DE10009030A1 - Hybrid cell, useful as therapeutic vaccine against cancer and viral, bacterial and protozoal infections, comprises allogeneic dendritic cells fused to autologous non-dendritic cells - Google Patents

Abstract

A hybrid cell (I) comprising a mature dendritic cell (DC) fused to a non-dendritic cell which is freshly isolated or obtained from a primary cell culture and where DC and non-DC are allogeneic to each other, is new. Independent claims are also included for the following: (1) making (M1) (I) comprising subjecting a population of mature DCs and non-DCs allogeneic to the DCs to conditions that promote cell fusion; (2) a kit comprising a sample containing a population of mature DCs in one or more containers and instructions for its use in treating or preventing cancer or an infectious disease or in making (I); (3) treating or preventing (M2) cancer or an infectious disease in a mammal comprising administering a therapeutically active amount of (I) which shares at least one class I major histocompatibility complex (MHC) allele with the mammal; and (4) preventing a cancer in an individual having a predisposition for the cancer comprising M2 where the non-dendritic cell displays the antigenicity of an antigen associated with the cancer.

Description

The present invention relates to a vaccine which is a is settable in patients whose immune system is certain pa not or not anymore or not yet works that the condition is manageable.

Specific immunotherapy or prevention of pathological Conditions like chronic infections, benign and malignant  Tumors or also family-specific dispositions for certain Diseases such as breast cancer are e.g. B. possible if the affected cells express antigenic determinants that distinguish them from healthy tissue. These cells escape nevertheless, their destruction often because they are insufficient Provide signals to activate the body's immune system.

It has now been recognized that u. a. lack of kostimu responsible molecules and / or modifying factors are literal. One approach, therefore, is through appliques tion of lymphokines or the introduction of genes into tumor cells len, for lymphokines or costimulating surface molecules encode le to replace the missing signals.

On the one hand, these measures are very complex and expensive, on the other hand, it shows very serious side effects gene.

Stuhler and Walden show Immunol in Cancer. Immunother. 1994, Volume 36, p. 342-345, a procedure involving the allogeneic MHC class II molecules in tumor cells are introduced to an immune system trigger response.

In the described method, electrofusion of Tumor cells and allogeneic B lymphocytes, the MHC class I and II express molecules, hybrid cells generated, the tumor antigen ne and allogeneic MHC class I and II molecules. These Hybrid cells have been shown to be potent stimulators of cyto toxic T lymphocytes (CTL).

The hybrid cells were irradiated to increase the Tu to prevent morocells, and then injected into mice. This Be action resulted in the rejection of an established tumor.

Control experiments showed that in the mouse model the antigens for both types of T cells, i.e. the helper T Cells and the naive T cells to be present on one cell  had to induce the activation of naive T cells to CTL adorn. In the same way it could also be shown that Have mice immunized against tumors if before application tion of tumor cells, the hybrid cells described are injected will.

Kugler et al. describe in Br J Urol 1998, volume 82, p. 487- 493, the vaccination of tumor patients with lethally irradiated Hybrid cells from allogeneic B lymphocytes and autologous or al logical tumor cells. Both hybrid cells led to the behan developed a comparable immune response. Everything However, 6 of 11 patients responded to those with the hybrid cells were vaccinated from allogeneic tumor cells, while in Ver use of autologous tumor cells only 3 out of 13 patients languages.

Gong et al. describe in Nature Medicine, 1997, Volume 3, p. 558-561 immunization of mice using auto lied to dendritic cells. Doing so became immature dendritic Cells (DC) from bone marrow cultures of the cloned syngeneic Mice fused with PEG with tumor cells to form hybrid cells induced tumor-specific CTLs in vivo in the mouse model.

The authors discuss against the background of their results the use of autolo derived from peripheral human blood gene dendritic cells for the fusion with tumor cells to in vivo to induce CTLs for fighting cancer. You know however point out the limitations of the animal model.

Against this background, the present invention is the Task based on a vaccine, a procedure for its Production and a method for therapeutic or before diffractive / immunizing treatment for patients with patholo to create genetically modified cells.  

According to the invention, this object is achieved by hybrid cells from allogeneic dendritic cells and patient-related, pa thologically modified cells.

According to the invention, a therapeutic / protective composition is produced by the steps:
Provision of dendritic cells of an allogeneic donor,
Provision of patient-related, pathologically modified cells, and
Fusion of the dendritic and pathologically changed cells into hybrid cells.

The object on which the invention is based is achieved in this way completely solved. The inventors of the present application ha ben recognized that through the use of allogeneic dendritic cells a more potent hybrid cell are generated can than with autologous DC. They are on autologous DC MHC Class II restricted antigens are unlikely while they are present on allogeneic DC per se. Well out for this reason, the inventors were able to show that when used of the hybrid cells provided according to the invention because of the mismatch between donor and patient with the MHC class II molecules very much potent CTLs are induced. In addition, allo gene DC, which come from healthy donors, easily z. B. from win peripheral blood and produce in almost any number gene.

Under patient in terms of registration, both humane and animal patients also understood the pathological change te cells or disposition for a certain Have disease associated with pathologically altered cells come here. The patient-related cells do not just fall into this category autologous cells from benign and malignant tumors but also autologist e.g. B. infected due to a chronic viral disease Cells that tract specific antigens for infection In addition to these autologous cells, the patient-related cells also allogeneic tumor cells from a cell line, for which the patient has a family disposition. This is e.g. B. in breast cancer or colon cancer the case where within a family often involves the same relevant genes are. So z. B. tumor cells of a family member used to hybrid cells for the Imp generation of another family member.

The inventors' findings were particularly surprising but in relation to malignant human tumors for which a high re mission rate was found when patients with hybrid cell len immunized from allogeneic DC and autologous tumor cells were. Protective immunity can also be achieved.

It is preferred if the DC used for the fusion are mature DC.

This is surprising in that mature DC is no longer in the are able to migrate in the body to the same extent as immature ones DC, so a better immune response with immature DC is expected which also Gong et al. have used.

However, the inventors have recognized that mature DC that de have a finite state that is reproducible to more potent ones Lead hybrid cells and beyond in handling have parts.

It is preferred if Mo Noocytes are isolated, which are then added with growth Differentiate factors into mature DC.

This is a robust, easy to use des and reproducible system. Before the merger, the ge primed DC can only be washed. This is a huge advantage e.g. B. versus B cells activated with toxic LPS Need to become.

It is further preferred if the hybrid cells are electrofu sion are produced, with the hybrid cells further preferred be lethally irradiated before using them for vaccination be det.

The advantage here is that after the electrofusion, none of the effort cleaning of the hybrid cells as required for fusion with PEG is harmful, with an increase by lethal radiation of the hybrid cells is prevented.

Against this background, the invention also relates to a vaccination substance for therapeutic and / or protective immunization ins special of human patients who the Hy contains brid cells, and the use of the invention Hybrid cells for the production of the vaccine, which is a patient is specific vaccine, and for therapy and / or pro tection of patients.

The invention further relates to the use of allogeneic DC for the production of the hybrid cells according to the invention and in the men of the therapeutic / protective treatment according to the invention patient development.

Furthermore, the invention relates to the use of autologous third-party cells for the production of hybrid cells in the frame of the invention for the therapeutic / protective treatment of human patient.

In the following pages, the invention is based on Experi elements and results described and discussed in more detail.  

A hyrid cell vaccination study will be presented with 17 patients animals. Using electrofusion techniques, autologous tumor and allogeneic dendritic cell hybrids produced the antigens expressed by the tumor together with the co-stimulating capabilities of dendritic cells present. After vaccination and a medium follow-up After 13 months, four patients encountered all metastatic Tumor lesions completely, one showed a mixed Ant word and in another two patients a tumor mass re production of more than 50% found. Induction of HLA-A2- restricted cytolytic T cells associated with the Muc1 tumor associated antigen are reactive, and the recruitment of CD8 lymphocytes in tumor exposure sites are demonstrated. The data presented here indicate that the Hy bridcell vaccination is a safe and effective therapy for RCC and having a broadly applicable strategy for other malignancies can provide unknown antigens.

For the rapid and large-scale production of hybrids, electrofusion was established as a two-step process. In a first step, tumor cells and DC were dielectrophorically aligned to form cell-cell conjugates. In a second step, a fusion pulse was administered which gave 10 to 15% hybrid cell formation, as demonstrated in FIG. 1a (tumor-DC fusion). Hybrids are characterized in that they emit both colors after the DC and tumor cells have been marked with red or green intracellular fluorescent dyes. Double fluorescent cells were neither detected in experiments in which a mixture of DC fused with DC and tumor cells fused with tumor cells (tumor-DC mixture) were analyzed, and without electrofusion (unfused). It is important that the uptake of apoptotic bodies from tumor cells by DC differs from the hybrid cell formation, because the incubation of DC with apoptotic tumor cells for 24 hours did not lead to double fluorescent cells (apoptotic tumor + DC). 10 ^X cells were processed simultaneously without the need for tumor cell cultures or fusion reagents.

Monocyte derived DCs were used as fusion partners for the tumor cells because of their unique ability to induce naive T lymphocytes both in vitro and in vivo. The phenotype of representative DC with high CD1a and CD83 expression and the lack of CD14 molecules is shown in Fig. 1b. While MHC class I (HLA ABC) antigens are expressed by Tu mor and dendritic cells, high amounts of the co-stimulating molecules CD80 and CD86 and MHC class II (HLA-DR) antigens were used to recruit helper T - Cells detected only on DC. The fusion of the populations could thus combine the unique and tumor-specific antigens with the co-stimulating abilities of the DC. The staining of tumor-DC fusions with fluorochrome-labeled antibodies directed against surface molecules or fluorescent membrane dyes led to a high number of false positive hybrid cells, because particles of membrane fragments formed during the electrofusion process or apoptosis , attach to unfused cells.

We used allogeneic DC to make allo-reactive helper T cells to recruit and thus avoid additional HLA Class II restricted tumor antigens are needed.

Seventeen patients were vaccinated subcutaneously very close to inguinal lymph nodes with fusions of 50 × 10 ⁶ autologous tumor cells and 50 × 10 ⁶ allogeneic DC. The vaccine was used immediately after electrofusion and 200 Gy irradiation without further selection. An identical, freshly prepared booster vaccine was given after six weeks, followed by a re-evaluation of the patients six weeks later. All patients who were not suffering from a progressive disease were continuously given immunizations every three months. To enable related TAA detection while avoiding dominant allo-specific responses, different HLA-different donors were recruited for each immunization.

Hybrid cell vaccination was well tolerated by all patients. There were no major adverse effects, nor any clinical signs of autoimmune reactions were observed. In egg In some cases, mild fever or appeared for a day or two a temporary reddening of the skin and hardening at the site of the Injection on. Six patients reported pain metastatic sites sufficient with non-opioid Analgesics have been treated. After multiple immunizations there were no significant changes in routine blood tests or in the ratios of lymphocyte populations in the peri pheren blood detected, which is indicated by flow cytometry Use of CD3, CD4, CD8, CD19 and CD56 specific Reagents have been assessed (not shown). We conclude from these findings that the hybrid cell vaccination under Ver use of electrofusion techniques without significant toxicity can be used repeatedly.  

In order to analyze the effector cells involved in tumor cell rejection and to define the specificity of the immune response induced, we made use of the previously identified HLA-A2-restricted Muc1 and HER2 / neu epitopes. Consistent with recently published data, we found Muc1 and HER2 / neu expression by the primary tumor in 82% (Table 1) and in 0% of the patients examined here. Interestingly, high HER2 / neu expression was demonstrated in tumor cell lines, which were established by patients No. 4, 5, 6, 7 and 16. The specific IFN-γ production by CD8-positive T-lymphocytes from HLA-A2-positive patients was determined before and after vaccination by ex vivo stimulation of peripheral T-lymphocytes with the respective tumor peptide antigens, and intra-cellular detection of the induced cytokines by means of flow cytometry Techniques determined. No reactivity to Muc1.1 and Muc1.2 or the HER-2 / neu derived antigens GP2 and E75 was observed prior to hybrid cell vaccination therapy. However, patient No. 7 showed a tumor-specific reaction after the fourth vaccination, since 0.8% of all CD8-positive cytolytic T lymphocytes were stained positive for IFN-γ for six hours after in vitro stimulation with Muc1.2 peptide antigens ( Fig. 2a). No reactivity was found against Muc1.1, E75 or GP2 epitopes. A similar response was observed with 0.4% Muc1.2-reactive CTL after hybrid cell vaccination in patient No. 4, whose disease is stable for 18 months after an initial decrease of less than 50% in some but not all lung metastases. These results thus clearly demonstrate the induction of specific CTL and the establishment of a tumor-directed immune response in vivo, which is in agreement with previous data showing a reversal of tolerance and Muc1-specific tumor immunity in HLA-A2 transgenic mice. Regardless of the clinical responses, however, Muc1- or HER-2 / neu-specific CTL were not observed in two other HLA-A2 positive patients (patient Nos. 9 and 12), which is an important role for T-lymphocytes with specificities for speaks different antigens than the epitopes tested here.

After receiving this information, we decided to determine the biological significance of the observed immune response. After in vitro stimulation with Muc1.1, Muc1.2 and the irrelevant HIV epitope, lymphocytes from patient No. 7 were tested for specific lytic activity in a ⁵¹ Cr release test. The HLA-A2 positive but Muc1 negative cell line Croft, which was pulsed with the peptide antigens, was used as the target. As shown in Fig. 2b, the CTL stimulated in the presence of Muc1.2 specifically lysed Muc1.2, but not HIV-pulsed Croft cells. Although induction of low-affinity CTL versus Muc1 was expected, high lysis of the HLA-A2 and Muc1-positive RCC line A498 was observed. The HLA-A2 restriction of the CTL response was confirmed by the finding that the HLA-A2-negative but Muc1-positive cell line SKOV was not lysed. Muc1.1-stimulated CTL did not lyse Muc1.1-coated Croft or A498 cells, which suggests that Muc1.2 is immunodominant over Muc1.1 in this patient. No lysis of the NK-sensitive line K562 was observed in any test. The in vitro induction of the CTL reactivity was excluded by the finding that CTL stimulated with HIV epitopes did not lyse HIV-covered CROFT target cells (not shown). Although CTL were found in vitro that were specific for the Muc1.2 self-antigen, patient No. 7 showed no clinical signs of an autoimmune disease and the in vivo role of this CTL remains unclear. It is important that the observation that only 60% to 80%, but not all tumor cells of patient # 7 express mucin antigens indicates a critical role for other antigens not tested here.

In order to monitor the migration of the lymphocytes into the tumor sites and the lymphocyte populations that are involved in the tumor cell destruction, DTH (Delayed Type Hypersensitivity) tests were carried out, in which 5 × 10 ⁵ irradiated autologous Tumor cells were injected intra-cutaneously into the forearm. No positive DTH response was obtained before vaccination. However, eleven out of seventeen patients who were tested after twelve weeks showed a positive reaction of more than 6 mm in diameter one to three days after tumor exposure. With the exception of patients Nos. 4, 9, 10 and 17, all patients who developed positive DTH responses showed significant objective responses and completely or partially rejected the metastatic lesions. Interestingly, patient No. 2, who did not develop a positive skin reaction after chemotherapy with IFN, interleukin-2 and 5-FU, reacted partially and with delay to the vaccination therapy.

Histological analyzes of skin biopsies showed before the hybrid Vaccination therapy cytokeratin-positive tumor cells and a mo derate lymphocytic infiltration corresponding to the primary Tumor lesion. Enrichment of CD8-positive lymphocytes the tumor exposure site was, however, only after the immunizations observed.

The study ended in November / December 1999. Seven out of seventeen patients (41%) who received the hybrid cell vaccination showed significant objective. Answers with four complete tumor remissions, two partial remissions and a mixed response. In addition, the multiple pulmonary lesions of two other patients (patients No. 4 and 9) were stabilized for 17 and 15 months, respectively. The middle ones. Examinations followed after 13 months (in the range of 21 to 3 months). Three of the four patients who showed complete remission successfully rejected all metastases within the first 12 weeks and with a total of two injections and remained free of any detectable tumor lesions for up to 21 months. Typically, the reduction in tumor masses was observed within the first few weeks after the first immunization. Metastases of the lungs, bones, lymph nodes and soft tissues were successfully destroyed. In general, patients suffering from a large tumor burden reacted weakly (patient nos. 6, 10, 11, 13 and 17). Large tumor masses, however, do not necessarily limit the success of the vaccination, since patients (No. 12 and 16 rejected locally recurrent tumor lesions that were larger than 500 ml). In three patients, an initial partial response with a tumor mass decrease of more than 50% was observed after 12 weeks, one patient remained in partial remission (patient no. 16) or in the case of patient no. 8 all metastatic tumor sites after 12 months The regression of the multiple metastatic lesions of the lungs and bones of patient No. 8 is shown in Fig. 3. An initial partial regression was observed in patient No. 14. The metastases of the liver progressed however, eventually moving forward and the patient died of the disease died of an advanced illness or died from her illness.

In summary, the ones presented in this communication indicate Data suggest that hybrid cell vaccination is safe and effective immunotherapy for human metastatic never is cell carcinoma. The hybrid cell vaccination is therefore an option play an individualized immunotherapy strategy, regardless of the genetic situation of the patient given ge common or unique antigens caused by the tumor to get presented. Given the heterogeneity of the tumor cells within a lesion could be a strategy that the Induction of against multiple and different tumor-associated Antigen-directed CTL aims for the results obtained be responsible. Furthermore, the use improves formation of allogeneic dendritic cells as fusion partner of the Tumor cells compared vaccination success to an earlier one Study using activated B cells.

Fig. 1: Characterization of the hybrid cell vaccine. a: Formation of the hybrids: DC and tumor cells were marked with red and green fluorescent dye. Tumor cells fused with DC (tumor-DC fusion) or a mixture of DC-DC fusions and tumor-tumor fusions (tumor + DC mixture) were analyzed by flow cytometry. The uptake of apoptotic bodies was assessed 24 hours after the incubation of apoptotic tumor cells with DC (apoptotic tumor + DC). Unfused cells are shown as controls (unfused). The percentage of cells per quadrant is given. b: The phenotype of the DC and tumor cells before the electrofusion was determined by flow cytometry analysis using FITC and PE-labeled mAbs as indicated.

Fig. 2: Data for the immunological response. A: The specificity of the T-lymphocytes of patient No. 7 for Muc1.1- and Muc1.2- tumor antigens was analyzed before and after vaccination. The lymphocytes were stimulated ex vivo with the peptide antigens for 6 hours and the production of IFN-γ was visualized by intra-cellular staining with specific PE-labeled antibodies (Ak). Correlation with lymphocyte subpopulations was achieved by three-color flow cytometry analysis using anti-CD3-PerCP and anti-CD8-FITC-Ab. Windows were set on CD3 positive T lymphocytes. b: HLA-A2 positive mononuclear cells from patient No. 7 were stimulated in vitro for 7 days with Muc1.2 (upper panel) or Muc1.1 (lower panel) epitopes and then for a further 5 days in the presence of the antigen and 20 U / ml IL-2 restimulated. The CTL activity was determined by specific lysis of the ⁵¹ Cr-labeled target cells (10 ⁴ cells) given variable effector-to-target ratios. HLA-A2 positive but Muc1 negative Croft target cells were coated with Muc1.1 (Croft Muc1.1), Muc1.2 (Croft Muc 1.2) or the irrelevant HIV (Croft HIV) peptide antigen. A498 is an HLA-A2 and Muc1 positive RCC tumor cell line. SKOV is HLA-A2 negative but Muc1 positive. K562 was used to analyze NK activity.

Clinical response and toxicity data

Metastatic lesions were by CT scintigrams, ultrasound scans and Bone scintigraphy before and 12 weeks after the first immunization valuation. Clinically non-progressive patients performed CT scintigraphy every 3 months. The date of the last CT scintigraphy of each patient is listed in Table 1. The WHO definitions for the complete (none detectable metastatic lesions) and the partial ant word (more than 50% reduction of all tumor sites) for at least applied for three months. A stable illness became with weni Defi less than 25% change in size and number for 6 weeks kidney. Adverse effects were re according to WHO criteria registered. Blood and urine have been routinely tested. CD4- too CD8 ratios and analyzes of the lymphocyte subsets were using CD3, CD4, CD8, CD19, CD56 reactive monoclonal antibodies using flow cytometry (Becton Dickinson, Heidelberg, Germany).

Tumor and dendritic cells

The tumor samples were internal half of 12 hours after tumor necrotomy or operati ven removal of the metastases processed. Single cell suspension by grinding the tumor and then ver last using 5.6 mg collagenase type VIII and 0.26 mg DNase type IV in 10 ml RPMI medium per gram of tumor (Sigma, Deisenhofen, Germany). To the epithelial Co-expression was used to confirm the origin of the tumor cells of vimentin and cytokeratin 8 and 18 by immunocytochemistry according to the manufacturer's instructions (Prolab Diagnostics, Kana da) evaluated. Before the electrofusion, the MHC class I Expression and ploidy of tumor cells using flow cytometry techniques determined. Dendritic cells were found in Brevity generated from peripheral blood monocytes. Leukocytes from ge healthy blood donors were collected by leukapheresis. To Ficoll-Hypaque density centrifugation was performed on the cells for 4 Adhesion to plastic trays at 37 ° C for hours. Non-adherent cells were removed and adherent mono Cytes were cultured in X-Vivo 15 medium (Bio Witthaker, Verviers, Belgium), the 0.1 µg / ml GM-CSF, 0.05 µg / ml Interleukin-4 contained. TNF-α has been in the past two Days of cultures added to 0.5 µg / ml (all cytokines are by Genzym, Rüsselsheim, Germany). The purity of so he maintained DC was greater than 85% by means of flow cytomas trie analysis (FacsCalibur, Becton Dickinson) was evaluated, using fluorochrome-labeled antibodies, the against HLA-ABC, HLA-DR, CD1a, CD3, CD14, CD19, CD40, CD54 and CD83 were directed (the antibodies were from Becton Dickin son-related, with the exception of Anti-CD83 and Anti-HLA-ABC, Immu notech, Marseille, France).

Generation of the hybrid cell vaccine

5 × 10 ⁷ autologous tumor lines and 5 × 10 ⁷ allogeneic DC were washed, re-suspended in 0.3 M glucose and transferred into an electroporation cuvette (BioRad, Munich, Germany). The electrofusion was carried out by first aligning the cells at 100 V / cm for 5 to 10 seconds to form cell-cell conjugates. Alignment was optimized by placing a drop of dielectric wax on one side of the electroporation cuvette to inhomogenize the electric field and thus direct the cells to the area of highest field strength. In a second step, a pulse of 1200 V / cm was applied at 25 µF using a Gene Pulser II (BioRad) to fuse the aligned cells.

The hybrids were irradiated (200 Gy) and without further selection injected on. To determine the fusion efficiency, the DC and tumor cells with red and green fluorescent cell Stain tracker dyes (Molecular Probes, Eugene, USA) and analyzed by flow cytometry (Beckton Dickinson). The apoptosis of the tumor cells was reduced by UV radiation Use of a Stratalinker device (Stratagene, Amsterdam, Netherlands) induced.

IMMUNIZATION THERAPY OF ADVANCED PATIENTS Renal cell carcinoma due to fusion of dendritic cells and TUMOR CELLS

A new tumor cell-based immunotherapy for patients with metastatic renal cell carcinoma (RCC) is presented. By electrofusion of autologous tumor cells and allogeneic dendri tic cells (DC) a hybrid cell vaccine is generated to to induce tumor-specific cytotoxic T lymphocytes (CTL) that even reject large tumor masses in vivo.

Tumor cells are from patients with metastatic RCC collected by nephrectomy. Dendritic cells are in in vitro after leukapheresis by stimulating monocytes with GM-CSF, Interleukin-4 and -α generated. Allogeneic DC are ver uses to improve the immunogenicity of the vaccine. Hybrid cells that are both co-stimulatory and DC-derived Antigen-presenting properties as well as tumor-derived Expressing antigens were by electrofusion of DC with Tu morcell generated. The efficiency of cell-cell fusion, evaluated by double fluorescence analysis, was 30%.

The generation of this type of tumor DC hybrid cell vaccine is described in detail below:

• 1. Planning the treatment
• 2. Preparation of autologous tumor cells
• 3. Preparation of allogeneic dendritic cells
• 4. Cell fusion
• 5. Quality control
• 6. Vaccination
• 7. Tumor-specific DTH response

1. Planning the treatment

Patients with metastatic renal cell carcinoma, pT 1-4 N1- and / or M1 classified are treated in our institute. Additional admission criteria are two-dimensionally measurable me traumatic lesions, ECOG scale <3, life expectancy of more than 3 months, no second malignancy. All patients and trained in our institute or an affiliate Hospital tumor nephrectomy or resection of meta stasis for tumor preservation and the tumor samples were taken on the day of the surgical procedure processed.

In order to assess the integrity of the cellular immune system conducted a Merieux multitest screening. A positive re Action in this test is mandatory for the patient be treated.

After initial vaccination, the patients continue 6 weeks later immunization is an immunization boost. A clinical one grading in connection with additional vaccinations in patients with objective reaction and stabilized sick is carried out every 3 months. Treatment will last until to objective signs of disease progression continued.

This form of treatment for. Advanced patients RCC was undertaken to track the efficiency of the vaccine point. In our patients, 11 out of 20 treated Pa provided an objective answer based on WHO criteria (mean follow-up examination = 8 months). Furthermore, glau we practice that patients with locally advanced disease (pT3N0M0) would benefit from this treatment.

2. Preparation of autologous tumor cells 2.1 material

• A) Fresh tumor tissue from the operating room (either Ge weave from a tumor nephrectomy sample or a resect tion of metastases, e.g. B. local tumor recurrence, lung, Le over-metastases).
• B) Type VIII collagenase (Sigma, Deisenhofen, Germany)
• C) DNase type IV (Sigma, Deisenhofen, Germany)
• D) cell culture medium. In our laboratory: RPMI 1640 without HEPES (Bio Whittaker), Glutamate (Bio Whittaker), RPMI- Amino acid solution (Sigma, Germany), RPMI vitamin (Sigma, Germany), Gly-Lys-His (Sigma, Germany), FCS (Bio Whittaker, Belgium).

Suggested dilution

RPMI 1640 500 ml Glutamine 5 ml RPMI amino acid solution 10 ml RPMI vitamin 5 ml Gly-Lys-His 1 ml FCS 75 ml (15%)

2.1.1 Preparation of aliquots for tumor tissue digestion 2.1.1.1 collagenase

• - 5 g collagenase type VIII in 40 ml TES buffer (stir in a 37 ° C water bath)
• - Fill up to 50 ml with TES buffer
• - Aliquot in 1400 µl units (can be stored at -70 ° C)

2.1.1.2 DNase

• - 11.2 ml collagenase / buffer (means 8 aliquots from 2.1.1 A) in 2000 ml RPMI
• - add 52 mg DNase type IV
• - Sterile filtration (nylon, 0.2 mm, Bibly Dunn, Coming Costar, Burmath, France)
• - Aliquot in 50 ml units (can be stored at -20 ° C).

2.2 Protocol

Sterile handling and GLP criteria are mandatory! The Tissue is picked up according to the recommendation of good laboratory practice and treated.

• A) Transport the sterile tumor tissue sample in RPMI from Operating room in the laboratory.
• B) Remove connective tissue, fat
• C) Cut the tumor tissue into 2-3 mm pieces
• D) Place the sample in a 37 ° C water bath and cover it with Collagenase / DNase mix (10 ml / g tumor).
• E) Stir for 2 hours, resulting in a tumor cell suspension leads
• F) Filter through a metal filter (60 mesh, 230 µm Breite, Sigma, Germany), the remaining tumor Webe has to be digested for another hour, again le filtration.
• G) Centrifuge at 800 / min for 10 min
• H) Wash with RPMI
• I) In the case of erythrocyte contamination: centrifuge and add 20 ml 0.2 M NaCl and resuspend, neutralize with 1.6 M NaCl and wash; repeat if necessary.
• J) Viability test (trypan blue exclusion). More than 70% must be vital.
• K) Store the tumor cells in cell freezing solution (Sigma, Germany) at -80 ° C.

In addition to this direct isolation of the tumor cells, primary cell cultures are established by sowing some tumor cells in culture bottles (Nunclon ^TM 25 cm ² , Nung, Wiesbaden, Germany), which are covered with 2-5 ml of RPMI medium and - after subconfluent growth of the Cells - the cells are passaged.

We usually isolate approximately 3 × 10 ⁸ tumor cells from fresh tumor tissue for three vaccinations. Cultured cells are used only in patients who receive multiple reinforcement shots (patients with an objective answer). When we do this, the origins of the tumor cells are confirmed by DNA fingerprinting (D1S80 allele match, Perkin Elmer, Branchburg, NJ, as described by the manufacturer) and microbiological contamination is excluded.

3. Production of the dendritic cells

Allogeneic leukocytes are collected by leukapheresis (approximately 10 ⁹ cells), donors without infectious diseases and known HLA status are selected. Alternatively, buffy coats can be used.

3.1 material

• - Ficoll-Hypaque (separation solution density 1.077, bio chrome / Seromed, Berlin, Germany)
• - HBSS (w / o Ca + Mg, Gibco BRL)
• - Cytokines: GM-CSF (0.1 µg%), IL-4 (0.05 µg%), TNF-α (0.5 µg%)
  All cytokines are from Genzyme (Rüsselsheim, Germany)
• - Culture medium: X-Vivo 15 (Bio Whittaker, Belgium)

3.2 Protocol

• A) 4-10 ml leukapheresis cells (or buffy coat) are included 4 × 20 ml HBSS mixed
• B) Carefully add this 30 ml (leukocyte + HBSS) to 15 ml Ficoll
• C) Centrifuge at 2000 rpm for 20 min
• D) Collect the cells in the interphase
• E) Wash 2x with PBS
• F) Sow the cells in bottles (Nunclon ^™ 80 cm ² ) and cover them with 2-5 ml culture medium
• G) Cell adhesion for 3 hours in an incubator (37 ° C)
• H) Wash 1x with PBS (and remove the non-adherent Cells),
• I) Bone 3 ml cell culture medium (+ cytokines GM- CSF (0.1 µg%) and IL-4 (0.05 µg%) in each bottle
• J) On day 3 of the incubation add another 3 ml of culture medium (+ Cytokines GM-CSF (0.1 µg%) and IL-4 (0.05 µg%), and am Day 5 add more TNF-α (0.5 µg%) until day 7. DC can be morphologically examined after day 5 in the light microscope and identified by FACS analyzes (see 5.).

4. Cell fusion 4.1 Material

• - Cuvettes (BioRad, Munich 2 mm condenser distance)
• - sterile paraffin
• - Gene Pulser (BioRad, Munich, Germany)
• - Power supply unit, DC voltage 40 V
• - 5% glucose

4.2 Protocol 4.2.1 DC preparation before cell fusion

• A) Coat a condenser of the cuvettes with paraffin to an inhomogeneous electric field in the capacitor produce. This is necessary for cell alignment such.
• B) Take the supernatant of the DC culture bottles and scrape the adherent DC from the bottom of the bottle.
• C) Wash the bottle with PBS to get the remaining DC hold.
• D) Centrifuge at 1000 rpm for 10 min
• E) Wash 4-5 times with PBS
• F) Count the cells and make a suspension of 10 ⁷ DC / ml in 5% glucose.

4.2.3 Tumor cell preparation before cell fusion

• A) Thaw the tumor cells (or collect the tumor cells Cultures as described in 2.2)
• B) Wash the tumor cells twice with PBS
• C) Count the cells and run a vitality test under Ver using the trypan blue exclusion
• D) Prepare a tumor cell suspension of 10 ⁷ cells / ml in 5% glucose.

Mix DC with tumor cells.

4.2.4 Cell fusion

• A) Fill a cuvette with the tumor / DC cell suspension.
• B) For cell alignment, a 40 V DC is used in the Gene Pulser voltage applied for 5 seconds.
• C) Fusion the cells using 1000 V / cm, 25 µF (BioRad Gene Pulser, Munich, Germany)

Repeat II. And III.

4.2.5 Irradiation

The vaccine is administered using a cobalt External blasting unit irradiated with 200 Gy.

5. Quality control 5.1 Dendritic cells

The purity of the DC obtained in this way is greater than 85%. The cell len show clear dendritic morphology and high expression from MHC Class I and Class II, CD1a, CD40, CD54, CD80, CD83 and CD86, however, are negative for CD14 molecules, which means Flow cytometry analysis (FacsCalibur, Becton Dickinson, Heidelberg, Germany) using fluorochrome labeled antibodies (all antibodies were from Becton Dickinson purchased Immuno with the exception of the anti-CD83 reagent tech, Marseilles, France).  

5.2 hybrid cells

After cell fusion, the hybrid cells cannot be identified by light microscopy alone. Cell clusters can be seen here. FACS analyzes give false positive results. In order to verify the effective fusion, double fluorescence staining must be carried out. Before the fusion, 1 × 10 ⁶ / ml tumor cells or 1 × 10 ⁶ / ml DC are stained separately with red or green fluorescent membrane dyes according to the manufacturer's instructions (Sigma, Deisenhofen, Germany). After the fusion, for example, the large red cell in the upper half is positive for green fluorescence (lower half) and is therefore considered to be fused. A fusion effectiveness of 30% is achieved.

6. Vaccination

After radiation, the vaccine (10-15 ml hybrid cells in 5% glucose) intra- / subcutaneously on various (<5) injecti sites injected near groin lymph nodes lie in the lower abdominal wall and the upper thigh. The vaccination process can be carried out in outpatient treatment the.

In addition to redness, fever for less than 48 hours (20% of pati ducks) and non-specific pain for less than a week, no side effects greater than WHO Class I were.

7. Tumor-specific DTH response

To monitor the specificity of the induced immune response against autologous tumor tissue, 1-5 × 10 ⁶ irradiated tumor cells were administered intracutaneously before and 7 days after each vaccination and the resulting delayed-type hypersensitivity response (DTH) was documented. Skin biopsies were taken one day after the tumor cell injection and CD8 ⁺ cells were stained using immunohistochemistry (monoclonal antibody, Boehringer, Ingelheim, Germany).

Before the vaccination, none of the patients developed a positive one Skin reaction against autologous tumor cells, what is considered a negative Test for tumor-specific delayed-type hypersensitivity (DTH) was interpreted. Positive DTH reactions could ever but in twelve out of 20 patients six to twelve weeks after the Hybrid cell vaccination therapy can be observed.

Claims (24)

1. Hybrid cells from allogeneic dendritic cell and patient cells related to pathology. 2. Hybrid cells according to claim 1, characterized in that the pathologically altered cells infected cells Patients who have anti specific for infection carry genes. 3. Hybrid cells according to claim 1, characterized in that the pathologically changed cells cells one for a Pa typical family of tumor cells for which the Patient has a disposition. 4. Hybrid cells according to claim 1, characterized in that the pathologically modified cells from benign tumors come. 5. Hybrid cells according to claim 1, characterized in that the pathologically modified cells from malignant tumors come. 6. Hybrid cells according to claim 4 or 5, characterized in that the pathologically altered cells are autologous tumor cells exhibit. 7. Hybrid cells according to one of claims 1 to 6, characterized ge indicates that by fusion, preferably by elec trofusion are generated.   8. Hybrid cells according to one of claims 1 to 7, characterized ge indicates that the dendritic cells are mature dendritic Cells are. 9. Hybrid cells according to one of claims 1 to 8, characterized ge indicates that they are lethally irradiated. 10. A method for producing a therapeutic / protective composition, comprising the steps:
Providing dendritic cells of an allogeneic donor,
Providing patient-related, pathologically changed cells, and
Fusion of the dendritic and pathologically modified cells into hybrid cells. 11. The method according to claim 10, characterized in that the pathologically altered cells are selected from the group pe, which includes: infected autologous cells, benign tumor cells len, maigne tumor cells, autologous tumor cells, typical Tu mor cells for which there is a disposition. 12. The method according to claim 10 or 11, characterized in that the dendritic cells from peripheral blood of an allo gene donors are isolated. 13. The method according to claim 12, characterized in that the isolated dendritic cells with the addition of growth factors can be differentiated. 14. The method according to claim 13, characterized in that the mature dendritic cells are washed prior to fusion. 15. The method according to any one of claims 10 to 14, characterized ge indicates that the hybrid cells are lethally irradiated. 16. The method according to any one of claims 10 to 15, characterized ge indicates that the fusion is done by electrofusion. 17. Vaccine for therapeutic / protective immunization of a Patients with hybrid cells according to one of claims 1 to 9. 18. Use of allogeneic dendritic cells for the manufacture treatment of hybrid cells and / or vaccine for therapeutic Protective immunization of a patient. 19. Use of hybrid cells according to one of claims 1 to 9 for the therapeutic / protective treatment of a human Patient. 20. Use of allogeneic dendritic cells for a ver drive according to one of claims 10 to 16 to therapeuti protective / protective treatment of a patient. 21. Use of autologous dendritic cells in the ver drive according to one of claims 10 to 16 to therapeuti protective / protective treatment of a human patient. 22. The method according to any one of claims 10 to 16, in the auto loge dendritic cells are used for fusion and the Composition is intended for human patients. 23. Hybrid cells according to one of claims 1 to 9, in which au tologe dendritic cells and pathologically altered cells of a human patient are fused. 24. Method for the therapeutic / protective treatment of a Patient, in which hybrid cells according to one of claims 1 to 9 or 23 and / or the vaccine according to claim 17 and / or a composition can be applied, which after Method from one of claims 10 to 16 or 22 herge was put.