JP2010508364A - Dendritic cell tumor injection therapy and related vaccines - Google Patents

Abstract

The present invention is an effective and safe comprehensive combination therapy that safely and effectively reduces the size of tumor cells in a patient's tumor tissue. The method involves collecting monocyte cells from a patient's tumor tissue, culturing the monocyte cells using one or more factors to form immature dendritic cells from the monocyte cells, and It involves reducing the size of the tumor cells in the patient's tumor tissue by introducing mature dendritic cells and an adjuvant and introducing activated T cells into the patient's tumor tissue. The method includes local chemotherapy, systemic chemotherapy, tumor radiotherapy, or local tumor radiotherapy prior to introducing immature dendritic cells, adjuvants and activated T cells into the patient's body. And further pretreating with systemic chemotherapy in combination. The present invention also provides a cancer vaccine for reducing the size of a patient's tumor. The cancer vaccine includes immature dendritic cells derived from monocytes collected from a patient, an adjuvant, Antigens from tumors and activated T cells are included.
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Description

<Cross-reference of related applications>
This application claims priority from US Patent Application No. 60 / 855,905, filed Oct. 31, 2006, and is hereby incorporated by reference.

  The present invention relates to a therapy for cancer patients, and more particularly to a combination therapy using conventional cancer therapy in conjunction with cancer immunotherapy.

<Description of prior art>
Dendritic cells (DCs) are sentinel antigen presenting cells of the immune system (Banchereau, J. et al, Ann NY Acad Sci, 987: 180-7, 2003). These cells have the ability to obtain antigenic material from their environment and then initiate an active immune response. Recognizing this possibility, DCs have been used as a platform to deliver candidate vaccines for cancer immunotherapy (den Brok, MB et al., Expert Rev Vaccines, 4: 699-710, 2005). In most of these studies, immature DCs are expressed in vitro from monocyte cells or stem cell precursors and carry candidate vaccines in the form of tumor cell lysates, proteins or HLA class I allelic signature peptides. . DCs mature with selected cytokines and are administered by intravenous or parenteral routes.

  As another approach, these cells at the injection site can theoretically enhance the generation of an immune response to a wide variety of antigenic determinants by injecting immature DCs together with an adjuvant directly into the patient's tumor tissue. A variety of tumor cell products can be obtained (Chi, KH et al., J Immunother, 28: 129-35, 2005).

  The combination of intratumoral injection of immature DCs and conventional treatments such as radiotherapy and chemotherapy has been proposed to increase immune response and improve clinical outcome (Hoffmann TK et al., Cancer Res , 60: 3542-9, 2000). Such treatments can induce apoptosis, create an environment that enhances the ability of DCs to utilize tumor antigens, and can mature and migrate lymph nodes that effectively display tumor antigens to T cells (Pierre, P et al., Nature, 388: 787-92, 1997). It is suggested that chemotherapy to control immunosuppressive regulatory T cells or injection with other cell products such as activated T (AT) cells can also help enhance immune responsiveness (Berd, D. et al., Cancer Res, 44: 5439-43, 1984). Cyclophosphamide, for example, has been shown to not only exert an immunoactive effect, but also reduce many regulatory T cells and increase apoptosis (Berd, D. et al., Cancer Res, 44: 5439-43, 1984). The injection of autologous AT cells, an inducer that expresses CD4OL and affects DC maturation, has shown a clinical stabilization situation. The injected cells can expand significantly in vivo and maintain a broad T cell spectrum (Berger C. et al., Blood, 101: 476-84, 2003). Therefore, administration of large amounts of AT cells may increase the T cell pool capable of responding to cancer. The anticancer effect can be further enhanced by the release of cytokines such as IFNγ by T cells.

  However, there is a need to provide more effective treatment protocols for cancer patients.

<Outline of the invention>
The present invention provides a comprehensive combination therapy that reduces the size of tumor cells in a patient's tumor tissue to meet this need.

  In one aspect of the present invention, a method for reducing the size of tumor cells in a patient's tumor tissue is provided, the method comprising collecting monocyte cells from the patient's tumor tissue and immature trees from the monocyte cells. Monocytes are cultured with one or more factors to form dendritic cells, immature dendritic cells and an adjuvant are introduced into the patient's tumor tissue, and activated T in the patient's tumor tissue Including introducing cells. A few days after the introduction of immature dendritic cells and adjuvant into the patient's body, activated T cells are introduced and antigen-specific T cells are generated.

  Introduction of immature dendritic cells, adjuvants and activated T cells can be effected by intratumoral injection.

  Patient monocytic cells, i.e., peripheral blood mononuclear cells (PBMCs) can be cultured using factors such as GM-CSF and IL-4, for example.

  T cell activation can be effected by exposing the T cell to an anti-CD3 antibody or an ionophore such as, for example, ionomycin.

  An activated lymphocyte solution can be prepared using CD3-CD28 beads.

  Adjuvants used in the methods of the present invention include, but are not limited to, activated lymphocyte solution (ALM), super lymphoid tissue extract, beta glucan or keyhole limpet hemocyanin. .

  In another aspect of the invention, a method for reducing the size of tumor cells in a patient's tumor tissue, wherein immature dendritic cells, adjuvants and activated T cells are introduced into the patient's tumor tissue. It further includes pre-treating the patient with local chemotherapy before.

  In another aspect of the invention, a method for reducing the size of tumor cells in a patient's tumor tissue, wherein immature dendritic cells, adjuvants and activated T cells are introduced into the patient's tumor tissue. The method further includes pre-treating the patient with systemic chemotherapy before.

  In another aspect of the invention, a method for reducing the size of tumor cells in a patient's tumor tissue, wherein immature dendritic cells, adjuvants and activated T cells are introduced into the patient's tumor tissue. The method further includes pre-treating the patient with systemic chemotherapy and local tumor radiation therapy.

  Another aspect of the present invention provides a cancer vaccine precursor that, when introduced into a tumor tissue, binds to an antigen and reduces the size of the tumor in the patient's body. The cancer vaccine is collected from a patient. Included are immature dendritic cells derived from monocytes, adjuvants, antigens from tumors in the patient's body and activated T cells.

  An object of the present invention is to provide a safer and more effective combination therapy protocol for cancer patients.

  It is a further object of the present invention to effectively reduce the size of tumor cells in a patient's body using intratumoral injection of immature dendritic cells, adjuvants and activated T cells in addition to conventional therapies. Is to provide a safe and secure method.

  It is a further object of the present invention to provide an effective and safe cancer vaccine for reducing the size of a patient's tumor cells, which comprises immature dendritic cells, adjuvants and activated T cells. Contains.

  The invention will be more fully understood by reference to the accompanying drawings in conjunction with the following description of the preferred embodiments.

FIG. 1 shows four treatment protocols for an embodiment of the present invention.

FIG. 2 is a PET CT image of recurrent upper laryngeal cancer recurrence in a 45-year-old woman, before AT cell treatment (A), after 3 weeks (B), after 3.5 months (C), and after 9 months (D ).

FIG. 3 is a graph showing that CEA blood concentration is decreased by the treatment using dendritic cells and AT cells (protocol IV).

<Description of preferred embodiments>
The present invention provides a safe and effective comprehensive combination therapy for reducing the size of tumor cells in a patient's tumor tissue.

  One embodiment of the present invention provides a method for reducing the size of tumor cells in a patient's tumor tissue, the method comprising collecting monocyte cells from the patient's tumor tissue and immature from the monocyte cells. Monocytic cells were cultured with one or more factors to form dendritic cells, immature dendritic cells and adjuvants were introduced into the patient's tumor tissue, and activated within the patient's tumor tissue Including introducing T cells. A few days after the introduction of immature dendritic cells and adjuvant into the patient's body, activated T cells are introduced and antigen-specific T cells are generated.

  Introduction of immature dendritic cells, adjuvants and activated T cells can be effected by intratumoral injection.

  Patient monocytic cells, i.e., peripheral blood mononuclear cells (PBMCs) can be cultured using factors such as GM-CSF and IL-4, for example.

  T cell activation can be effected by exposing the T cell to an anti-CD3 antibody or an ionophore such as, for example, ionomycin.

  An activated lymphocyte solution can be prepared using CD3-CD28 beads.

  Adjuvants used in the methods of the present invention include, but are not limited to, activated lymphocyte solution (ALM), super lymphoid tissue extract, beta glucan or keyhole limpet hemocyanin. .

  In another embodiment of the present invention, a method for reducing the size of tumor cells in a patient's tumor tissue, wherein immature dendritic cells, adjuvants and activated T cells are introduced into the patient's tumor tissue. Further comprising pre-treating the patient with local chemotherapy prior to.

  In another embodiment of the present invention, a method for reducing the size of tumor cells in a patient's tumor tissue, wherein immature dendritic cells, adjuvants and activated T cells are introduced into the patient's tumor tissue. Further comprising pre-treating the patient with systemic chemotherapy prior to.

  In another embodiment of the present invention, a method for reducing the size of tumor cells in a patient's tumor tissue, wherein immature dendritic cells, adjuvants and activated T cells are introduced into the patient's tumor tissue. Further comprising pre-treating the patient with systemic chemotherapy and local tumor radiation therapy prior to.

  Another embodiment of the present invention provides a cancer vaccine precursor that, when introduced into tumor tissue, binds to an antigen and reduces the size of the tumor in the patient's body. Included are immature dendritic cells derived from harvested monocyte cells, adjuvants, antigens from tumors in the patient's body and activated T cells.

  As used herein, “patient” refers to mammals including humans.

  The present invention is more specifically described in the following non-limiting examples, which are for illustrative purposes only and that many modifications and changes can be made by those skilled in the art. It will be clear.

1. INTRODUCTION A safe pilot test was conducted to investigate whether the blood circulation state was stabilized by combining DC cells and AT cell therapy with normal therapy. Several protocols were developed, in which immature DCs and AT cells were generated from peripheral blood and injected into the metastatic lesions of patients in the advanced stage of malignancy. On day 0, patients were first injected with autologous immature DCs and adjuvant. Immediately, immature DC injections were confirmed not to cause rejection and additional treatment modalities known to enhance immune responses that were substantially non-toxic were incorporated into the treatment group. Radiation therapy or chemotherapy was given before immature DC administration. Certain tumor sites were affected by radiation therapy (20-50 Gy) and induced antigenicity derived from degradation of tumor DNA. In some cases, chemistries including cisplatin (CDDP), cyclophosphamide (Cytoxan), fluorouracil (5-FU), docetaxel (DTX) or adriamycin (ADM), which are used for conventional treatment of specific malignancies Therapeutic agents were administered to reduce the presence of cells that can induce apoptosis or suppress immune responses. AT cell therapy was performed for 3 days following immature DC and adjuvant treatment.

II. Patients and methods Patient treatment 37 patients aged 41-83 years were enrolled in the DC and AT cell therapy study protocol. All patients had relapsed after undergoing standard chemotherapy and / or radiation therapy for cancer and failing. Table 1 shows the types of cancer and the number of cases included in the study. Patients were included in the study protocol and summarized as in Table 2.

All patients will receive immature DCs (range 4.9 × 10 ^{6 to} 5.9 × 10 ⁹ per injection) and adjuvant, either alone or with conventional chemotherapy, radiation therapy and / or AT Administration in combination with cells (4.1 × 10 ^{1 to} 7.5 × 10 ⁸ ). Prior to treatment, tumors were biopsied and blood tests were performed. FIG. 1 shows the courses of each of the five protocols that were followed. All patients received immature DCs and adjuvant on day 0. Patients enrolled in Protocol I received immature DCs and adjuvant. Protocol II patients were pretreated with local chemotherapy prior to administration of immature DCs and adjuvant. Other groups of patients received AT cells 3 days after injection of immature DC and adjuvant, with or without pretreatment with chemotherapy and radiation therapy. Chemotherapy was performed intratumorally (Plutocol III), systemically (Protocol IV), or in combination with radiation therapy (Plutocol V). The patient had signs of rejection locally and / or systemically. Cell biopsies and blood tests were performed 3-4 weeks after treatment. Local or metastatic tumor regression was assessed by PET-CT imaging. Treatment was repeated when tumor regression was observed.

2. Collection and isolation of peripheral blood mononuclear cells (PBMCs) Leukopheresis is a manual adjustment of the COBE Spectra blood separator (Gambro KK, Tokyo, Japan) using the monocyte nucleus (MNCs) collection program (version 7.1). It was carried out by using together with the plasma pump. Anticoagulation was performed using ACD-A (ratio 12: 1, Baxter, Deerfield, IL). The injection rate was 40-60 ml / min, the collection rate was 1 ml / min, and the separation factor was 700. MNCs were subjected to cell separation and stored at a low temperature of 1.5 × 10 ⁸ cells / ml / vial in 10% DMSO in an AIM-V solution (Gibco, Invitrogen, Tokyo) at −80 ° C. for 1 to 3 months. Saved with. Prior to use in DC and AT cell cultures, MNCs were thawed in a 3V water bath, washed twice in AIM-V solution and counted.

3. Generation of immature DCs (iDCs) Thawed MNCs (approximately 6 × 10 ⁸ ) were resuspended in 20 ml of AIM-V solution, each of 44-T-75 cm ² containing 1 Oral of AIM-V solution. 5 ml aliquots were dispensed into polystyrene flasks. After culturing at 37 ° C. for 2 hours, non-adherent cells were removed by pipette, transferred to a conical tube, and stored for AT cell preparation (see below). 15 ml of DC growth solution (AIM-V solution supplemented with 800 IU / ml GM-CSF (CellGenix, Germany) +500 Uml IL4 (BD Pharmingen)) was added to each flask containing adherent cells. The flask was incubated at 37 ° C. with 5% carbon dioxide. The growth solution was renewed on day 3 and DCs were collected on day 7 by pipetting. The collected cells were counted, resuspended in an AIM-V frozen solution containing 20% autologous serum + 10% DMSO, and stored frozen in a BICELL container (Nihon Freezer Co., Tokyo, Japan). The BICELL container is capable of gradual cell freezing with a programmed freezing process (freezing rate of 1 ° C./min). Cells were stored at −80 ° C. until injected into patients (0.5-3 months).

4). Generation of AT cells Non-adherent T cells (approximately 6-9 × 10 ⁸ cells) collected after monocyte adherence were washed and resuspended with 20 ml of AIM-V solution for DC generation. It became cloudy. 5 ml of this cell suspension and 35 ml of AT cell solution were added to four T-225 cm ² flasks each coated with an anti-CD3 antibody (Yamazaki, T. et al, Neurol Med Chir, Tokyo, 32: 255-61, 1992). The flask was then incubated at 37 ° C. with 5% carbon dioxide. Three hours before harvesting, 1 ug / ml of ionomycin (Sigma, USA) was added to the solution to stimulate T cells (Sato, T. et al., Cancer Immunol Immunother, 53: 53-61, 2004). Since the AT cell solution consists of an AIM-V solution with IL2 and autologous serum, each flask will contain a final level of 1000 IU / ml, 10% autologous serum. Anti-CD antibody coating was performed by adding 10 ml of 5 ug / ml anti-CD3 antibody (Orthoclone, OKT3 injection. Janssen Pharmaceutical, KK) in DPBS to the flask and let stand at room temperature for 2 hours, before adding cells The flask was washed 3 times with 15 ml DPBS. Harvested cells were cryopreserved and stored at −80 ° C. (0.5-3 months) prior to injection into patients.

5). Cell culture sterility and purity test A microbial test was performed by culturing the cultured cells on agar at 37 ° C. 7 days before collecting the cells. Only cells with negative results were used clinically. Cell purity was determined using a commercially available chromogenic endotoxin test kit (Toxicolor system LS-50M, Seikagaku Corp., Tokyo, Japan) according to the manufacturer's instructions and endotoxin level (<0.5 EU / ml). It was evaluated by measuring. The concentration of endotoxin in each sample was measured by the Limulus Amoeba Lysate Test (LAL). Briefly, 50 ul of each sample was transferred to an endotoxin-free 96-well plate (Toxipet LP; Seikagaku), 50 ul of LAL reagent was added to each well, and the mixture was incubated at 37 ° C. for 30 minutes. To stop the reaction, 0.8 M acetic acid was added to each well, mixed and measured at 405 nm. Endotoxin levels were measured against the reference endotoxin E. coli 011 1: B4 LPS (Toxicolor system CSE; Seikagaku).

6). Cell culture characteristics Standard flow cytometry labeling protocols were used to determine the expression of cell surface markers (Parks, D. et al., Flow Cytometry and Fluorescence-Activated Cell Sorting; Raven Press Ltd, New York, 1989). DCs were cultured for 7 days, characterized for surface marker expression, thawed, and then using fluorochrome labeled monoclonal antibodies against CD11c, CD14, CD40, CD80, CD83, CD86 and HLA-DR (BD Pharmingen) DC injection was performed. AT cells were evaluated for CD3, CD4, CD8, CD11c, CD14, CD19, CD25, CD45, CD56, CD154 (CD40L) and HLA-DR after culture. A minimum of 10,000 events was obtained with a BD FACscan (BD Biosciences) and data was analyzed using Cell Quest analysis software. Marker expression is expressed as the average level of fluorescence in the standard deviation of the patient's cell culture.

7). Cell preparation for injection DCs and AT cell vials were thawed in a 37 ° C. water bath for 1 hour prior to the planned injection. 1 ml of AIM-V solution was added to each thawed vial and the vials were left for 2 minutes, transferred to 50 ml of solution and centrifuged (300 × g, 7 minutes) to remove DMSO. Cells were resuspended and counted in fresh solution and samples were removed for sterility testing on agar. The remaining cells were distributed into two labeled microtubes (500 ul each) and placed on ice for transport to the hospital for injection into the patient's metastatic lesion. Cells were injected based on CT scan information.

8). Adjuvant generation The protocol included different adjuvants as follows. Commercially available (1) Super lymphoid tissue extract (S-LTE, 0.5 ml, Shukokai, Inc., Japan, Protocol I and Protocol II), (2) β-glucan (0.5 ml-1.0 ml at 50 ug / ml in saline, Wako, Japan, protocol II and protocol III), (3) Keyhole limpet hemocyanin (KLH) 1 mg (Immucothel, protocol IV). These were prepared as recommended by the manufacturer. Also, (4) Activated lymphocyte solution (ALM) (Protocols III, IV and V, prepared in our clinical laboratory from ertriated lymphocytes (Gambro BCT, Colorado, USA)). To make ALM, lymphocytes were suspended at 1 × 10 ⁶ cells / ml in 50 ml of XVIVO solution (Cambrex, Walkersville, MD) per T-75 cm ² flask and CD3-CD28 T cell expander beads (Dynal. Norway). (Levine, BL et al., J Immunol, 159: 5921-30, 1997) was added at a ratio of 1 bead to 1 cell and cultured at 37 ° C. and 5% carbon dioxide for 2 days. did. Supernatants were collected by centrifugation (300 × G, 7 minutes) and stored at 4 ° C. for later use. All adjuvants were delivered intratumor simultaneously with the delivery of DCs.

9. Chemotherapy Chemotherapy types were selected based on standard treatments for specific cancers. Chemotherapy was given intratumorally or systemically according to the protocol. 21 patients received cyclophosphamide (CPM, 5-800 mg, Shinogi), 5 patients received cisplatin (CDDP, 2.5-5.0 mg, Nihon Kayaku), and 1 patient Was given docetaxel (DTX, 55 mg, Aventis) and one patient was given doxorubicin (ADM, 20 mg, Kyowa Hakko). CDDP was given systemically to one patient with upper pharyngeal cancer in combination with fluorouracil (5-FU, 900 mg / day, Kyowa Hakko).

10. Radiotherapy Radiotherapy (20-50 Gy) was given to two patients with Protocol V. 50 Gy was given to one metastatic upper pharyngeal cancer patient and 20 Gy was given to other recurrent metastatic esophageal cancer patients. Radiotherapy was given to the patients according to a predetermined protocol (Shimamura, H. et al., Eur Surg Res, 37: 228-34, 2005). The radiation range of the tumor site was designated in advance, and the radiation was given 2 to 2.5 Gy per dose for 3 to 4 weeks. Radiation therapy was given during the same period as chemotherapy. DCs are injected intratumor 7 days after radiation therapy is completed, AT cells are injected intratumor 10 days after radiation therapy is completed, ie, DC cells are injected in the applied protocol. And then injected on the third day.

11. Assessment of response Carcinoembryonic antigen (CEA) levels elevated in cancers (eg, large intestine, rectum, stomach, breast, lung and pancreatic cancer) were used to monitor patient response to treatment and disease recurrence. Tumor size was evaluated before injection and 3 to 4 weeks after treatment using PET-CT imaging and a solid cancer therapeutic effect determination method (RECIST) for both injection and irradiation sites and metastatic sites. Baseline sum was assessed as the largest diameter in the measurable lesion. The efficacy of the treatment is defined as complete response (CR, complete disappearance of measurable lesions and defined as new lesions not appearing or maintained for more than 4 weeks), partial Response (PR, defined as the tumor size injected is reduced by at least 30%), disease stable (SD, size reduced by less than 30% or enlarged by less than 20%, no new lesion development) Defined as progressive disease (PD, defined as an increase of more than 20% from the original measurement). Side effects were recorded using common WHO toxicity criteria.

12 Statistical measurements Tumor size data were compared using Student's t-test and paired t-test to measure changes before and after treatment. Statistical significance was p <0.05.

III. Results 1. Characteristics of DCs and AT cells Adherent PBMCs can be distinguished against DCs. Adherent cells from various cancer patients cultured for 7 days show costimulatory markers and low CD83 surface antigen, indicating immature DCs (Table 3). Cell viability remained as high as 85-95% after 7 days of culture, similar to the following cryopreservation. Lymphocytes treated with anti-CD3 and activated T-cell ionomycin showed CD25 and CD4OL (Table 3). The viability of the stimulated cells ranged from 75 to 90% before and after cryopreservation.

2. Toxicity Blood and urinalysis performed during and after treatment include total protein, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), lactate dehydrogenase (LDH), alkaline phosphatase, total bilirubin, blood There were no changes in medium urea nitrogen (BUN), creatinine, total cholesterol, triglycerides, blood glucose, hematuria, or proteinuria.

All treatments were generally well tolerated and only a few of the groups receiving AT cell treatment were associated with rejection (Table 4). Patients receiving DCs + adjuvant with Protocols I and II did not observe local or systemic rejection immediately after injection or during the follow-up period. In patients receiving AT cell therapy, fever (38 ° C.) was observed immediately after injection in four patients with protocol III, and two patients with protocol V had swelling, lymphadenopathy and pain at the injection site, I experienced a fever.

3. Patient Response Clinical efficacy was observed by comparing tumor size before treatment with tumor size 3-4 weeks after the first DC injection (Table 5).

  Of 7 patients (19%) out of 37 patients, the therapeutic effect of complete cure (1 patient in Protocol V) or partial regression (6 patients in Protocol I and Protocol II) at the tumor site observed for at least 3 months It was seen. Complete tumor regression was diagnosed with recurrence of upper laryngeal cancer and effectively received DCs, AT cells, and both chemotherapy and radiotherapy (protocol V, FIG. 2) at both treatment and metastasis sites It was seen in.

  In 25 of 37 patients (67%) of all protocols, tumor size remained the same or unchanged for at least 3 months. Serum tumor markers followed by the patient showed a specific tumor and remained stable over this period. However, one metastatic gastric adenocarcinoma patient showed a dramatic reduction in CEA levels after injection of DCs and AT cells (FIG. 3). The patient had been diagnosed for approximately 3 years before enrolling in our protocol and experienced multiple chemotherapy and gastrectomy. Despite these standard therapeutic approaches, CEA levels continued to increase and the size of the peraortic lymph nodes increased. The patient received 800 mg of cyclophosphamide before injecting DCs following the AT cells. Serum CEA levels showed a steady decline reaching a level of 6.7 in the most recent study.

  Progressive disease in 4 patients who received DCs alone or DCs with chemotherapy (protocols I and II) and 1 patient who received DCs and AT cells with chemotherapy and radiation therapy (protocol V) Was observed. In the latter, stable symptoms were observed for 1 month, and progression to untreated tumors was observed in 3 months.

IV. DISCUSSION Anti-cancer treatments including cancer radiotherapy, chemotherapy and adoptive cell therapy have shown efficacy by themselves. The primary objective of this study is to treat patients with various advanced cancers with their own immature DCs directly into the tumor and other approaches such as injecting activated T cells directly into metastatic foci. It was to determine the safety and effectiveness of the method and combination with pre-treatment such as local chemotherapy, radiation therapy, and systemic chemotherapy at the injection site. This study has shown that combining different treatment methods with unique properties significantly increases the anti-cancer response within a single protocol.

  DCs serve as important antigen-presenting cells for specific T cell immunity induction (Banchereau, J. et al., Nature 392: 245-52; 1998), eg, many clinical trials for various cancers Establishing the foundation of is established. Some trials have used DSc generated by the addition of tumor-specific antigens in vitro, which leads to the induction of anti-tumor immunity when returned to the host (Hsu, FJ. et al., Nat. Med., 2: 52-58, 1996). However, in cancers where the antigen associated with the tumor is not well defined, the entire tumor is used in the form of apoptotic cells, as in this study. Apoptotic cells effectively cross-prime T cell responses and induce strong immunity.

  Ionizing radiation therapy or chemotherapy is used to induce apoptosis. Tumor radiation therapy generally involves releasing intracellular contents into the surrounding environment that accompany the activation of (but not limited to) cytokines, prostaglandins and heat shock proteins. Causes inflammation. Local delivery of chemotherapy creates an environment similar to the tumor site. Such strategies not only create an environment that promotes antigen acquisition, processing, and maturation, but also stimulate the migration of DCs to draining lymph nodes that interact with a wide range of potential effector cells.

  Combining conformal radiation therapy and chemotherapy with DC intratumoral injection improves the immune responsiveness that appears in both human and murine models and, from a practical standpoint, antigens added on DCs by direct injection A sufficient amount can be secured.

  Since the primary objective of this study was to evaluate the safety and efficacy of different therapies using immature DCs as basic immunity, the group of patients was initially treated directly with an untreated tumor. DCs were injected (protocol I). No rejection was observed using this injection method, some patients actually showed a local response to the tumor site (25%, 2/8) and some had stable symptoms (50%, 4/8), while several patients (25%, 2/8) showed disease progression. In the next group of patients, no rejection was observed when local cancer chemotherapy was added to enhance antigen uptake by DCs (protocol II). In fact, adding local treatment slightly improved the local partial tumor response (40%, 4/10). The patient also became stable (40%, 4/10) and progressive disease (20%, 2/10).

  Tumor cells that are resistant to apoptosis may not induce immunity due to the lack of local secretion of immunosuppressive factors such as TGFO and IL10 and sufficient systemic DCs to reach the tumor periphery (Bodey, B. et al., Anti cancer Res., 20: 2665-76, 2000). The tumor environment determines the maturity state that will induce resistance or immunity. Therefore, it may be necessary to combine other pro-inflammatory cytokines and immuno-inducing substances with immature DSc to complete DC activation at the site of injection.

  Therefore, combining DSc with other cell types such as AT cells is a promising treatment. DSc activates T cells and these interactions are important for establishing an effective immune response. Activated T cells can influence antigen presentation by DCs by a number of mechanisms. AT cells express surface molecules that physically interact with immature DCs and, in addition, affect the maturation and migration of DCs to partial lymph nodes where an adaptive immune response to the resulting antigen occurs. Producing cytokines and chemokines. As a result, a tumor-specific immune response may be generated and will be established in long-term memory.

  There are different strategies to generate adaptive therapeutic T cells that affect not only antigen specificity, tumor affinity and cell phenotype, but also properties such as in vivo lifespan, trafficking and antitumor effects give. T cells proliferate by a variety of methods, including cells that are stimulated in vitro and designed for antigen presentation (eg, peptide-pulsed DCs, transfected RNA) , Using antigen-presenting cells to derive antigen-specific T cells from peripheral blood. By collecting infiltrating lymphocytes from tumor cells, a group of polyclonal T cells having a broad response to the tumor to the tumor is generated. Infusion of these cells again after growth in vitro is used as a method of immunotherapy treatment. Another approach is to activate and proliferate peripheral blood lymphocytes by triggering T cell receptors and costimulatory molecules with antibodies and / or using cytokines that promote T cells. It is.

  In this study, AT cells are proliferated from peripheral blood lymphocytes and up-regulated CD4OL by anti-CD3-IL2 culture and ionomycin activity. A few days after DC injection, cultured AT cells were injected intratumor based on the idea that antigen-specific T cells would be generated. Patients undergoing AT cell therapy may have 1) local chemotherapy (protocol III), 2) systemic chemotherapy (protocol IV), or 3) systemic chemotherapy and partial tumor radiotherapy (protocol V) ) Was pretreated.

  When local tumor radiotherapy was added to the third AT cell protocol (protocol V), after AT cell injection, two patients were given temporary fever, lymphadenopathy, metastatic site pain, and AT cell injection. A swelling of the site appeared. These symptoms improved within a week, and notably a complete tumor regression was observed in one pharyngeal cancer patient at both the treated and untreated sites. During the first 3 weeks after treatment, there was regression in all areas, including the pharynx, larynx, and cervical lymph nodes, except for the injection site, and until 9 months, the tumor was completely regressed, including the injection site . A second patient in this treatment group who was in a highly advanced esophageal cancer stage appeared to respond early but developed progressive disease. It is not clear which part of the treatment contributes to the response by a strong inflammatory response, control or lack of immunosuppressive factors, or in combination with these or other events. Fever is associated with two groups that received both AT cells and local tumor treatment with either chemotherapy (Protocol III) or radiation therapy (Protocol V) and causing such a response. Suggests release of known inflammatory cytokines. Indeed, these rejections appear to be necessary for the immune response that occurs in the tumor.

Chemotherapeutic agents were administered systemically or locally to determine if these agents could be used safely and effectively with immunotherapeutic treatments. The administration of this chemotherapy was not generally the optimal amount to treat the patient's malignancy, but rather as an aid in coping with the immune response to cancer. Chemotherapy can induce apoptosis that increases antigen uptake in tumor cells. More importantly, these drugs may produce regulatory T cells. Regulatory cells, commonly defined as CD4 ⁺ CD25 ⁺ , have been found to be present in most human solid tumors, and their high number and frequency appear to correlate with overall survival. (Beyer, M. et al., Blood 108: 804-11, 2006). Their removal or selective destruction by directly targeting CD25 at the cell surface is known to be the result of an enhanced tumor-specific response to immunotherapy. It has been recognized that the use of low doses of cyclophosphamide not only reduces the number of regulatory cells, but also exerts an immunostimulatory effect. No adverse events were seen when chemotherapy was given systemically prior to DC and AT cell injection. The purpose of this study is safety and does not necessarily require therapeutic effects, but the obvious advantages observed by combining these approaches are that each treatment (chemotherapy, radiotherapy, immature DC and AT cells Injection) should be considered with the recognition that it was able to affect the tumor. In fact, two patients (head and neck cancer (protocol V), gastric cancer (protocol VV)) both failed standard radiotherapy / chemotherapy, but received radiotherapy and / or chemotherapy and then In addition, since DCs and AT cells were injected intratumorally, a sufficient response was shown.

  Overall, in this study, all of the treatment protocols are relatively well tolerated, and conventional tumor injections of immature DCs and AT cells are used in order to enhance or reverse resistance to immunogenicity. It has been demonstrated that combination plus treatment can be safely and effectively incorporated into a single protocol.

  The invention has been specifically described and described with reference to preferred embodiments, but those skilled in the art will recognize, without departing from the spirit and scope of the invention, the form and details as defined in the appended claims. It should be understood that various modifications can be made.

Claims (20)

A method for reducing the size of tumor cells in a patient's tumor tissue, comprising:
Collecting monocyte cells from a patient;
Culturing the monocyte cells with one or more factors to form immature dendritic cells from the monocyte cells;
Introducing immature dendritic cells and an adjuvant into the patient's tumor tissue; and
Introducing activated T cells into the patient's tumor tissue;
A method for reducing the size of tumor cells in a tumor tissue of a patient comprising:   2. The method of claim 1, wherein the introduction of immature dendritic cells, adjuvants and activated T cells is effected by intratumoral injection. The method according to claim 1, wherein the activated T cells are introduced after introducing immature dendritic cells and an adjuvant into the body of a patient to generate antigen-specific T cells.   The method according to claim 1, wherein the monocytic cells are cultured using GM-CSF and IL-4.   The method of claim 1, wherein the adjuvant is selected from the group consisting of activated lymphocyte solution, superlymphoid tissue extract, beta glucan and keyhole limpet hemocyanin.   6. The method of claim 5, wherein the activated lymphocyte solution is made using CD3-CD28 beads.   2. The method of claim 1, wherein activation of activated T cells is effected by exposing the T cells to an anti-CD3 antibody and an ionophore.   8. The method of claim 7, wherein the ionophore is ionomycin.   The method of claim 1, further comprising pre-treating the patient with local chemotherapy prior to introducing the adjuvant and activated T cells into the patient's tumor tissue.   2. The method of claim 1, further comprising pre-treating the patient with systemic chemotherapy prior to the step of introducing immature dendritic cells, adjuvants and activated T cells into the patient's tumor tissue. Method.   Further comprising pre-treating the patient with systemic chemotherapy and local tumor radiotherapy prior to introducing the immature dendritic cells, adjuvant and activated T cells into the patient's tumor tissue. The method of claim 1.   The method of claim 1, wherein the patient is a human. A cancer vaccine that, when introduced into tumor tissue, binds to an antigen and reduces the size of the patient's tumor,
A cancer vaccine comprising immature dendritic cells derived from monocyte cells taken from a patient, an adjuvant, an antigen from a tumor in the patient's body and activated T cells.   The cancer vaccine according to claim 13, which is introduced into the tumor tissue of a patient by intratumoral injection.   The cancer vaccine according to claim 13, wherein the monocytic cells are cultured using GM-CSF and IL-4.   The cancer vaccine according to claim 13, wherein the adjuvant is selected from the group consisting of an activated lymphocyte solution, a superlymphoid tissue extract, beta-glucan, and keyhole limpet hemocyanin.   The cancer vaccine according to claim 16, wherein the activated lymphocyte solution is prepared using CD3-CD28 beads.   The cancer vaccine according to claim 13, wherein activation of activated T cells is effected by exposing the T cells to an anti-CD3 antibody and an ionophore.   The cancer vaccine according to claim 18, wherein the ionophore is ionomycin.   The cancer vaccine according to claim 13, wherein the patient is a human.

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