KR20060116850A - Intratumoral delivery of dendritic cells - Google Patents

Abstract

Methods included herein describe the treatment of a tumor by administering dendritic cells either directly into the same or into its surrounding tissue. Further methods describe the induction of immune cell infiltration into tumors and the treatment of tumors with unprimed dendritic cells by administering dendritic cells in a similar fashion. The methods of the present invention are particularly advantageous in the treatment of brain tumors and other solid tumors disposed throughout the body of a mammal that are difficult or impossible to treat by conventional surgical means. Dendritic cell-based compositions effective in the treatment of such tumors are also described.

Description

Intratumoral delivery of dendritic cells {INTRATUMORAL DELIVERY OF DENDRITIC CELLS}

The present invention relates to methods of treating tumors by administering dendritic cells and compositions effective therein. More specifically, the method includes directly administering dendritic cells to a tumor or surrounding tissue located in the body of a mammal. The composition is based on dendritic cells.

Cancer is one of the leading causes of death in the United States and the world. Various cancers are treated differently depending on the location of the tumor to be treated. One particularly difficult group of tumors to treat is in or around the brain. Treatment of brain tumors presents many problems as well as inherent risks in any surgical operation, including tissue located in proximity to the brain region. There is room for prognosis and error of small surgical accidents that may adversely affect the patient (which may cause brain damage or even death). However, surgery remains the preferred method for treating most brain tumors and is often performed in combination with radiation and chemotherapy. However, mainly concerned medical authorities recommend that patients with brain tumors be left to centers specializing in research treatment (primary treatments are not overwhelmingly successful).

Glioblastoma multiforme and anaplastic astrocytoma are classified into a class of brain tumors, commonly known as malignant glioma. Although they are not particularly common tumors themselves, they represent the type of tumors associated with significant mortality and morbidity. Currently, the treatment of malignant gliomas consists of surgical resection with radiotherapy and chemotherapy. However, the treatment generally fails to sufficiently change the outcome seen in the patient (appears to be lower than the survival rate due to medical procedures per year).

In particular, inducing the human immune system to kill tumor cells may eventually have to completely remove them. Such an immunotherapy approach was attempted decades ago by treating cancer patients with recombinant human interleukin-2 (IL-2) and lymphokine activated killer cells (LAK cells). Although patients withstand the immunotherapy well, treatment attempts to do so do not confirm superior treatment.

More recent research has focused on initiating and publishing an immune response by activating T-cells and targeting them against infiltrating tumor cells. In general, the immune response of the human body is initiated when antigen-transmitting cells (APCs) digest the antigen into fragments and then the digested antigen is present in T-cells. T-cells recognize digested sections and connect to APCs; It activates T-cells and produces an immune response.

Some studies suggest that tumor cells themselves may contain immunogenic antigens. However, these studies note that tumor cells are weak in APC; They process / represent T-cells without effectively absorbing tumor antigens [S. Constant et al. "Peptide and Protein Antigens Requiring a Clear Antigen Cell Subpopulation for Priming CD4 + T-Cells", J. lummunol . 154: 4915-4923 (1995); D. Levin et al., “The Role of Dendritic Cells in Priming of CD4 + Lymphocytes as Peptide Antigens In Vivo”, J. lummunol . 151: 6742-6748 (1993). Therefore, additional support and / or stimulation are needed to elicit more essential and effective immune responses.

Vaccination with various cytokine expressing cells that have been genetically engineered has been attempted to aid in the promotion of such immune responses and to increase tumor cell immunogenicity. Cytokines are known to induce immunity and induce inflammatory responses and there have been a variety of studies showing reactions with anti-tumor effects [G. Dranoff et al. "Inoculation of irradiated tumor cells treated to secrete mouse granulocyte macrophage colony stimulating factors that stimulate potential, specificity and long-lasting anti-tumor immunity", Proc . Natl. Acad . Sci . USA 90: 3539-3543 (1993). In particular, such anti-tumor effects are known to be initiated by the role of cytokines in the presence of antigen through the increase of APCs such as phagocytes and B-cells that can activate sensitized T-cells. For example, containing interleukin-4, migration of growth factor-β and GM-CSF treatment techniques have been successfully implemented for anti-tumor effects. In fact, GM-CSF has been found to be the most effective cytokine for obtaining brain tumor specific immunity when the cytokine is peripherally inoculated (Dranoff et al . 3539). Although isolation of ubiquitous tumor antigens has been difficult to capture, peripheral inoculation of cells expressing various cytokines triggers the body's immune response and can be targeted against intracranial tumors. However, intracranial inoculation with cytokine-expressing cells is not therapeutically performed as intracranial cytokine expression, such as induction of undesirable inflammatory responses in brains not targeted to tumor cells, is present in potential complication hosts. .

B-cells, macrophages, and other APCs that are augmented by cytokines can help activate the immune response, but the most effective APCs in the body are dendritic cells. Dendritic cells are "professional APCs" that can uniquely activate both sensitized T-cells (as in the case of macrophages and B-cells) and simple T-cells (ie, those that have never encountered an antigen before). In fact, dendritic cells are the only APCs known to process exogenous antigens through the Class I pathway. Thus, efforts have been made to determine whether dendritic cells with tumor associated antigens can mediate significant anti-tumor responses; Potentially anti-tumor responses are stronger than those induced by cytokine-existing cells.

In mouse models and two known clinical trials, cytokine-promoting dendritic cells were pulsed with tumor antigens in vitro and have been successfully used as anti-tumor vaccines for extracranial tumor models (J. Mayorodomo et al. , "Protection of Synthetic Tumor Peptide-induced and Treated Anti-tumor Immunity and Pulsed Bone Marrow-derived Dendritic Cells," Nature Med. 1: 1297-1302 (1995); J. Young and K. Inaba, Dendritic Cells and Adjuvant for I ”, J. Exp . Med . 183: 7-11 (1996); L. Zivogel et al. ,“ Treatment of Mouse Tumors with Tumor Peptide-Pulse Dendritic Cells: T Cells, B7 Balls Palpation and dependence on T-helper cell 1-related cytokines ", J. Exp . Med . 183: 87-97 (1996); F. Hsu et al. ," B-cells with autologous antigen-pulsed dendritic cells Vaccination of patients with lymphoma ", Nature Med . 2: 52-58 (1996); and FO Nestle et al. ," Pulsed into dendritic cells Vaccination of Melanoma Patients with Peptide- or Tumor-Soluble ", Nature Med. 4: 328-332 (1998). In addition, another study found that the use of tumor peptide-pulse APCs resulted in a mouse model of a metastatic intracranial tumor model. Successful treatment has been demonstrated [DMAshley et al. , "Induction of anti-tumor immunity against central nervous system tumors of myeloid producing dendritic cells pulsed with tumor extract or tumor RNA", J. Exp . Med . 186: 1177-1182 (1997)] In addition, the inoculation of dendritic cells in human brain tumor patients has demonstrated encouraging results [JSYu et al. , “Systemic Cytotoxicity and Intracranial T- of Malignant Glioma Vaccination with Peptide Pulsed Dendritic Cells. Inducing cell infiltration ", Cancer Res. 61: 842-847 (2001).

The main limitation of the vaccine strategy based on each of these dendritic cells is that they rely on the acquisition of tumor tissue as a protein source for use in sensitizing dendritic cells in vitro. In general, sensitization involves culturing tumor cells and dendritic cells for use. This process provides dendritic cells access to tumor proteins and thus involves antigens preparing for the presence of digested antigens on T-cells to be administered to the patient. However, sensitizing dendritic cells in this field precludes the use of this therapeutic type when tumor tissue cannot be readily obtained; Disadvantages are that they often encounter various types of tumors because different environments may or may not make sensitization ex vivo. For example, a tumor may be surgically inaccessible, or its surgical procedure may be unreasonably dangerous to the health and safety of the patient. Even if the tumor tissue is easily accessible and can be sampled, the patient's tissue is required for surgical harvesting to be obtained by other procedures, preferably avoided.

Therefore, there is a need for supplementing vaccination strategies based on dendritic cells for these limitations and practical purposes. More specifically, there is a need for methods and compositions that supplement a vaccination strategy based on dendritic cells to treat tumors without the need to prime dendritic cells prior to administration to the patient in vitro.

The present invention provides compositions and methods based on dendritic cells for treating tumors. The method involves administering dendritic cells directly to a tumor or surrounding tissue located around the tumor and is effective in treating tumors that spread through the body of a mammal. The methods and compositions do not require dendritic cells to be used sensitized in vivo prior to administration to or incorporation into a patient. The method of the invention induces immune cell infiltration into tumors.

Other features and advantages of the invention will be apparent from the following examples, which are set forth in various embodiments. Certain embodiments may be effective in the treatment of brain tumors and other solid tumors that have been difficult to treat by various conventional methods.

The present invention relates to a method and a composition for treating a tumor by administering or injecting dendritic cells into a tumor or surrounding tissue (hereinafter the tumor and surrounding tissue collectively referred to as a "tumor site"). More specifically, the method involves the direct administration or injection of dendritic cells into a solid tumor. Tumor treatment in accordance with an embodiment of the present invention provides dendritic cells with more direct access to the tumor than was possible with conventional methods (particularly conventional methods where dendritic cells are administered peripherally). The technique of the invention is particularly useful when the tumor is surgically inoperable, when surgery is undesirable or when there is no part of the tumor that can be recovered as ex vivo sensitized dendritic cells for the tumor.

Because of the difficulty in accessing and treating brain tumors by conventional surgical methods, the method of the present invention is particularly useful for the treatment of tumors located in the brain of a mammal; It is particularly useful in the treatment of high- or low-grade malignant glioma, especially in the treatment of anaplastic astrocytoma or glioblastoma multiforme. However, many other types of tumors, particularly solid tumors that are located variously throughout the body of a patient, can be treated according to the methods of the present invention. Such other locations may include, but are not limited to, skin (eg, melanoma), chest, gastrointestinal tract, or respiratory tract.

The methods and compositions of the present invention are based, in particular, on the surprising discovery that sensitized dendritic cells are delivered to a patient's tumor site and are thus effective for the treatment of a tumor. Dendritic cells administered in this way essentially establish themselves in vivo contact or biochemical communication with target tumor cells and their homologous antigenic proteins, essentially priming themselves.

Desensitized dendritic cells include dendritic cells that do not rely on the acquisition of tumor tissue, such as protein sources, and the following to be cultured with them. In the conventional method, as mentioned above, dendritic cells are primed ex vivo . This generally involves culturing tumor cells and dendritic cells for use in the following. This process provides dendritic cells with access to tumor proteins, thereby allowing the cells to have associated tumor antigens and preparing the presence of digested antigens in T-cells for the introduction of dendritic cells into the patient's body. However, in various embodiments of the present invention, dendritic cells are delivered directly to the tumor layer or tumor site without first sensitizing out of the living body; Dendritic cells have tumor antigens only in vivo.

Dendritic cells suitable for use in accordance with the present invention may be obtained from any tissue that is independent or where cells are found, or may be cultured and supplied. In particular, the antigen-present dendritic cells are preferably used according to the method of the present invention. Such dendritic cells are found in the skin and introduced according to the methods of the invention, for example, having a certain quality similar to mammalian bone marrow or peripheral blood mononuclear cells (PMBCs), spleen, skin (ie, Langerhans cells, dendritic cells). Within the scope of "dendritic cells" as used herein. In the most preferred embodiment of the invention, cells obtained from the bone marrow of a mammal can be used. Thus, in one embodiment of the present invention, bone marrow can be harvested from mammals and cultured in medium. Any suitable medium that promotes the growth of dendritic cells can be used in accordance with the present invention and can be readily identified by one skilled in the art without undue experimentation.

In one embodiment of the invention, GM-CSF and / or IL-4 may be included in the medium described above. The medium is replenished at least partially every few weeks (eg 2-4 weeks) during the culturing process. After an appropriate time, the strains of dendritic cells are clearly visible in the medium and recovered from them in each strain group or in other easy amounts. Quantitative dendritic cells are passaged and preferably produced in the same amount so far.

Dendritic cells used in connection with the methods of the invention are delivered from the receiving site to any tumor site (eg, around a brain tumor or brain tissue) by any suitable method. The delivery method may include, but is not limited to, injection, infusion, inoculation, direct surgical treatment, or a combination thereof. In a preferred embodiment of the invention, dendritic cells can be administered to a mammal by direct inoculation through surgical operations; Standard inoculation procedures are techniques known to neurosurgery. In addition, suitable mechanisms for delivering dendritic cells to tumors or surrounding tissues are contemplated within the scope of the present invention and will be apparent to those skilled in the art without undue experimentation.

The composition of the present invention may comprise desensitizing dendritic cells in a pharmaceutical carrier. Any conventional pharmaceutical carrier can be used in accordance with the methods or compositions of the present invention and the appropriate carrier can be selected by one skilled in the art without undue experimentation. In one embodiment of the present invention the pharmaceutical carrier is saline, and other carriers may be used depending on the nature of the composition, as appropriate. For example, different compositions can be adapted to the patient's physiological differences (e.g., gender, weight, age, etc.), other tumor types (e.g., brain, chest, lungs, etc.), and other delivery techniques for other factors. Can be formed.

Dendritic cells administered to a patient in accordance with the methods and compositions of the present invention can be delivered in any combination of various additional substances and compounds. For example, the dendritic cells of the present invention can be administered to a patient with appropriate carriers, mediators, adducts, excipients, pharmaceutical supplements or other suitable agents that can be readily identified and recognized by those skilled in the art. In addition, the dendritic cells of the present invention can be administered in connection with other therapeutic compositions or formulations useful for the treatment of a tumor or for the relief or treatment of pain therein.

Quantitative dendritic cells are appropriately administered to a patient by the most convenient route, such as to form a composition of the present invention based on a variety of factors in order for the method of the present invention to be effective. Such factors include, but are not limited to, physical characteristics of the patient (eg, age, weight, gender, etc.), physical characteristics of the tumor (eg, location, size, growth rate, accessibility, etc.) and methods of the invention Expansion to other treatment methods that are performed concurrently with (eg, chemotherapy, radiation therapy, etc.).

Notwithstanding a variety of factors, in the preferred embodiment of the invention in practicing the method of the present invention for treating a tumor, 10 ⁵ to 10 ⁷ dendritic cells are administered to a patient in a single dose of 0.30 mL to 0.30 mL of saline. can do. Additional administration may be necessary depending on the technique and other factors, such as the severity of the tumor pathology. In a preferred embodiment of the present invention, 10 ⁶ dendritic cells were administered 1 to 5 times at intervals of two weeks.

In a preferred embodiment of the invention the mammal is treated with dendritic cells in combination with radiotherapy. Without being bound by any theory, it is believed that previous or concurrent treatment with radiation therapy makes dendritic cell vaccination more effective in killing tumor cells. Similarly, chemotherapy is administered prior to or concurrent with dendritic cell vaccination induced by apoptosis (ie, programmed cell death) tumor cells that may be beneficial.

Here, a tumor "treatment" is not limited thereto, but is therapeutically effective for improving tumors, alleviating the severity of complications, causing reductions in amount and / or size, suppressing expression, reinhibiting, exacerbating or any of the effects mentioned above Include effort.

1 shows a phenotypic diagram of dendritic cells according to an embodiment of the invention. Bone marrow cultures the resulting cells expressing cell surface phenotypic markers in which dendritic cells commonly express.

Figure 2 shows the inhibition of tumor growth by intratumoral dendritic cell vaccination according to an embodiment of the present invention. The dendritic cell treated group (column B) and saline treated group (column A) were vaccinated subcutaneously with 9L glioma tumors on the instep.

Figure 3 shows intratumoral vaccination with dendritic cells inducing T-cell infiltration into brain tumors according to an embodiment of the invention. Dendritic cells vaccinated with brain tumors compared to the saline treated control group (B) induce increased CD4 + T-cell infiltration (A).

4 shows the sustained survival of intracranial dendritic cell vaccination according to an embodiment of the present invention. Fishermen with 9L LacZ brain tumors vaccinated with intracranial dendritic cells survived longer than 90 days with 73% (n = 18) survival and longer survival compared to 8% (n = 12) saline-inoculated controls. .

Figure 5 shows the sustained survival of intracranial dendritic cell vaccination in animals with established intracranial neuroglioma according to an embodiment of the present invention. When intracranially inoculated with dendritic cells, mice with 9L neuroglioma survived longer than mononuclear cell-inoculated controls.

Figure 6 shows the promotion of T-cell infiltration in 9L intracranial neuroglioma inoculated with dendritic cells according to an embodiment of the present invention. Strong T-cell infiltration in dendritic cells inoculated (FIG. 6A), weak infiltration in mononuclear cells (FIG. 6B) and saline treated controls (FIG. 6C) were observed.

Figure 7 shows the migration of dendritic cells to systemic lymph nodes in accordance with an embodiment of the present invention and was performed in color. GFP expressing dendritic cells were found to be scattered within the main tumor mass (FIG. 7A) and deep neck lymph nodes (FIG. 7B) like the implanted site; However, GFP positive did not appear in lymph nodes (FIG. 7C) or in opposing neck lymph node tissues (not shown) in animals not inoculated with dendritic cells.

8 shows the enhancement of tumor specific cytotoxic T-cell activity by intratumoral inoculation of dendritic cells into intracranial brain tumors according to an embodiment of the present invention. T-cells of animals inoculated with dendritic cells showed a 1.48-fold increase in IFN-gamma RNA messages compared to mononuclear (1.12-fold) and saline (1.20-fold) treated controls.

Figure 9 shows the enhancement of tumor specific cytotoxic T-cell activity by intratumoral inoculation of dendritic cells into intracranial brain tumors according to an embodiment of the present invention. T-cells of animals inoculated with dendritic cells showed a 1.31-fold increase in secreted IFN-gamma compared to mononuclear (0.70-fold) and saline (1.10-fold) treated controls.

The examples described below show that when dendritic cells are transplanted directly into tumors, they inhibit the growth of brain tumors and that such treatment can extend the life of patients with brain tumors. The examples also show that vaccination of dendritic cells induces immune cell infiltration into brain tumors and systemic lymph nodes. In addition, the examples show that direct transplantation of dendritic cells into tumors placed throughout the mammal's whole body is effective for treating tumors.

Example 1 Intracranial Dendritic Cell Vaccination of Brain Tumors

Bone marrow was harvested from the femur and tibia of adult rats. Cells were obtained from RMP1 1640 medium (Gibco BRL) in medium containing GM-CSF and IL-4 (both available from R and D Systems; Minneapolis, MN; hereafter “R and D”) in a 24 well plate (Gaibersburg, MD; hereinafter "Gibco") at a concentration of 1 million per well. Medium was partially replenished every three days. After 8 days, strain groups of expanded suspended / partially adherent dendritic cells were clearly visible. Each of the cells was pooled and their phenotype profile evaluated using a flow immunocytometer. They were positive for MHC class II and B-7 co-stimulatory molecules; This confirmed that the cells were actually dendritic (FIG. 1).

Example 2 Inhibition of Tumor Growth of Dendritic Cells During Inoculation into Tumors

Dendritic cells were inoculated subcutaneously into the right instep of adult rats as a mixture of irradiated and viable 9L glioma cells. After two weeks of this treatment, two doses of dendritic cells were inoculated into each growth tumor. After two weeks of dendritic cell vaccination for 8 weeks, tumor size was measured using precision calipers. Compared with the control group treated with saline only, tumor size was significantly reduced in the intra-tumoral dendritic cell vaccination group (Fig. 2).

Example 3 Induction of Immune Cell (T-Cell) Infiltration by Brain Tumors of Dendritic Cell Vaccination

Dendritic cells were intracranially inoculated into the right striatum (basal ganglia) of adult rats as a mixture of irradiated and viable 9L glioma cells. After two weeks of this treatment, two doses of dendritic cells were inoculated into each growth tumor. After two weeks of dendritic cell vaccination, animals were euthanized and their brains harvested. The brains were immediately frozen and sliced into frozen micro cutters (available from Janis Research Company, Inc .; Wilmington, Mass.). Thinly sliced sample pieces were stained with T-cell markers (ie, CD4 and CD8). Tumors of animal groups vaccinated with dendritic cells showed an increase in the amount of T-cell infiltration compared to tumors of control animals treated with saline only (FIG. 3).

Example 4 Prolonged Survival of Dendritic Cell Vaccination in Mice with Brain Tumors

Dendritic cells were intracranially inoculated into the right striatum (basal ganglia) of adult rats as a mixture of irradiated and viable 9L glioma cells. After two weeks of this treatment, two doses of dendritic cells were inoculated into each growth tumor. Control animals were treated with intracranial saline inoculation at similar time points. Survival rates of animals are as follows. Mice treated with intracranial dendritic cell vaccination survived longer than 90 days after initial tumor transplantation, with longer survival compared to 5% for saline treated controls (FIG. 4).

Example 5 Prolonged Survival of Dendritic Cell Vaccination in Mice with Established Brain Tumors

A mixture of irradiated and viable 9L glioma cells was introduced into the right striatum (basal ganglia) of adult rats. Two days later, dendritic cells were inoculated into tumors of mice. Control animals were treated with intracranial monocyte / macrophage inoculation at similar time points. Survival rates of animals are as follows. Mice treated with intracranial dendritic cell vaccination survived longer than 90 days after initial tumor transplantation and survived longer compared to 10% for monocyte / macrophage treated controls (FIG. 5).

Example 6 Promoting T-Cell Infiltration into Brain Tumors of Dendritic Cell Vaccination

A mixture of irradiated and viable 9L glioma cells was introduced into the right striatum (basal ganglia) of adult rats. Mice were vaccinated with intratumoral inoculation with immature dendritic cells, monocytes or saline on days 2 and 16 post tumor implantation. The tumors were then harvested and stained with CD4 + and CD8 + T-cells twice weekly. The results show strong T-cell infiltration (FIG. 6A) in the dendritic cells inoculated and drug infiltration (FIG. 6B) in mononuclear cells and saline treated with control (FIG. 6C).

Example 7 Migration of Dendritic Cells to Systemic Lymph Nodes

To assess whether dendritic cells inoculated into intracranial brain tumors can be excreted into lymphatic system, deep neck lymph nodes from partially irradiated 9L glioma cells and from rats receiving intracranial transplantation of GFP expressing dendritic cells 4 days ago. Harvested. Brain fragments bearing tumors from the animals that turned out to be GFP positive dendritic cells were interspersed in the main tumor mass (FIG. 7A). Deep neck lymph nodes, like the implanted site, were infiltrated with multiple GPF expressing cells (FIG. 7B). On the other hand, lymph nodes from opposite neck lymph node tissues (not shown) that were not inoculated with dendritic cells or from mice with 9L glioma showed no GFP positive response.

Example 8 Enhancement of Tumor Specific Cytotoxic T-cell Activity with Dendritic Cell Inoculation Measured by Increased IFN-Gamma Message

Rats with brain tumors were treated with intratumor inoculation on Days 2 and 16 with the following tumor transplantation with dendritic cells, monocytes or saline (n = 4 per group). T-cells were isolated from spleen two weeks after the second intratumoral inoculation. Harvested T-cells were quadrupled restimulated with 9L glioma cells that were irradiated in vitro . Each of the two re-stimulations lasting 7 days was applied to each of the T-cell ends that were not exposed or reexposed to the newly irradiated 9L glioma cells (“targets” in FIG. 8) (“notarget” in FIG. 8). The sample was carried out.

After 4 hours of re-exposure, RNA was harvested from T-cells and analyzed by quantitative PCR to track IFN-gamma messages.

In addition, all samples were analyzed with CD8 RNA messages used as internal controls for normalizing IFN-gamma message levels. Differences in IFN-gamma message levels were compared based on PCR cycles at the point where a particular cycle passes the established threshold. A cycle difference of 1 was assumed to indicate a 2-fold difference in the message. After normalization to the CD8 message, the fold-increase in the IFN-gamma message in each treatment group was calculated by comparing the target and no target.

As can be seen in FIG. 8, T-cells of animals inoculated with dendritic cells showed a 1.48-fold increase in IFN-gamma RNA messages compared to monocytes (1.12-fold) and saline (1.20-fold).

Example 9 Enhancement of Tumor Specific Cytotoxic T-cell Activity with Dendritic Cell Inoculation Measured by Increased IFN-gamma Secretion

Rats with brain tumors were treated with intratumor inoculation on Days 2 and 16 with the following tumor transplantation with dendritic cells, monocytes or saline (n = 4 per group). T-cells were isolated from spleen two weeks after the second intratumoral inoculation. Harvested T-cells were quadrupled restimulated with 9L glioma cells that were irradiated in vitro . Two re-stimulations each lasting 7 days were applied to each of the T-cell ends that were not exposed or reexposed to the newly irradiated 9L glioma cells (“target” in FIG. 8) (“notarget” in FIG. 8). The sample was carried out.

After 24 hours of re-exposure, media was harvested from T-cell culture and analyzed by ELISA to quantify IFN-gamma protein secretion. For control purposes, media were also analyzed from re-promoted T-cells of animals that were not transplanted into brain tumors and therefore not manipulated (“non-tumor” group in FIG. 9).

Fold-increasing of IFN-gamma levels in each treatment group was calculated by comparing the target and no target. As can be seen in FIG. 9, T-cells of animals inoculated with dendritic cells showed a 1.31-fold increase in secreted IFN-gamma compared to mononuclear cells (0.70-fold) and saline (1.10-fold).

While the above description is directed to specific embodiments of the invention, it is obvious that various modifications may be made without departing from the spirit of the invention. For example, the protease inhibitors of the present invention can be used for the treatment of any condition in which inflammation is observed to be readily recognized by those skilled in the art without undue experimentation. The claims are intended to cover such modifications within the spirit and scope of the invention.

Therefore, the disclosed embodiments are described in the scope of the invention as indicated by the claims rather than the above description, but are not limited thereto and all modifications are possible within the scope and meaning of the claims.

Administer dendritic cells to the tumor or surrounding tissue if the tumor is surgically inoperable, if surgery is undesirable or if there is no part of the tumor that can be recovered as ex vivo sensitized dendritic cells for the tumor. Or a method of treating a tumor by injection.

Claims (51)

A method of treating a tumor in a mammal, comprising administering dendritic cells to a site selected from the group consisting of a tumor, a tumor surrounding tissue, and both the tumor and the tumor surrounding tissue. The method of claim 1, wherein said dendritic cells are desensitizing dendritic cells. The method of claim 1, wherein the tumor is a tumor surrounding tissue comprising a brain tumor and at least a portion of a mammalian brain. The method of claim 1, wherein the tumor is a tumor surrounding tissue comprising at least a portion of a breast tumor and a mammalian breast. The method of claim 1, wherein the tumor is a tumor surrounding tissue comprising a gastrointestinal tract tumor and at least a portion of the mammalian gastrointestinal tract. The method of claim 1, wherein the tumor is a tissue surrounding a tumor comprising at least a portion of a respiratory tumor and a mammalian respiratory tract. The method of claim 1, further comprising harvesting the dendritic cells from the mammal prior to administering the dendritic cells. 8. The method of claim 7, wherein harvesting dendritic cells from the mammal further comprises harvesting dendritic cells from a source selected from the group consisting of mammalian bone marrow, peripheral blood mononuclear cells (PMBC), spleen and skin. Way. 8. The method of claim 7, further comprising culturing the dendritic cells in the medium after harvesting the dendritic cells. 10. The method of claim 9, wherein the medium comprises granulocyte macrophage colony stimulating factor (GM-CSF). 10. The method of claim 9, wherein the medium comprises interleukin-4 (IL-4). The method of claim 9, wherein culturing the dendritic cells in the medium further comprises periodically supplementing the medium. The method of claim 9, wherein culturing the dendritic cell type strain group and the dendritic cell in the medium further comprises collecting the strain group. The method of claim 1, wherein administering the dendritic cells further comprises using a dosing technique selected from the group consisting of injection, infusion, inoculation, direct surgical treatment, direct inoculation via surgical operation, and combinations thereof. The method of claim 1, wherein administering the dendritic cells further comprises administering dendritic cells that are 10 ⁵ to 10 ⁷ dendritic cells in 0.05 mL to 0.30 mL saline. The method of claim 1, wherein administering the dendritic cells further comprises multiple administrations of the dendritic cells. 17. The method of claim 16, wherein the annual administration is every two weeks. The method of claim 1, wherein the tumor is surgically inoperable. The method of claim 1, wherein the tumor is a solid tumor. The method of claim 1, further comprising administering to the mammal by radiation therapy. The method of claim 1, further comprising administering to the mammal by chemotherapy. A method of inducing immune cell infiltration into a tumor of a mammal comprising administering dendritic cells to a site selected from the group consisting of a tumor, a surrounding tumor, and a tumor and a surrounding tumor. 23. The method of claim 22, wherein said dendritic cells are desensitizing dendritic cells. 23. The method of claim 22, wherein the tumor is a tumor surrounding tissue comprising a brain tumor and at least a portion of a mammalian brain. 23. The method of claim 22, wherein the tumor is a tumor surrounding tissue comprising at least a portion of a breast tumor and a mammalian breast. 23. The method of claim 22, wherein the tumor is a tumor surrounding tissue comprising a gastrointestinal tract tumor and at least a portion of the mammalian gastrointestinal tract. 23. The method of claim 22, wherein the tumor is a tissue surrounding tumor comprising at least a portion of a respiratory tumor and a mammalian respiratory tract. The method of claim 22, further comprising harvesting the dendritic cells from the mammal prior to administering the dendritic cells. The method of claim 28, wherein harvesting the dendritic cells from the mammal further comprises harvesting the dendritic cells from a source selected from the group consisting of mammalian bone marrow, peripheral blood mononuclear cells (PMBC), spleen, and skin. 29. The method of claim 28, further comprising culturing the dendritic cells in medium after harvesting the dendritic cells. 31. The method of claim 30, wherein the medium comprises granulocyte macrophage colony stimulating factor (GM-CSF). 31. The method of claim 30, wherein the medium comprises interleukin-4 (IL-4). 31. The method of claim 30, wherein culturing dendritic cells in the medium further comprises periodically supplementing the medium. 31. The method of claim 30, wherein culturing the dendritic cell type strain group and the dendritic cell in the medium further comprises collecting the strain group. The method of claim 22, wherein administering the dendritic cells further comprises using a dosing technique selected from the group consisting of injection, infusion, inoculation, direct surgical treatment, direct inoculation via surgical operation, and combinations thereof. The method of claim 22, wherein administering the dendritic cells further comprises administering dendritic cells that are 10 ⁵ to 10 ⁷ dendritic cells in 0.05 mL to 0.30 mL saline. The method of claim 22, wherein administering the dendritic cells further comprises administering dendritic cells consecutively. 38. The method of claim 37, wherein the annual administration is every two weeks. The method of claim 22, wherein the tumor is surgically inoperable. The method of claim 22, wherein the tumor is a solid tumor. The method of claim 22, further comprising administering to the mammal by radiation therapy. The method of claim 22, further comprising administering to the mammal by chemotherapy. A composition for treating tumors comprising an sensitized dendritic cell and a pharmaceutical carrier. 44. The composition of claim 43, wherein said desensitizing dendritic cells are harvested from a source of mammal. 45. The composition of claim 44, wherein said source is selected from the group consisting of mammalian bone marrow, peripheral blood mononuclear cells (PMBC), spleen and skin. 44. The composition of claim 43, wherein said pharmaceutical carrier is saline. 47. The composition of claim 46, further comprising 0.05 mL to 0.30 mL saline and 10 ⁵ to 10 ⁷ sensitized dendritic cells. 44. The composition of claim 43, wherein the nonsensitized dendritic cells are cultured in medium. 49. The method of claim 48, wherein the medium comprises granulocyte macrophage colony stimulating factor (GM-CSF). 49. The method of claim 48, wherein said medium comprises interleukin-4 (IL-4). 44. The method of claim 43, further comprising an additional component selected from the group consisting of carriers, mediators, adducts, excipients, adjuvants, therapeutic compounds or therapeutic agents useful for treating tumors, and therapeutic compounds or therapeutic agents useful for pain relief. Composition.

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