dendritic cell differentiation inducing agent under conditions suitable for dendritic cell attachment to the tissue culture substrate and suitable for activation; and a predetermined antigen. The dendritic cell population can produce an antigen-specific immune response similar to that induced by the mature dendritic cell population produced by the methods described above. The dendritic cell population is generated by isolating immature dendritic cells from a prior culture medium and added cytokines and then contacting the isolated immature dendritic cells with a tissue culture substrate under conditions suitable for attachment of the DCs to the tissue culture substrate without the addition of a dendritic cell maturation agent. Dendritic cells, upon contact with a tissue culture substrate under conditions suitable for attachment of the dendritic cells to the tissue culture substrate, are triggered to undergo activation and maturation to form active mature dendritic cells. Predetermined soluble or particulate antigens added to the tissue culture substrate simultaneously with or during maturation (i.e., after activation but before complete maturation) with immature dendritic cells can be taken up and processed by dendritic cells and presented in the context of appropriate cell surface receptors (available upon contact with T cells) to induce an antigen-specific immune response.

In another aspect, an isolated, activated dendritic cell population is provided. The cell population comprises and is enriched for mature activated monocytic dendritic cells prior to contact with a tissue culture substrate and a dendritic cell differentiation inducing agent under conditions conducive to dendritic cell attachment to the tissue culture substrate. The resulting activated dendritic cells can take up and process antigen and, after continued culture in vitro, can acquire the cell surface phenotype of mature dendritic cells. The cell population may optionally include predetermined antigens and/or isolated T cells, such as naive T cells. The T cells may optionally be present in a preparation of isolated lymphocytes.

Methods of producing activated T cells are also provided. The methods generally include providing a cell population enriched for isolated immature dendritic cells; contacting the immature dendritic cells with a predetermined antigen and contacting the immature dendritic cells with a tissue culture substrate and a dendritic cell differentiation inducing agent under conditions suitable for attachment of the immature dendritic cells to the tissue culture substrate. The cells are cultured for a time sufficient to induce activation of the immature dendritic cells to form activated dendritic cells. The activated dendritic cells can be contacted with naive T cells, thereby forming activated antigen-specific T cells. Suitable antigens can include, for example, tumor specific antigens, tumor associated antigens, viral antigens, bacterial antigens, tumor cells, bacterial cells, recombinant cells expressing antigens, cell lysates (including tumor cell lysates), membrane preparations, recombinantly produced antigens, peptide antigens (e.g., synthetic peptide antigens), or isolated antigens. The predetermined antigen may be a soluble antigen or a particulate antigen. Agents that direct the maturation of DCs to shift the response toward Th1 and/or Th2 responses, such as interferon gamma, may also be added.

The cell population enriched for immature dendritic cells can be contacted with the predetermined antigen and the tissue culture substrate and the dendritic cell differentiation inducing agent simultaneously, or the cells can be contacted with the predetermined antigen before, during, or simultaneously with or after the contacting with the tissue culture substrate and the dendritic cell differentiation inducing agent. In certain embodiments, the method can further comprise obtaining a cell population enriched for monocytic dendritic cell precursors; and culturing the precursor in vitro in the presence of a dendritic cell differentiation inducing agent to induce the formation of immature dendritic cells. Suitable differentiation inducing agents include, for example, GM-CSF, interleukin 4, interleukin 13, or interleukin 15, or mixtures thereof. Monocytic dendritic cell precursors can optionally be obtained from a human subject. In particular embodiments, the monocytic dendritic cell precursor cells, immature dendritic cells and/or T cells are autologous to each other.

The activated antigen-specific T cells can be administered to an animal, particularly a mammal, in need of stimulation of an antigen-specific immune response. Suitable antigens include, for example, tumor specific antigens, tumor associated antigens, viral antigens, bacterial antigens, tumor cells, bacterial cells, recombinant cells expressing antigens, cell lysates, membrane preparations, recombinantly produced antigens, peptide antigens (e.g., synthetic peptide antigens), or isolated antigens. In particular embodiments, the cell lysate and/or membrane preparation is derived from a tumor or tumor cell line of the patient. The immature dendritic cells can optionally be contacted with the predetermined antigen, and the tissue culture substrate, dendritic cell differentiation inducing agent simultaneously, or the immature dendritic cells can be contacted with the predetermined antigen prior to contacting with the new, unused, clean tissue culture substrate.

In certain embodiments, the method may further comprise isolating the monocytic dendritic cell precursors from the animal; and culturing the precursor in vitro in the presence of a differentiating agent to form the immature dendritic cells. The differentiating agent may be, for example, GM-CSF, interleukin 4, interleukin 13, interleukin 15, or a mixture thereof.

The monocytic dendritic cell precursor immature dendritic cell population and/or T cells can be autologous to the animal or allogeneic to the animal. Alternatively, the monocytic dendritic cell precursors, immature dendritic cells and/or T cells can have the same MHC haplotype as the animal, or share an MHC marker. In certain embodiments, the animal may be a human or may be a non-human animal.

Detailed Description

The present invention provides methods for inducing or triggering the activation and maturation of immature Dendritic Cell (DC) populations and priming these cell populations for antigen-specific immune responses. The immature dendritic cell population can be obtained from a preparation medium (including isolation medium, culture medium, and the like), and the cell population enriched for immature monocytic dendritic cells is contacted with a tissue culture substrate and a dendritic cell differentiation inducing agent under conditions suitable for attachment of DCs to the tissue culture substrate in the absence of the dendritic cell maturation agent. Immature dendritic cells can be contacted with a predetermined antigen under conditions suitable for in vitro cell culture to allow uptake processing and presentation by the DCs. The contacting with the antigen of interest can be performed during, before, or after the initiation of activation. Alternatively, immature monocytic dendritic cells that have been exposed to an antigen (e.g., in vivo) can be obtained and contacted with a tissue culture substrate under similar conditions suitable for cell culture. The resulting activated mature dendritic cells present the antigen of interest and are thereby primed to activate the T cells towards an antigen-specific response. The direction of the response, i.e. the propensity to support a Th-1 or Th-2 response, can be influenced by the addition of a directional maturation agent, such as interferon gamma (IFN γ) or the like.

In another aspect, a cell population enriched for monocytic dendritic cell precursors can be obtained from a subject or from a donor. The cell population can be contacted with a dendritic cell differentiation inducing agent, such as one or more cytokines (e.g., without limitation, GM-CFS, and mixtures of GM-CSF with IL-A, GM-CSF with IL-13, GM-CSF with IL-15, etc.) to obtain immature dendritic cells. The immature dendritic cells can then be contacted with a predetermined antigen, either with a tissue culture substrate and a dendritic cell differentiation inducing agent or, with a cytokine or other directed maturation agent, to activate and/or partially mature the dendritic cells. Mature dendritic cells can be used directly to induce an antigen-specific immune response in a subject or cells can be used to induce an antigen-specific immune response in T cells. In certain embodiments, MHC class I antigen processing is stimulated, which is used to elicit a CTL response against cells displaying a predetermined antigen. The direction of the induced response can be influenced by the addition of a targeted maturation agent such as interferon gamma. As used herein, a targeted maturation agent is an agent that, when used alone, can affect the final state of a mature DC but does not induce DC maturation. For example, a targeted maturation agent may polarize maturation of a DC population to favor a Th1 response over a Th-2 response, or vice versa.

Dendritic cells are distinct populations of antigen presenting cells found in a variety of lymphoid and non-lymphoid tissues.(See Liu, Cell 106: 259-containing 262 (2001); Steinman, Ann. Rev. Immunol.9: 271-296 (1991)). Dendritic cells include lymphoid dendritic cells of the spleen, langerhans cells of the epidermis and veil cells in the blood circulation. In general, dendritic cells are classified as a class of cells based on their morphology, high levels of surface MHC-class II expression, and the absence of other surface markers expressed on certain T cells, B cells, monocytes, and natural killer cells. In particular, monocyte-derived dendritic cells (also referred to as monocytic dendritic cells) typically express CD11c, CD80, CD83, CD86 and are HLA-DR⁺But is usually, but not always, CD14^-。

Conversely, the monocytic dendritic cell precursor (typically a monocyte) is typically CD14⁺And express no or very low levels of HLA-DR, CD83, and CD 86. Monocytic dendritic cell precursors can be obtained from any tissue in which they are present, particularly lymphoid tissues such as spleen, bone marrow, lymph nodes and thymus. Monocytic dendritic cell precursors can also be obtained from the circulatory system. Peripheral blood is a readily available source of monocytic dendritic cell precursors. Cord blood is another source of monocytic dendritic cell precursors. Monocytic dendritic cell precursors can be obtained from a variety of organisms in which an immune response can be elicited. Such organisms include animals, including, for example, humans and non-human animals, such as primates, other mammals (including, but not limited to, dogs, cats, mice, and rats), birds (including chickens), and transgenic species thereof.

In certain embodiments, the cell population enriched for monocytic dendritic cell precursors and/or immature dendritic cells can be obtained from a healthy subject or alternatively from a subject in need of immune stimulation, such as a cancer patient (e.g., brain cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, colon cancer, etc.) or another subject for which cellular immune stimulation may be beneficial or desirable (e.g., a subject having a bacterial or viral infection, etc.). Dendritic cell precursors and/or immature dendritic cells can also be obtained from an HLA-matched healthy individual for administration to an HLA-matched subject in need of immunostimulation.

Dendritic cell precursors and immature dendritic cells

Methods for isolating cell populations enriched for dendritic cell precursors and immature dendritic cells from different sources, including blood and bone marrow, are known in the art. For example, the heparinized blood can be collected by apheresis or leukapheresis, by buffy coat preparation, rosetting, centrifugation, density gradient centrifugation (e.g., using FICOLL (e.g., FICOLL-PAQUE))、PERCOLL(colloidal silica particles (15-30mm diameter) coated with non-dialyzable polyvinylpyrrolidone (PVP), sucrose, etc.), differential lysis of cells, filtration, etc. to obtain a population of cells enriched for dendritic cell precursors and immature dendritic cells. In certain embodiments, the leukocyte population can be prepared, for example, by collecting blood from a subject, centrifuging (defribrinating) to remove platelets, and lysing red blood cells. Optionally, by passage through a density gradient material (e.g., PERCOLL)Gradient) to enrich the monocytic dendritic cell precursors with dendritic cell precursors and immature dendritic cells.

All populations enriched for dendritic cell precursors and immature dendritic cells can optionally be prepared in, for example, a closed, sterile system. As used herein, the term "closed, sterile system" or "closed system" refers to a system in which exposure to non-sterile, ambient or circulating gases or other non-sterile conditions is minimized or eliminated. Closed systems for obtaining dendritic cell precursors and immature dendritic cells typically do not include density gradient centrifugation in open-top tubes, open air transfer of cells (open air cells), cell culture in tissue culture dishes or unsealed culture flasks, and the like. In a general embodiment, the closed system allows the sterile transfer of dendritic cell precursors and immature dendritic cells from an initial collection vessel to a sealable tissue culture vessel without exposure to non-sterile air.

In certain embodiments, the monocytic dendritic cell precursors are obtained by attaching monocytes to a monocyte-binding substrate as described in WO 03/010292 (the disclosure of which is incorporated herein by reference). For example, a population of leukocytes (e.g., isolated by leukapheresis) can be contacted with a substrate to which monocytic dendritic cell precursors are attached. When the leukocyte population is contacted with the substrate, the monocytic dendritic cell precursors in the leukocyte population preferentially adhere to the substrate. Other leukocytes (including other potential dendritic cell precursors) exhibit reduced binding affinity to the substrate, allowing preferential enrichment of monocytic dendritic cell precursors on the surface of the substrate.

Suitable substrates include, for example, substrates having a large surface area to volume ratio. Such a matrix may be, for example, a particulate or fibrous matrix. Suitable microparticle matrices include, for example, glass, plastic microparticles, glass-coated polystyrene microparticles, and other microbeads suitable for protein adsorption. Suitable fibrous microparticles include microcapillaries and microvillous membranes. The particulate or fibrous substrate generally allows the attached monocytic dendritic cell precursors to be eluted without significantly reducing the viability of the attached cells. The particulate or fibrous substrate may be substantially non-porous, thereby facilitating elution of the monocytic dendritic cell precursors or dendritic cells from the substrate. A "substantially non-porous" matrix is one in which at least a majority of the pores present in the matrix are smaller than the cells, thereby minimizing the capture of cells in the matrix.

Optionally, the attachment of the monocytic dendritic cell precursors to the substrate can be enhanced by the addition of a binding medium. Suitable binding media packIncluding monocytic dendritic cell precursor medium (e.g., AIM-V)、RPMI 1640、DMEM、X-VIVO 15Etc.), either alone or in any combination, supplemented with cytokines (e.g., granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin 4(IL-4), or interleukin 13(IL-13)), plasma, serum (e.g., human blood, such as autologous or allogeneic serum), purified proteins such as serum albumin, di-cations (e.g., calcium and/or magnesium dissociated), and other molecules that aid in the specific attachment of monocytic dendritic cell precursors to a substrate or prevent the attachment of non-monocytic dendritic cell precursors to a substrate. In certain embodiments, the plasma or serum can be heat inactivated. The heat inactivated plasma may be autologous or heterologous to the leukocytes.

After the monocytic dendritic cell precursors attach to the substrate, the unattached leukocytes can be separated from the monocytic dendritic cell precursor/substrate complex. The unattached cells can be separated from the complex using any suitable method. For example, a mixture of unattached leukocytes and the complex can be allowed to settle, and then the unattached leukocytes and the culture medium can be decanted or drained away. Alternatively, the mixture can be centrifuged, and the supernatant containing unattached leukocytes decanted or drained from the precipitated complex.

In another method, all populations enriched for monocytic dendritic cell precursors can be obtained from cell populations enriched in leukocytes prepared by using a tangential flow filtration device. A tangential flow filtration device for obtaining a cell population enriched for monocytic dendritic cell precursors may comprise a remover unit (remover unit) with a cross flow chamber, a filtration chamber and a filter placed in between (see WO 2004-. The filter communicates with the cross-flow chamber in the liquid on one side (retentate surface) and with the filter chamber on the other side (filtrate surface). The cross flow chamber has an inlet adapted to introduce a sample of blood components containing leukocytes into the cross flow chamber and is parallel to the retention surface of the filter. An outlet is also provided in the cross-flow chamber, located in a central portion of the chamber, opposite the retentate surface of the filter. Filters suitable for use in tangential flow filtration devices typically have an average pore size in the range of about 1 to 10 microns. The filter may have an average pore size of about 3 to about 7 microns. Methods for providing a predetermined input rate of sample into the inlet of the cross-flow chamber and methods for controlling the filtration rate of filtrate through the filter and into the filter chamber may also be included. Filtration rate control means that the rate of filtration is limited to be lower than the unopposed filtration rate of the filter. The sample comprising the blood component may be provided by a source device such as a leukocyte separation device or a container containing the sample collected from the leukocyte separation device.

Dendritic cell precursors can be cultured in vitro or ex vivo for differentiation, maturation and/or expansion. As used herein, isolated immature dendritic cells, dendritic cell precursors, T cells and other cells refer to cells that exist by artificial isolation from their natural environment and are thus non-natural products. Isolated cells can exist in a purified form, a semi-purified form (e.g., an enriched cell population), or in a non-native environment. Briefly, ex vivo differentiation generally involves culturing dendritic cell precursors or populations of cells bearing dendritic cell precursors in the presence of one or more dendritic cell differentiating agents. Suitable differentiation inducing agents may be, for example, but are not limited to, cell growth factors (e.g., cytokines such as (GM-CSF), interleukin 4(IL-4), interleukin 13(IL-13), interleukin 15(IL-15), and/or mixtures thereof). In certain embodiments, the monocytic dendritic cell precursors are induced to differentiate into monocyte-derived immature dendritic cells.

The dendritic cell precursors can be cultured and induced to differentiate under suitable culture conditions. Suitable tissue cultureThe nutrient group includes, for example, AIM-V、RPMI 1640、DMBM、X-VIVO 15And the like. The tissue culture medium may be supplemented with serum, amino acids, vitamins, cytokines such as GM-CSF and/or I L-4, IL-13 or IL-15, divalent cations, and the like, to promote differentiation of the cells into immature dendritic cells. In certain embodiments, dendritic cell precursors can be cultured in vitro in serum-free media. Such culture conditions may optionally exclude any animal-derived products. A typical cytokine mixture in a typical dendritic cell culture medium contains approximately 500 units/ml each of GM-CSF and IL-4. Dendritic cell precursors, when differentiated to form immature dendritic cells, have a phenotype similar to skin langerhans cells. The immature dendritic cells are typically CD14^-And CD11c⁺Expressing low levels of CD86 and CD83, soluble antigens can be captured by specialized endocytosis.

Typically, in prior methods, immature dendritic cells are matured in vitro or ex vivo to form mature dendritic cells prior to administration to a patient or prior to contact with T cells. In these methods, a dendritic cell maturation agent is added to an in vitro culture comprising immature dendritic cells prior to or with a predetermined antigen. Once mature, DCs gradually and progressively lose the ability to take up antigen, they often display up-regulated expression of costimulatory cell surface molecules and various cytokines. In particular, mature DCs expressing higher levels of MHC class I and class II antigens than immature dendritic cells, which are generally identified as exhibiting CD80⁺、CD83⁺、CD86⁺And CD14^-. Higher MHC expression leads to an increase in antigen density on the DC surface, while upregulation of the co-stimulatory molecules CD80 and CD86 potentiates the T cell activation signal by the counterpart of the co-stimulatory molecule on T cells, such as CD 28.

Unlike prior methods, activation of immature dendritic cells in the methods of the invention is initiated or triggered by contacting the isolated immature dendritic cells with a tissue culture substrate and a dendritic cell differentiation inducing agent under conditions suitable for dendritic cell attachment to a tissue culture surface. In the usual method of the invention, activation is accomplished without the addition of a maturation agent. Removing and isolating the immature dendritic cells from the culture medium or purification medium. The isolated immature dendritic cells are then counted and frozen for later use with freshly prepared media (without addition of dendritic cell maturation factors for the remainder of the process). In an alternative method, a directional maturation agent may be added during activation to bias the dendritic cells so that they can polarize the T cell response towards a Th-1 or Th-2 response. For example, but not by way of limitation, interferon gamma may be added to bias the T cell response towards a Th-1 response. Interferon gamma added to monocytic dendritic cell precursors or immature DCs does not itself induce DC differentiation and/or maturation.

Tissue culture substrates for use in the methods of the invention may include tissue culture wells, flasks, bottles, bags, or any substrate used in bioreactors, such as fibers, beads, plates, and the like. Typically, the tissue culture substrate comprises a plastic such as polystyrene, Teflon(polytetrafluoroethylene, PTFE) and the like. These tissue culture substrates, which are most commonly used for ex vivo culture of dendritic cells (for immunotherapy), include tissue culture flasks, bags, or chamber sections (cell fractions) consisting of multiple superimposed layers of plastic, etc.

The isolated immature dendritic cells can be cultured and matured in suitable maturation culture conditions. Suitable tissue culture media include AIM-V、RPMI 1640、DMEM，X-VIVO 15And the like. The tissue culture medium may be supplemented with amino acids, vitamins, cytokines, human serum, e.g., about 1% to about 10% human AB serum, divalent cations, etc., to promote maturation of the cells.

The maturation or activation of dendritic cells is monitored by methods known in the art. Cell surface markers can be detected in assays familiar to the art, such as flow cytometry, immunohistochemistry, and the like. Cells can also be monitored for cytokine production (e.g., by ELISA, FACS, or other immunoassay). In the DC population activated according to the invention, cells can also be assayed for the presence of the typical cell surface markers CD83, CD86, and HLA-DR. Some of these antigens, such as CD83, are only expressed on mature DCs, whereas the expression of other antigens is significantly upregulated after maturation. Mature DCs also lose the ability to take up antigen by pinocytosis, which can be analyzed by uptake assays well known to those skilled in the art. Antigen-contacted or antigen-uncontacted dendritic cell precursors, immature dendritic cells and mature dendritic cells can be cryopreserved for later use. Methods for refrigerated preservation are well known in the art. See, for example, U.S. Pat. No. 5,788,963, which is incorporated herein by reference in its entirety.

Antigens

Mature, activated dendritic cells of the invention can present antigen to T cells. Mature, activated dendritic cells can be formed by contacting immature dendritic cells with a predetermined antigen during or after activation. Alternatively, immature dendritic cells that have been contacted with an antigen (e.g., prior to isolation in vivo) can be contacted with a tissue culture substrate and a dendritic cell differentiation inducing agent, thereby generating activated mature dendritic cells to induce a cytotoxic T cell response.

Suitable predetermined antigens may include any antigen for which activation of T cells is desired. Such antigens may include, for example, bacterial antigens, tumor-specific or tumor-associated antigens (e.g., intact cells, tumor cell lysates, e.g., lysates from glioblastoma, prostate, or ovarian, breast, colon, brain, melanoma, or lung tumor cells, etc.), isolated antigens from tumors, fusion proteins, liposomes, etc., viral antigens, and fragments of any other antigens or antigens, such as peptide or polypeptide antigens. In certain embodiments, the antigen may be, for example, but not limited to, Prostate Specific Membrane Antigen (PSMA), Prostate Acid Phosphatase (PAP), or Prostate Specific Antigen (PSA). (see, e.g., Pepsidero et al, Cancer Res.40: 2428-32 (1980); McCormack et al, Urology 45: 729-44 (1995)). The antigen may also be a bacterial cell, a bacterial lysate, a membrane fragment from a cell lysate, or any other source known in the art. The antigen may be expressed or recombinantly produced or even chemically synthesized. The recombinant antigen may also be expressed on the surface of a host cell (e.g., a bacterial, yeast, insect, vertebrate, or mammalian cell), may be present in a lysate, or may be purified from a lysate. The antigen may be a soluble antigen or a particulate antigen.

The antigen may also be present in a sample from the subject. For example, a tissue sample from a hyperproliferative (hyperproliferative) or other condition in a subject may be used as a source of antigen. Such samples may be obtained, for example, by biopsy or by surgical resection. Such a sample may for example be used as a lysate or as an isolated preparation. Alternatively, a membrane preparation of cells or an established cell line of a subject (e.g., a cancer patient) may also be used as an antigen or source of an antigen.

In exemplary embodiments, glioblastoma cell lysate recovered from surgical samples may be used as a source of antigen. For example, a tumor sample of a cancer patient's own, which may be obtained at the time of biopsy or surgical resection, may be used directly for antigen presentation to dendritic cells or to provide cell lysates for antigen presentation. Alternatively, a membrane preparation of tumor cells of a cancer patient may be used. The tumor cell may be a glioblastoma, prostate, lung, ovary, breast, colon, brain, melanoma, or any other type of tumor cell. Preparations of the lysate and membrane can be prepared from isolated tumor cells by methods well known in the art.

In one embodiment, monocytic dendritic cell precursors can be isolated on a substrate, the cells eluted from the substrate, transferred to a bioreactor or other closed system such as a tissue culture bag. Suitable tissue culture bags include, for example, STERICELL culture containers (Nexell therapeutics Inc.) or TEFLON bags (American Fluoroseal Corp.) and the like. The closed system may have any suitable size or volume, as will be appreciated by those skilled in the art. Suitable volumes include, for example, volumes of about 0.01 liters to about 5 liters, or about 0.01 liters to about 0.05 liters, and even larger or smaller volumes are also possible and within the scope of the invention.

Monocytic dendritic cell precursors can also be cultured on the substrate. For example, the monocytic dendritic cell precursors on the substrate can be cultured in a bioreactor (including a fermentor) or in a tissue culture flask (culture vessel), e.g., a tissue culture flask, bag, or plate. The tissue culture flasks, bags, or plates may be of any suitable size, as will be appreciated by those skilled in the art. Bioreactors generally have a volume of about 0.01 to about 5 liters or about 0.01 to about 0.05 liters, although larger or smaller volumes are possible and within the scope of the present invention. Typically, bioreactors used for culturing monocytic dendritic cell precursors have a volume of about 0.01 to about 0.1 liters. Any suitable amount, e.g., about 10, can be used⁵Cell to about 5X 10⁶Monocytic dendritic cell precursors of individual cells/ml of substrate are seeded into the bioreactor. Monocytic dendritic cell precursors on a substrate can also be cultured in a closed, sterile system.

Culturing and differentiating the monocytic dendritic cell precursors to obtain immature dendritic cells. Suitable tissue culture media include AIM-V, RPMI 1640, DMEM, X-VIVO15, and the like. The tissue culture medium may be supplemented with amino acids, vitamins, cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF) and/or interleukin 4(IL-4), interleukin 7(IL-7) or interleukin 13(IL-13), divalent cations, etc., to promote differentiation of the monocytic dendritic cell precursors into immature dendritic cells. A commonly used combination of cytokines is about 500 units/ml each of GM-CSF and IL-4. Generally, if monocytic dendritic cell precursors are cultured on a substrate, the number of mature dendritic cells recovered as cells shed from the surface of the substrate is primary mature dendritic cells. The monocytic dendritic cell precursors can be cultured for any suitable time.

In certain embodiments, a suitable culture time for differentiating the precursor into immature dendritic cells can be about 4 to about 7 days. Differentiation of immature dendritic cells from precursors can be monitored by methods known to those skilled in the art, for example by monitoring the presence or absence of cell surface markers such as CD14, CD11c, CD83, CD86, HLA-DR using labeled monoclonal antibodies. The phenotype of the dendritic cells can also be determined by analyzing the pattern of gene expression using methods well known in the art. The general cell surface phenotype of immature dendritic cells is CD14^-、CD11c⁺、CD83^-、CD86^-And HLA-DR⁺. Immature dendritic cells can also be cultured in a suitable tissue culture medium to expand the cell population and/or maintain the immature dendritic cells in a naive state for further differentiation or antigen uptake, processing and presentation. For example, immature dendritic cells can be maintained and/or expanded in the presence of GM-CSF and IL-4.

Immature dendritic cells may be preferred for some applications because they retain the ability to process new antigens. (see, e.g., Koch et al, J.Immunol.155: 93-100 (1995)). In contrast, mature dendritic cells (e.g., CD 14)^-、CD11c⁺、CD83⁺、CD86⁺And HLA-DR⁺) Mature dendritic cells, which have been exposed to antigen and to process the antigen and to appropriate maturation conditions, typically have lost the ability to efficiently process the neoantigen. Mature dendritic cells can be contacted with peptides capable of binding to MHC class I and/or MHC class II molecules for presentation on the cell surface.

During culture, the immature dendritic cells can optionally be exposed to a predetermined antigen. Suitable predetermined antigens may include any antigen for which activation of T cells is desired, as described above. In one embodiment, the immature dendritic cells are cultured in the presence of a tumor lysate (e.g., a lysate of a glioma, prostate, ovarian, breast, colon, brain, melanoma, or tumor cells, etc.) for cancer immunotherapy and/or tumor growth inhibition. Other antigens may include, for example, bacterial and viral antigens, tumor cells, purified tumor cell membranes, tumor specific or tumor associated antigens (e.g., isolated antigens from tumors, fusion proteins, liposomes, etc.), bacterial cells, bacterial antigens, viral particles, viral antigens, and any other antigens. In addition, the antigen may be expressed on the surface of a transformed or transfected host cell expressing the antigen, or in a purified membrane or cell lysate of a transfected or transformed cell expressing the antigen of interest.

After contact with an antigen, e.g., a tumor cell lysate, the cells can be cultured for any suitable time to allow antigen uptake and processing, to allow expansion of a population of antigen-specific dendritic cells, and the like. Immature dendritic cells can also be matured into activated dendritic cells that present antigen in the context of MHC class I or MHC class II molecules. This maturation can be performed, for example, by culturing on a tissue culture substrate and using a dendritic cell differentiation inducing agent under conditions conducive to the attachment of dendritic cells to the tissue culture substrate in the absence of other known maturation agents. Generally, the dendritic cell differentiation inducer is GM-CSF, IL-4, IL-13, IL-15, or the like.

According to another aspect, dendritic cells can be exposed to a predetermined antigen and a targeted maturation agent. When used alone, the directed maturation agent does not induce differentiation of monocytic dendritic cell precursor cells or does not induce maturation of immature dendritic cells, but, in general, when combined with an activation method, directs the maturation of DCs towards cells that can induce Th-1 or Th-2 responses. Interferon gamma is an example of an agent that can induce a bias in the induced T cell response towards a Th-1 response.

In another embodiment of the invention, non-mature dendritic cells exposed to a predetermined antigen can be used to activate anti-antigen T cells in vitro. The dendritic cells can be used to stimulate T cells immediately after exposure to the antigen. Alternatively, the dendritic cells can be maintained in the presence of a mixture of cytokines (e.g., GM-CSF and IL-4) prior to exposure to antigen and T cells, or can be cryopreserved for later use by methods well known in the art. In particular embodiments, human dendritic cells are used to stimulate human T cells.

T cells or T cell subsets can be obtained from different lymphoid tissues for use as responder cells. Such tissues include, but are not limited to, spleen, lymph nodes and peripheral blood. Isolated or purified T cells can be co-cultured with dendritic cells exposed to a predetermined antigen, either as a mixed T cell population or as a purified T cell subpopulation.

For example, purified CD8 can be used⁺T cells are co-cultured with antigen-exposed dendritic cells to elicit antigen-specific CTL responses. In addition, CD4⁺Early clearance of T cells prevents CD4⁺The cells are in CD8⁺And CD4⁺Overgrowth in mixed cultures of T cells. Purification of T cells can be achieved by positive or negative selection including, but not limited to, the use of antibodies to CD2, CD3, CD4, CD6, and/or CD 8.

Alternatively, CD4 may be used⁺And CD8⁺The mixed population of T cells is co-cultured with dendritic cells to elicit a response specific to the antigen to which cytotoxicity and T helper immune response are obtained. In certain embodiments, activated CD8 may be generated according to the methods of the present invention⁺Or CD4⁺T cells. Generally, mature, antigen-contacted dendritic cells used to generate antigen-reactive, activated T cells are syngeneic with (e.g., obtained from) a subject to which the dendritic cells are administered. Alternatively, non-cancerous cells from an HLA matched donor may be usedCells (e.g., normal cells) produce dendritic cells in vitro that have the same HLA haplotype as the intended recipient subject. In particular embodiments, antigen-reactive T cells, including CTL and Th-1 helper cells, are expanded in vitro as a source of cells for immune stimulation.

In vivo administration of cell populations

In another aspect of the invention, methods are provided for administering mature, antigen-contacted dendritic cells or activated, e.g., polarized, T cells or cell populations comprising such cells to a subject in need of immune stimulation. Such cell populations may include mature, activated dendritic cell populations and/or activated, e.g., polarized, T cell populations. In certain embodiments, the methods are performed by obtaining dendritic cell precursors or immature dendritic cells, differentiating and maturing the cells by contact with a tissue culture substrate, a dendritic cell differentiation inducing agent, and a predetermined antigen, thereby forming a mature, activated dendritic cell population that elicits an induction of an antigen-specific T cell response. The immature dendritic cells can be contacted with the antigen before, during, or after activation. Mature or activated dendritic cells can be administered directly to a subject in need of immunostimulation.

In related embodiments, activated or mature dendritic cells can be contacted with lymphocytes from the subject to stimulate T cells within the lymphocyte population. Activated, polarized lymphocytes (optionally followed by antigen reaction CD4⁺And/or CD8⁺Clonal expansion in cell culture of T cells) to a subject in need of immune stimulation. In certain embodiments, the activated, polarized T cells are autologous to the subject.

In another embodiment, the dendritic cells, T cells and recipient subject have the same mhc (hla) haplotype. Methods for determining the HLA haplotype of a subject are known in the art. In related embodiments, the dendritic cells and/or T cells are allogeneic to the recipient subject. For example, dendritic cells may be allogeneic to T cells and recipients having the same mhc (hla) haplotype. Allogeneic cells are typically matched for at least one MHC allele (e.g., share at least one but not all MHC alleles). In a less common embodiment, the dendritic cells, T cells, and recipient are all allogeneic with respect to each other, but all share at least one common MHC allele.

According to one embodiment, the T cells are obtained from the same subject from which the immature dendritic cells were obtained. After in vitro maturation and polarization, autologous T cells are administered to the subject, thereby eliciting and/or enhancing an existing immune response. For example, by a factor of, for example, about 10⁸To about 10⁹Cell/m²Doses of body surface area T cells are administered by intravenous infusion (see, e.g., Ridell et al, Science 257: 238-241(1992), incorporated herein by reference). The infusion may be repeated at desired intervals, for example, once a month. Recipients can be monitored during and after T cell infusion for any signs of adverse effects.

According to another embodiment, dendritic cells matured by contact with new, unused clean tissue culture substrates of the present invention can be injected directly into a tumor or other tissue containing the target antigen. Such partially mature cells can take up antigen in vivo and present the antigen to T cells.

Examples

The following examples are provided merely as illustrations of various aspects of the invention and should not be construed as limiting the invention in any way.

Example 1: monocytic dendritic cell pre-treatment by contact with tissue culture substrate Maturation of bodies

In this example, monocytic dendritic cell precursors in a cell population enriched for precursors are differentiated to form immature dendritic cells in the presence of GM-CSF and IL-4. Immature dendritic cells are collected from the tissue culture system, washed, counted and mixed with predetermined antigens in a new clean tissue culture flask. The cells are cultured in the presence of predetermined soluble or particulate antigens under conditions generally suitable for dendritic cell maintenance, with the usual dendritic cell culture media supplemented with GM-CSF and IL-4. No dendritic cell maturation agent was added to the medium. Determining that the dendritic cells have matured without the addition of the dendritic cell maturation agent by determining the presence of cell surface marker features of the mature dendritic cells.

Briefly, by OptiMEM supplemented with 1% human plasmaImmature DCs were prepared by contacting peripheral blood mononuclear cells with plastic in the presence of medium (Gibco-BRL). Unbound monocytes can be removed by washing. In the presence of 500 units of GM-CSF per ml and 500 units of IL-4 in X-VIVO15The bound monocytes are cultured in medium for about 6 to 7 days.

Immature DCs were collected by rinsing and cells were washed with medium. The cells are counted and the washed DCs are mixed with tumor cell lysate from, for example, a glioblastoma surgically removed from the patient receiving treatment. The above DCs and lysate in medium with GM-CSF and IL-4 were added to a fresh, clean, unused tissue culture dish and cultured for about 12 to about 20 hours. In an alternative embodiment, interferon gamma may be added to induce a Th-1 enhanced response.

The activated DCs are collected, washed and prepared for administration to a patient. Samples of activated DCs were used to detect the phenotype of cells by labeling with antibodies specific for CD14, CD83, CD86, and HLA-DR, respectively. The labeled cells were analyzed by flow cytometry to determine their phenotype. Activated DCs showed little or no CD14, were positive for CD83, and expressed high levels of CD86 and HLA-DR, which is the expected phenotype of mature dendritic cells.

The remaining part of the activated DC is 1 × 10⁶1X 10 per cell¹⁰Individual cells/injected amount are administered to the patient. Induction of antigen-specific immune responses was assessed by measuring T cell responses using MHC tetramer analysis and/or measuring antibody responses to antigen by ELISA.

Example 2: determination of cell surface phenotype of monocytic dendritic cell precursor cells activated in the absence of maturation agent

In this example, monocytes were purified by attachment to plastic tissue culture flasks, harvested, washed, and resuspended in a new tissue culture flask for additional culture time. Cell surface expression of the mature dendritic cell marker CD83 was determined.

To generate mature DCs, monocytes were purified by attachment to plastic and then cultured in dendritic cell culture media with GM-CSF and IL-4 and 1% human AB serum for approximately 7 days. After about 7 days, cells that were almost completely free-floating in culture medium with GM-CSF and IL-4 and about 1% human AB serum were harvested and then replated in unused clean polystyrene tissue culture flasks containing dendritic cell culture medium. Visual observation of the recoated cells under an inverted microscope showed that most of the cells were tightly attached to the tissue culture surface. At 20 hours after re-plating, the cells displayed induction of the DC marker CD 83. The following table provides CD83 staining of different samples of isolated monocytes.

TABLE 1

Sample (I)	％CD83+
		A	56.7
B	47.6
		C	41
D	21
		E	15.4
F	24.1
		G	40.5
H	19.6
		I	20.5
J	19.4
		K	17.9
L	11.6
		M	12.5
N	23.6
		O	17.1
P	13.8

Example 3: clinical use of dendritic cells activated without maturation agents

In this example, the safety and efficacy of dendritic cell compositions obtained from glioblastoma multiforme patients without the use of a dendritic cell differentiating agent and contact with tumor lysate prepared from the patients were tested.

Tumor lysates were prepared from surgically excised tumor tissue. The separated tumor tissue is minced and put into a container containing a buffer solution containing collagenase to separate the tissue. The mixture was allowed to stand at room temperature overnight. After filtration to ensure tissue digestion, the released tumor cells were centrifuged into a pellet. The cell pellet was resuspended in a small volume of RPMI 1640 and subjected to 3 freeze-thaw cycles. After freezing and thawing, the tumor lysate was clarified by centrifugation and the supernatant containing the protein was filtered through a 0.22 micron filter for sterilization.

The cell population enriched for monocytic dendritic cell precursors was prepared by purifying the leukapheresis product on a FICOLL gradient and then attaching it to plastic. Adherent cells are monocytic dendritic cell precursors cultured in RPMI 1640 supplemented with 10% AB human serum and 500U/ml each of hGM-CSF and hIL-4 for 7 days under standard dendritic cell culture conditions.

After 7 days, the differentiated immature dendritic cells were harvested, washed, frozen for later use or mixed with patient tumor lysate and replated in fresh tissue culture flasks in the presence of 1% to 10% human AB serum and 500U/ml each of hGM-CSF and IL-4. The final concentration of DCs is 0.5 to 2 million cells/ml (typically 1X 10)⁶Individual cells/ml), the final concentration of tumor lysate is 10 to 1000 μ g/ml (typically 100 μ g/ml).

In a first trial, the patient received dendritic cells contacted with tumor lysate derived from autologous cultured tumor cells. The study was a dose escalation study in which 3 human subjects received 1X 10⁶DC, 3 subjects received 5X 10⁶DC, 6 subjects received 10X 10⁶And (6) a DC. The immune response is assessed by measuring the delayed hypersensitivity (DTH response), the cytotoxic T cell response (CTL) and by determining the presence of infiltrating lymphocytes during reoperation in case of disease recurrence. Each assay measures a different aspect of the immune response induced. DTH responses are often evidence of helper T cell (Th) activity, whereas CTL responses measure whether the induced response has the effect of causing the generation of T cells with the ability to kill tumor cells. Infiltrating T cells detected in the tumor after the reaction measure whether activated T cells have the ability to migrate to the tumor site, thereby killing tumor cells in situ.

TABLE 2 summary of immune response-phase I assay

Patient numbering	DC dose (# cells/injection)	Increase in DTH skin test	Increased peripheral CTL Activity	Intratumoral T cells (at reoperation)a
					1	1×106	Positive for	Positive for	++++
2	1×106	Negative of	Negative of	--
					3	1×106	Positive for	Positive for	+++
4	5×106	Negative of	Positive for	++
					5	5×106	Positive for	Positive for	n.a.
6	5×106	Negative of	Positive for	n.a.
					7	10×106	Negative of	Negative of	--
8	10×106	Negative of	Negative of	++
					9	10×106	Positive for	Positive for	+++
10	10×106	Negative of	Negative of	n.a.
					11	10×106	Negative of	Negative of	n.a.
12	10×106	Negative of	Negative of	n.a.

^aScoring by semi-subjective assessment of the number of infiltrating lymphocytes.

n.a., inapplicable because there is no surgery.

The second trial was also a dose escalation study, with 4 subjects receiving 1X 10⁶One DC/injection, 6 subjects received 5X 10⁶One DC/injection, 6 subjects received 10X 10⁶One DC/injection. In this study, tumor lysates were derived from tumors excised from each particular patient, and the lysates were contacted with DCs derived from that patient. By antigen specific CD8 after vaccination⁺T cells are tetrameric stained and, if applicable, the immune response is assessed by determining the presence of infiltrated intratumoral T cells at the time of re-surgery. Typically, reoperation can be performed after the disease has recurred. 13 of the patients had a recently diagnosed glioblastoma multiforme (GBM), 2 had recurrent GBM, and 1 had recurrent grade III oligoastrocytoma.

Assessment of the immune response by tetramer staining of antigen-specific T cells allows determination of the presence of circulating T cells expressing T cell receptors specific for a given antigen and quantification of the level of circulating T cells. Such T cells, if they also express CD8, are considered to be representative of CTL populations directed against these antigens. The antigen selected for this study is known to be a tumor antigen that has been shown by histological methods to be present in the initially resected tumor. The reactivity of tumor-associated antigens, including Gp100 (melanoma-associated tumor antigen, also found in GBM), Trp-2 (tyrosinase-related protein 2), Her-2 (epidermal growth factor receptor-related receptor-enzyme-tyrosine kinase), and CMV (cytomegalovirus) was tested using standard methods.

Attempts for antigen-specific CD8 in 7 patients⁺Tetramer staining of T cells, a positive response was detected in 5 patients. Of the 5 responding patients, 2 had used 1X 10⁶One DC was immunized, and 3 had been used 10X 10⁶Individual DCs were immunized. These results indicate that DCs matured by the methods of the invention are capable of inducing immune responses in a large fraction of patients. In most cases, the response includes an antigen-specific cytotoxic T cell response.

The foregoing examples are provided to illustrate and not to limit the scope of the invention. Other variations of the invention will be apparent to those skilled in the art and are encompassed by the appended claims. All publications, patents, patent applications, and other references cited herein are also incorporated by reference in their entirety.

Claims (31)

1. A method for producing a cell population comprising an enriched population of activated dendritic cells, the method comprising: contacting a cell population comprising an enriched population of immature dendritic cells with a tissue culture substrate and a culture medium having a dendritic cell differentiating agent under conditions suitable for attachment of the immature dendritic cells to the tissue culture substrate and for in vitro culture of the dendritic cells for a time sufficient to mature the activated dendritic cells.

2. The method of claim 1, wherein the predetermined antigen is contacted with the population of cells prior to, simultaneously with, or after the contacting with the tissue culture substrate.

3. The method of claim 2, wherein the predetermined antigen is a tumor specific antigen, a tumor associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide, or an isolated antigen.

4. The method of claim 3, wherein the cell lysate or membrane preparation is obtained from a brain tumor, a prostate tumor, prostate tissue, ovarian tumor, breast tissue, a population of leukemia cells, a lung tumor, melanoma, bladder tumor, or a tumor cell line.

5. The method of claim 4, wherein the brain tumor is glioblastoma multiforme or oligoastrocytoma.

6. The method of any one of claims 1 to 5, wherein the dendritic cell differentiating agent is GM-CSF, IL-4, IL-13, IL-15, or a combination thereof.

7. The method of any one of claims 1 to 6, wherein the immature dendritic cells are derived from a cell population enriched for monocytic dendritic cell precursors.

8. The method of claim 7, wherein the cell population enriched for monocytic dendritic cell precursors is contacted with a dendritic cell differentiation inducing agent.

9. The method of claim 8, wherein the dendritic cell differentiation inducing agent is GM-CSF, IL-4, IL-13, or IL-15, and combinations thereof.

10. The method of any one of claims 7 to 9, wherein the monocytic dendritic cell precursors are from a patient or from an HLA-matched individual.

11. The method of any one of claims 3 to 9, wherein the tumor cells and dendritic cells are from the patient.

12. The method of any one of claims 1 to11, wherein the immature dendritic cells are further contacted with a directional maturation agent.

13. The method of claim 12, wherein the targeted maturation agent is interferon gamma.

14. A method for activating immature dendritic cells, the method comprising contacting immature dendritic cells with a tissue culture substrate and a dendritic cell differentiation inducing agent under conditions suitable for in vitro culture of the immature dendritic cells and for attachment of the immature dendritic cells to the tissue culture substrate.

15. The method of claim 14, further comprising contacting the immature dendritic cells with a predetermined antigen prior to, simultaneously with, or after contacting the immature dendritic cells with the tissue culture substrate.

16. The method of claim 15, wherein the predetermined antigen is a tumor specific antigen, a tumor associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide, or an isolated antigen.

17. The method of claim 16, wherein the cell lysate or membrane preparation is derived from tumor tissue or a tumor cell line.

18. The method of claim 17, wherein the tumor tissue or tumor cell line is obtained from a population of brain tumor, prostate tumor, ovarian tumor, breast tumor, lung tumor, melanoma, bladder tumor, or leukemia cells.

19. The method of claim 18, wherein the brain tumor is glioblastoma multiforme or oligoastrocytoma.

20. The method of any one of claims 14 to 19, wherein the dendritic cell differentiation inducing agent is GM-CSF, IL-4, IL-13, IL-15, or a combination thereof.

21. The method of any one of claims 14 to 20, wherein the immature dendritic cells are further contacted with a directional maturation agent.

22. The method of claim 21, wherein the targeted maturation agent is interferon gamma.

23. A method for activating T cells, the method comprising: contacting the T cells with a dendritic cell population that is matured by contacting the tissue culture substrate and a dendritic cell differentiation inducing agent under conditions conducive to the attachment of immature dendritic cells to the tissue culture substrate and a predetermined antigen.

24. A method for producing activated antigen-specific T cells, the method comprising: contacting the isolated immature dendritic cells with a predetermined antigen; contacting the isolated immature dendritic cells with a tissue culture substrate and a dendritic cell differentiation inducing agent to activate the immature dendritic cells under conditions conducive to attachment of the immature dendritic cells to the tissue culture substrate; and contacting the activated dendritic cells with naive T cells to form activated antigen-specific T cells.

25. The method of claim 24, wherein the predetermined antigen is a tumor specific antigen, a tumor associated antigen, a viral antigen, a bacterial antigen, a tumor cell, a bacterial cell, a recombinant cell expressing an antigen, a cell lysate, a membrane preparation, a recombinantly produced antigen, a peptide antigen, or an isolated antigen.

26. The method of claim 25, wherein the cell lysate or membrane preparation is obtained from a brain tumor, a prostate tumor, prostate tissue, ovarian tumor, leukemia cell population, a lung tumor, a breast tumor, or a bladder tumor.

27. The method of claim 26, wherein the brain tumor is glioblastoma multiforme or oligoastrocytoma.

28. The process of any one of claims 24 to 27, comprising as a first step: the monocytic dendritic cell precursors are cultured in the presence of a differentiation inducing agent to form immature dendritic cells.

29. The method of claim 28, wherein the differentiation inducing agent is GM-CSF, interleukin 4, a mixture of GM-CSF and interleukin 4, or interleukin 13.

30. The method of claims 28 and 29, wherein the monocytic dendritic cell precursors are isolated from a human subject.

31. The method of claim 30, wherein the immature dendritic cells and the T cells are autologous with respect to each other.