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Phase I/II study of immunotherapy using autologous tumor lysate-pulsed dendritic cells in patients with metastatic renal cell carcinoma
Abstract
This phase I/II study was conducted to evaluate the feasibility, safety and efficacy of immunotherapy using tumor lysate (TL)-pulsed dendritic cells (DC) in patients with metastatic renal cell carcinoma (RCC). DC were generated by culturing peripheral blood mononuclear cells in the presence of GM-CSF and IL-4 and were pulsed with autologous TL and keyhole limpet hemocyanin (KLH). Maturation of DC was induced by a combined treatment of CD40 ligand, IFN and monocyte-conditioned medium. The patients were administered two cycles of TL-pulsed DCs vaccination, each of which comprised of four doses injected subcutaneously at biweekly intervals. Nine patients were included. The immunotherapy was well tolerated without severe side effects. One patient achieved an objective partial response (PR). Five patients showed stable disease (SD), and the remaining three had progressive disease (PD). With a median follow-up of 17.5 months, the median time to progression was 5.2 months and the median overall survival was 29 months. In the antigen-specific lymphocyte proliferation assay, eight patients showed a proliferative response, which tended to be stronger in patients with SD or PR than in patients with PD. The ELISPOT assay was performed in two patients and indicated that one patient with PR showed a much stronger response than another with PD. Our results suggest that TL-pulsed DC immunotherapy in combination with nephrectomy affect the natural course of RCC and are associated with clinical benefits for patients with metastatic diseases.
... Vaccines as a tool for treating cancer have been studied to augment and maintain anticancer immune response in patients with cancer. Cancer vaccines have been tried preferentially in highly immunogenic type of cancers, such as renal clear cell carcinoma (RCC) [11], malignant melanoma [12], or non-small cell lung cancer (NSCLC) [13]. Peptide, protein, whole tumor cells or dendritic cell (DC)-based vaccines have been also tried in gastrointestinal cancers [14]. ...
... DCs are the most potent antigen-presenting cells which activate T cells and express immune stimulatory molecules. To enhance antitumor immune response, tumor lysate-pulsed DCs have been tried in patients with various cancers including malignant melanoma, CRC, NSCLC, prostate cancer, RCC [11], and brain tumor [17]. The positive results with survival benefit were reported by a phase III IMPACT trial with sipuleucel-T, an autologous DC therapeutic vaccine designed to enhance the immune T cell response to prostatic acid phosphatase in patients with metastatic castration-resistant prostate cancer [18]. ...
Cancer immunotherapy is at dawn of the Renaissance after the Medieval Dark Ages. Recent advances of understanding tumor immunology and molecular drug development are leading us to the epoch of cancer immunotherapy. Some types of immunotherapy have shown to provide survival benefit for patients with solid tumors such as malignant melanoma, renal cell carcinoma, or non-small cell lung cancer. Several studies have suggested that immune checkpoint inhibition might be effective in some patients with gastrointestinal cancers. However, the era of cancer immunotherapy in gastrointestinal cancers is still in an inchoate stage. Here we briefly review the current status and perspective of immunotherapeutic approaches in patients with gastrointestinal cancers.
... Peripheral blood mononuclear cells (PBMCs) were obtained from the patients with HCC through leukapheresis at Seoul National University Hospital. Dendritic cells were generated from blood monocytes, as described previously (Kim et al, 2007), with minor modifications. DCs were prepared in a Good Manufacturing Practice-compliant facility at JWCreaGene, Inc. Peripheral blood mononuclear cells isolated by Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation were resuspended in RPMI1640 medium (Lonza, Basel, Switzerland) supplemented with autologous heat-inactivated plasma, and then incubated in CellSTACK Culture Chambers (Corning, Corning, NY, USA). ...
... We defined TD instead of maximum TD (MTD), because most HCC patients have cytopenias, and thus repeated leukapheresis may not be feasible. We found that 5 Â 10 7 cells per injection was well tolerated as revealed in our phase I/II study of DC vaccine in renal cell carcinoma (Kim et al, 2007), thus this dose was designated as TD. The dosages of the DC-based cancer vaccines registered on the NIH website https://clinicaltrials.gov are in the range of 1 Â 10 6 and 1 Â 10 8 , and mostly 1B3 Â 10 7 cells per injection. ...
Background:
To date, no adjuvant treatment has been shown to have a clear benefit in patients with hepatocellular carcinoma (HCC). In this prospective phase I/IIa study, we evaluated the safety and efficacy of adjuvant dendritic cell (DC) therapy in HCC patients who received primary treatment for HCC.
Methods:
Twelve HCC patients who had no viable tumour after primary treatments were included. Dendritic cell vaccines pulsed with cytoplasmic transduction peptide-attached alpha-fetoprotein, glypican-3 and melanoma-associated antigen 1 recombinant fusion proteins were injected subcutaneously near to inguinal lymph nodes. Adverse effects, time to progression (TTP), and associated immune responses were evaluated after DC vaccination.
Results:
Nine of 12 patients had no tumour recurrence up to 24 weeks after DC vaccination. Among a total of 144 adverse events, 129 events (89.6%) were regarded as adverse drug reactions, all of which were grade 1 or 2. The majority of patients showed enhanced anti-tumour immune responses after DC vaccination. Recurrence-free patients exhibited relatively stronger anti-tumour immune responses than patients who developed recurrence after DC vaccination, as evidenced by lymphocyte proliferation and IFN-γ ELISPOT assays. The median time of TTP was 36.6 months in the DC-vaccination group and 11.8 months in the control group (hazard ratio, 0.41; 95% confidence interval, 0.18-0.95; P=0.0031 by log-rank test).
Conclusions:
Adjuvant DC vaccine for HCC was safe and well tolerated in phase I/IIa study, and preliminary efficacy data are encouraging to warrant further clinical study in patients with HCC after primary treatments.British Journal of Cancer advance online publication, 10 December 2015; doi:10.1038/bjc.2015.430 www.bjcancer.com.
... Dendritic cells (DCs) are primary antigen-presenting cells (APCs) that play a key role in regulation of immune responses to a variety of antigens [15,16], representing an attractive tool for therapeutic immunization of cancer [17]. Collective results of DC vaccines have demonstrated the safety and effectiveness of this approach against RCC [18][19][20]. However, vaccination of DCs pulsed with CA9-derived peptides did not induce a strong CA9-specific anti-tumour immune Clinical and Experimental Immunology ORIGINAL ARTICLE doi:10.1111/j. ...
... Therapeutic DC vaccines are a promising approach to treatment of RCC and a number of studies have evaluated their anti-tumour efficacy in murine models and in human clinical trials [18][19][20]32,33]. CA9 has been identified as a potential target for immunotherapy specific for RCC. ...
Carbonic anhydrase IX (CA9), a specific molecular marker for renal cell carcinoma (RCC), serves as a potential target for RCC-specific immunotherapy using dendritic cells (DCs). However, pulsing of DCs with CA9 alone is not sufficient for generation of a therapeutic anti-tumour immune response against RCC. In this study, in order to generate a potent anti-tumour immune response against RCC, we produced recombinant CA9-Acinetobacter baumannii outer membrane protein A (AbOmpA) fusion proteins, designated CA9-AbOmpA, and investigated the ability of DCs pulsed with CA9-AbOmpA fusion proteins in a murine renal cell carcinoma (RENCA) model. A recombinant CA9-AbOmpA fusion protein was composed of a unique proteoglycan-related region of CA9 (1-120 amino acids) fused at the C-terminus with transmembrane domain of AbOmpA (1-200 amino acids). This fusion protein was capable of inducing DC maturation and interleukin (IL)-12 production in DCs. Interaction of DCs pulsed with CA9-AbOmpA fusion proteins with naive T cells stimulated secretion of IL-2, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in T cells. Lymphocytes harvested from mice immunized with DCs pulsed with CA9-AbOmpA fusion proteins secreted IFN-γ and showed a specific cytotoxic activity against CA9-expressing RENCA (RENCA-CA9) cells. Administration of CA9-AbOmpA-pulsed DC vaccine suppressed growth of RENCA-CA9 cells in mice with an established tumour burden. These results suggest that DCs pulsed with CA9-AbOmpA fusion proteins generate a specific anti-tumour immune response against RCC, which can be utilized in immunotherapy of RCC.
... DC vaccines pulsed with tumor lysates have been reported as effective vaccines for the treatment of tumors 14,15,16 . Therefore, in this study, we engineered B. pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and CpG (C) as a tumor vaccine (SB-LC), and investigated its antitumor effects in murine models. ...
Due to its size, shape, and inherent expression of pathogen-associated molecular patterns and invasion-assistant adhesion proteins, Burkholderia pseudomallei can easily attach to, and then be internalized by, dendritic cells (DCs), leading to more efficient antigen cross-presentation if modified as carrier. Herein, we engineered Burkholderia pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and CpG (C) to be used as a tumor vaccine (SB-LC). We found that the adhesion proteins of Burkholderia pseudomallei promote internalization of the SB-LC vaccine by DCs, and result in enhanced DC maturation and antigen cross-presentation. SB-LC induces robust cellular and humoral antitumor responses that synergistically inhibit tumor growth with minimal adverse side effects in several tumor models. Moreover, SB-LC vaccination reverses the immunosuppressive tumor microenvironment, apparently as a result of CD8 ⁺ -induced tumor ferroptosis. Thus, SB-LC is a potential model tumor vaccine for translating into a clinically viable treatment option.
... The average callus formation score was significantly higher in the experimental group at 1 and 2 months of follow up, while the osteoblast injection response was not statistically different between younger and older patients. No adverse effects were observed in association with the osteoblast injection (Kim et al., 2007). ...
The introduction of advanced therapy medicinal products (ATMPs) to the global pharma market has been revolutionizing the pharmaceutical industry and has opened new routes for treating various types of cancers and incurable diseases. In the past two decades, a noticeable part of clinical practices has been devoting progressively to these products. The first step to develop such an ATMP product is to be familiar with other approved products to obtain a general view about this industry trend. The present paper depicts an overall perspective of approved ATMPs in different countries, while reflecting the degree of their success in a clinical point of view and highlighting their main safety issues and also related market size as a whole. In this regard, published articles regarding safety, efficacy, and market size of approved ATMPs were reviewed using the search engines PubMed, Scopus, and Google Scholar. For some products which the related papers were not available, data on the relevant company website were referenced. In this descriptive study, we have introduced and classified approved cell, gene, and tissue engineering-based products by different regulatory agencies, along with their characteristics, manufacturer, indication, approval date, related regulatory agency, dosage, product description, price and published data about their safety and efficacy. In addition, to gain insights about the commercial situation of each product, we have gathered accessible sale reports and market size information that pertain to some of these products.
... Therefore, the antigens are administered together with adjuvants, such as GM-CSF and DC-targeting antibodies, against DEC-205 (4, 5). In some clinical trials, GM-CSF was administered with autologous tumor-cell lysates or lysate-pulsed DCs in mesothelioma and metastatic RCC patients, respectively (90,91). NY-ESO-1, a cancer/testis antigen expressed in the testicular germ cells of normal adults, was engineered to be fused with an antibody targeting DEC-205 to induce cross-presentation of DCs and activation of NY-ESO-1-specific T cells from PBMCs of cancer patients (92). ...
Dendritic cells (DC), which consist of several different subsets, specialize in antigen presentation and are critical for mediating the innate and adaptive immune responses. DC subsets can be classified into conventional, plasmacytoid, and monocyte-derived DC in the tumor microenvironment, and each subset plays a different role. Because of the role of intratumoral DCs in initiating antitumor immune responses with tumor-derived antigen presentation to T cells, DCs have been targeted in the treatment of cancer. By regulating the functionality of DCs, several DC-based immunotherapies have been developed, including administration of tumor-derived antigens and DC vaccines. In addition, DCs participate in the mechanisms of classical cancer therapies, such as radiation therapy and chemotherapy. Thus, regulating DCs is also important in improving current cancer therapies. Here, we will discuss the role of each DC subset in antitumor immune responses, and the current status of DC-related cancer therapies.
... In a different approach, tumor lysates were tested to increase the availability of immunogenic antigens, comprising lysates from tumor cell lines (e.g., KATO-III [65,93]) or autologous patient tumor lysates [46,47,94]. Using cell lines provides the advantage of preparing readily available lysates in advance; however, again antigen loss of the patient's tumor cannot be addressed. ...
Dendritic cells (DCs) are pivotal regulators of immune responses, specialized in antigen presentation and bridging the gap between the innate and adaptive immune system. Due to these key features, DCs have become a pillar of the continuously growing field of cellular therapies. Here we review recent advances in good manufacturing practice strategies and their individual specificities in relation to DC production for clinical applications. These take into account both small-scale experimental approaches as well as automated systems for patient care.
... De Vries et al. [14] found that CD83 alone as a DC maturation marker is correlated with improved clinical efficacy of the vaccine in melanoma patients, while Heiser et al. used CD83 as a DC maturation marker for their study in prostate cancer, with a total CD83 expression~11% in DC [49]. HLA-DR and CD86 were found to be good surrogates of DC maturation by Kim et al. in renal cell carcinoma, with about 90% matured DC [50], while Su et al. indicated CD83 and CD86 as maturation markers [51]. Other studies suggest the use of a combination of CD80, CD83, CD86, and HLA-DR, along with CCR7, for a more reliable assessment of DC maturation, for example Waeckerle-Men et al. described that~50% CD83, 90% CD86, 40% CD80, and 80% HLA-DR expression had clinical benefits [52]. ...
Background
We developed and clinically test a new method to manufacture mature dendritic cells for autologous cell therapy of solid and hematological malignancies.
Methods
Peripheral blood monocytes are matured into dendritic cells with GM-CSF/IL-4 mixture, and then activated through IL1b, TNFa, IFNa-2A, and Poly(I:C), then viably frozen until use. DC are injected intra-dermally for a total of 6 injections.
Results
High-grade mDC with high pre- and post- freezing yields could be generated. These DC spontaneously produce IL12p70 and they can be further stimulated via CD40. In a patient with advanced pancreatic cancer treated with our mDC formulation, we observed no toxicity but remarkable immune response and objective response in terms of tumor shrinking.
Conclusion
We describe a new method to manufacture GMP-grade dendritic cells for autologous therapeutic cancer vaccines. We also show proof-of-principle efficacy in a patient with advanced pancreatic cancer.
Trial registration
NCT02705703.
... Immunotherapy has demonstrated some clinical effectiveness in tumors that are associated with an inflammatory or immune response, such as liver cancer, melanoma, and renal cell carcinoma [5][6][7]. It has also shown effects on chronic viral infection, including chronic hepatitis B (CHB) [8]. ...
Restoring antiviral immunity is a promising immunotherapeutic approach to the treatment of chronic hepatitis B virus (HBV) infection. Dendritic cells play a crucial role in triggering antiviral immunity. In this study, we identified immunodominant epitopes prevalent in CD8⁺ T cell responses. We characterized the hierarchy of HBV epitopes targeted by CD8⁺ T cells following autologous monocyte-derived dendritic cell (moDC) expansion in HBV-infected subjects with distinct disease stages: treatment-naïve (TN group, n = 168), treatment with complete virological response (TR group, n = 72), and resolved HBV infection (RS group, n = 28). T cell responses against 32 HBV epitopes were measured upon moDC expansion. Several subdominant epitopes that triggered HBV-specific CD8⁺ T cell responses were identified. These epitopes’ responses varied in individuals with different disease stages. Moreover, the most immunodominant and immunoprevalent epitope included the envelope residues 256-270 (Env256-270), corresponding to amino acid residues 93-107 in the small HBV surface protein (SHBs) across three patient groups. The frequency of Env256-270-specific interferon-γ-producing T cells was the highest in the RS group and the lowest in the TN group. In addition, individuals with HLA-A*02:03/02:06/02:07 were capable of responding to Env256-270. Env256-270-specific CD8⁺ T cells tolerated amino acid variations within the epitope detected in HBV genotypes B and C. This suggests that Env256-270 in SHBs is crucial in HBV-specific T cell immunity following autologous moDC expansion. It might be a potential target epitope for dendritic-cell-based immunotherapy for CHB patients with complete viral suppression by long-term NAs treatment.
Electronic supplementary material
The online version of this article (10.1007/s00705-018-4095-0) contains supplementary material, which is available to authorized users.
... The efficacy and success of the cellular immunotherapy, using sipuleucel-T, in the treatment of metastatic prostate cancer 28 has prompted the evaluation of DC vaccines to treat metastatic renal cell cancer. Phase I studies of vaccines containing DCs transfected with tumor RNA or pulsed with tumor lysate showed that these were safe and effective in treating RCC either alone [29][30][31][32] or in combination with cytokines. 33,34 Recent studies have shown that CSCs can be used as antigen sources to elicit DC-mediated CSC-specific humoral and cellular immune responses, leading to efficient antitumor immunity. ...
Cancer stem cells (CSCs) are responsible for tumor initiation, progression, and resistance to therapeutic agents; they are usually less sensitive to conventional cancer therapies, and could cause tumor relapse. An ideal therapeutic strategy would therefore be to selectively target and destroy CSCs, thereby preventing tumor relapse. The aim of the present study was to evaluate the effectiveness of dendritic cells (DCs) pulsed with antigen derived from CD105+ human renal cell carcinoma(RCC) CSCs against renal cancer cells in vitro and in vivo. We identified "stem-like" characteristics of CD105+ cells in two human RCC cell lines: A498 and SK-RC-39. Loading with cell lysates did not change the characteristics of the DCs. However, DCs loaded with lysates derived from CD105+ CSCs induced more functionally specific active T cells and specific antibodies against CSCs, and clearly depressed the tumor growth in mice. Our results could form the basis for a novel strategy to improve the efficacy of DC-based immunotherapy for human renal cell carcinoma. This article is protected by copyright. All rights reserved.
... In the setting of adjuvant immunotherapy, the long-term use of the cytokines interleukin (IL)-2 [7] and IFNα [8] has been extended to IL-21 [9], while in parallel new immune modulators have been developed, such as check point inhibitors acting on the PD1/PDL1 axis [10]. In the setting of active immunotherapy patients have been vaccinated with "undefined" tumor specificities using autologous or allogeneic tumor cells whose immunogenicity has been enhanced by transfection of immune stimulatory molecules [11][12][13], IFNγ treatment [14] or by loading their lysate / fusing them with dendritic cells (DC) [15][16][17]. With the identification of antigenic epitopes expressed by RCC [18][19][20][21][22], various clinical trials have evaluated their functionality [23,24]. ...
A major requirement for cancer immunotherapy is the development of biomarkers for prognosis and for monitoring therapy response. In an attempt to evaluate the immune response of renal cell carcinoma (RCC) patients, tumor lesions and / or blood samples from 12 RCC patients underwent deep T cell receptor (TCR) sequencing. Despite the low number of samples, different TCR distribution patterns could be detected. Most of the RCC patients presented "patient-specific" TCR sequences, and those clonotypes were present at higher frequency in tumor lesions suggesting a specific extravasation from the blood. Comparison among the tumor samples revealed also "patient-shared" TCR patterns. Indeed, a central core of 16 different TCRs were shared by 3 patients, whereas other 6 patients shared between 4 and 6 TCR sequences, with two sub-groups sharing 12 to 17 different clonotypes. The relative frequencies of shared clonotypes were very different varying from < 1% to a maximum of 37% of the total TCR repertoire. These data confirm the presence of tumor-specific TCR within the cancer tissue and suggest the existence of shared epitopes among different patients that might be used as targets for tumor immunotherapy.
... Lastly, in trials for renal cell carcinoma, DC vaccines improved the overall survival of patients 3.9-17.0 months [264][265][266][267][268]. Thus, there is discordance between overall survival and objective response rate. ...
Blood transfusion is the second most used medical procedures in health care systems worldwide. Over the last few decades, significant changes have been evolved in transfusion medicine practices. These changes were mainly needed to increase safety, efficacy, and availability of blood products as well as reduce recipients' unnecessary exposure to allogeneic blood. Blood products collection, processing, and storage as well as transfusion practices throughout all patient populations were the main stream of these changes. Health care systems across the world have adopted some or most of these changes to reduce transfusion risks, to improve overall patients' outcome, and to reduce health care costs. In this article, we are going to present and discuss some of these recent modifications and their impact on patients' safety.
Copyright © 2016 Elsevier Masson SAS. All rights reserved.
... In the past 20 years, CIK-based immunotherapies have been proved to be effective in a number of cancer types such as metastatic renal cell carcinoma, 30 gastric or colorectal adenocarcinoma, 2 and lung cancer. 31 Cytokine-induced killer cells are a heterogeneous cell population, including CD3 þ CD56 þ cells, CD3 þ CD56 À cells, and CD3 À CD56 þ cells. ...
Objective:
To investigate the efficacy of cytokine-induced killer cell-based immunotherapies in patients with advanced malignant solid tumors and the difference in clinical efficiency among 3 kinds of cytokine-induced killer cell-based immunotherapies.
Methods:
One hundred forty-six cases with advanced solid tumor, 230 cycles of cytokine-induced killer cell-based immunotherapies, were involved in this study. T-lymphocyte subsets, carcinoembryonic antigen, and adverse reactions were recorded.
Results:
CD3(+) T lymphocyte, Th, NKT, and Th/Tc were increased after cytokine-induced killer cell-based treatment, from 55.67 ± 3.64 to 84.12 ± 5.15, 26.56 ± 4.47 to 42.76 ± 3.68, 1.82 ± 0.58 to 7.08 ± 0.92, 0.79 ± 3.64 to 1.35 ± 0.20, respectively (P < .001). Carcinoembryonic antigen was decreased from 398.39 ± 219.16 to 127.26 ± 153.41 (P < .001). Difference values were greater than 0 (P < .001). Difference value of carcinoembryonic antigen was obviously less than 0 (P < .001). There was no obvious difference in all variations between cytokine-induced killer cell and DC+CIK groups (P > .05). The highest amount of CD3(+) T lymphocyte and Th was recorded after at least 4 cycles of immunotherapy. And CD8(+) T/CD4(+) T also began to decrease after 4 cycles of immunotherapy. Difference value of T lymphocyte and Tc of patients with surgery is higher than that of patients without surgery.
Conclusion:
Cytokine-induced killer cell-based immunotherapy is capable of increasing T-lymphocyte subsets, recovering cellular immunity without severe side effects, and is suitable for different kinds of solid cancer. Clinical efficiency of cytokine-induced killer cell-based immunotherapy is influenced by many factors such as surgery, stage.
... Therapeutic vaccines have also been explored in RCC. Phase I studies of vaccines containing DCs transfected with tumour RNA or pulsed with tumour lysate found them to be safe and effective in RCC, either as a monotherapy [41,42] or in combination with cytokines [43]. The tumour-associated peptide (TUMAP)-containing vaccine IMA901 specifically activates cytotoxic CD8+ T cells to recognize HLA molecules on the surface of RCC tumours [44]. ...
Recent advances in immuno-oncology have the potential to transform the practice of medical oncology. Antibodies directed against negative regulators of T cell function (checkpoint inhibitors), engineered cell therapies, and innate immune stimulators such as oncolytic viruses are effective in a wide range of cancers. Immune-based therapies have had a clinically meaningful impact on the treatment of advanced melanoma and the lessons regarding use of single agents and combinations in melanoma may be applicable to the treatment of urological cancers Checkpoint inhibitors, cytokine therapy and therapeutic vaccines are already showing promise in urothelial bladder cancer, renal cell carcinoma and prostate cancer. Critical areas of future immuno-oncology research include the prospective identification of patients who will respond to current immune-based cancer therapies, and the identification of new therapeutic agents that promote immune priming in tumors, and increase the rate of durable clinical responses. This article is protected by copyright. All rights reserved.
... To constitute these vaccines DCs are allowed to undergo maturation ex vivo in the presence of tumor antigens and then infuse into the tumor bearing host. Phase I studies of vaccines containing DCs transfected with tumor RNA or pulsed with tumor lysate found them to be safe and effective in RCC either alone [57][58][59] or in combination with cytokines [60,61]. ...
Localized renal cell carcinoma (RCC) is often curable by surgery alone. However, metastatic RCC is generally incurable. In the 1990s, immunotherapy in the form of cytokines was the mainstay of treatment for metastatic RCC. However, responses were seen in only a minority of highly selected patients with substantial treatment-related toxicities. The advent of targeted agents such as vascular endothelial growth factor tyrosine kinase inhibitors VEGF-TKIs and mammalian target of rapamycin (mTOR) inhibitors led to a change in this paradigm due to improved response rates and progression-free survival, a better safety profile, and the convenience of oral administration. However, most patients ultimately progress with about 12% being alive at 5 years. In contrast, durable responses lasting 10 years or more are noted in a minority of those treated with cytokines. More recently, an improved overall survival with newer forms of immunotherapy in other malignancies (such as melanoma and prostate cancer) has led to a resurgence of interest in immune therapies in metastatic RCC. In this review we discuss the rationale for immunotherapy and recent developments in immunotherapeutic strategies for treating metastatic RCC.
... In conclusion, our findings along with other studies [41,42] demonstrated that tumor cell lysate pulsed autologous dendritic cells could elicit T cell mediated immune responses in patients with breast cancer in vitro and in vivo. This approach may have important implication for the treatment of residual or resistant disease with active or adoptive immunotherapy after standard surgical and cytotoxic treatments, this modality particularly recommended to patients who induce type 1 immune response, however, the future design and implementation for clinical trial will ultimately determine the validity of this approach. ...
... CD27, a member of the tumor necrosis factor receptor family, is expressed on most peripheral blood T cells. 15 Upon T-cell activation, CD27 expression is strongly enhanced. 16 Ligation of CD27 by anti-CD27 monoclonal antibody provides a costimulatory signal to T cells and promotes T-cell proliferation and activation. ...
In the current study, we investigated whether anti-CD27 monoclonal antibody can enhance the antitumor efficacy of a dendritic cell-based vaccine in prostate cancer-bearing mice. The overall therapeutic effect of a dendritic cell-based vaccine for prostate cancer remains moderate. A prostate cancer model was established by subcutaneous injection of RM-1 tumor cells into male C57BL/6 mice on day 0. After 4 days, tumor-bearing mice were treated with RM-1 tumor lysate-pulsed dendritic cells (i.e., dendritic cell-based vaccine), anti-CD27 monoclonal antibody, or a combination of RM-1 tumor lysate-pulsed dendritic cells with anti-CD27 monoclonal antibody. Mice were killed at 21 days after tumor cell implantation. Tumor size was measured for assessment of antitumor effect. Spleens were collected for analysis of antitumor immune responses. The antitumor immune responses were evaluated by measuring the proliferation and activity of T cells, which have the ability to kill tumor cells. The combination therapy with RM-1 tumor lysate-pulsed dendritic cells and anti-CD27 antibody significantly enhanced T-cell proliferation and activity, and significantly reduced tumor growth, compared with monotherapy with RM-1 tumor lysate-pulsed dendritic cells or anti-CD27 antibody. Our results suggest that combined treatment can strengthen antitumor efficacy by improving T-cell proliferation and activity.
... In conclusion, our findings along with other studies [41,42] demonstrated that tumor cell lysate pulsed autologous dendritic cells could elicit T cell mediated immune responses in patients with breast cancer in vitro and in vivo. This approach may have important implication for the treatment of residual or resistant disease with active or adoptive immunotherapy after standard surgical and cytotoxic treatments, this modality particularly recommended to patients who induce type 1 immune response, however, the future design and implementation for clinical trial will ultimately determine the validity of this approach. ...
... The safety and feasibility of anti-cancer DC vaccines have been confirmed in several clinical trials. [5][6][7][8][9] The clinical response of DC vaccines targeting OvCa has recently been published. [10][11][12][13] These reports investigated the frequency of regulatory T cells (Tregs) as an effective means to reveal the efficacy of the vaccine. ...
While ovarian cancer (OvCa) responds well to surgery and conventional chemotherapy, a high recurrence rate of advanced OvCa is observed. In this phase I/II study, 10 OvCa patients with minimal residual disease were treated with autologous dendritic cells (DCs) and IL-2 to evaluate the safety and feasibility of this therapeutic strategy and to characterize the antigen-specific immune alterations induced through this treatment. Approximately 4 months after initial debulking and chemotherapy, patients received two subcutaneous doses of autologous monocyte-derived DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin (KLH) at 4-week intervals. After each DC inoculation, low-dose (200 mIU) IL-2 was introduced for 14 consecutive days as an immune adjuvant. The vaccination was well tolerated. In three out of 10 patients, the inclusion status after the initial therapy showed the maintenance of complete remission (CR) after DC vaccination for 83, 80.9 and 38.2 months without disease relapse. One patient with stable disease (SD) experienced the complete disappearance of tumor after DC vaccination, and this status was maintained for 50.8 months until tumor recurrence. In two patients with partial response (PR) was not responding to DC vaccination and their disease recurred. In the three patients with disease free long-term survival, significant immune alterations were observed, including increased natural killer (NK) activity, IFN-γ-secreting T cells, immune-stimulatory cytokine secretion and reduced immune-suppressive factor secretion after DC vaccination. Thus, in patients with NED status and increased overall survival, DC vaccination induced tumor-related immunity, potentially associated with long-term clinical responses against OvCa.Cellular & Molecular Immunology advance online publication, 30 June 2014; doi:10.1038/cmi.2014.40.
... Though not firmly established, the first generation DC vaccines were also some what effective in the induction of antitumor immunity, leading to tumor prevention in a mouse challenge model and to inhibition of tumor recurrence or metastasis after surgery in tumor-bearing mice [5]. Some clinical trials of early DC vaccines demonstrated significant tumor responses in patients with renal cell carcinoma, non-Hodgkin's lymphoma or melanoma [4,6]. DCs derived from CD34 + hematopoietic progenitor cells showed better clinical outcome than those by MoDCs. ...
... This is due to their cationic property and has been found to be influenced by pH and ionic strengths. 86 Furthermore, the DDMC/ DNA formation reaction is influenced by the Coulomb forces. The hydrophobic bonding strength as well as the hydrogen bonding strength have a role due to the hydrophobicity of the grafted MMA sections. ...
Lung cancer still remains to be challenged by novel treatment modalities. Novel locally targeted routes of administration are a methodology to enhance treatment and reduce side effects. Intratumoral gene therapy is a method for local treatment and could be used either in early-stage lung cancer before surgery or at advanced stages as palliative care. Novel non-viral vectors are also in demand for efficient gene transfection to target local cancer tissue and at the same time protect the normal tissue. In the current study, C57BL/6 mice were divided into three groups: (a) control, (b) intravenous and (c) intatumoral gene therapy. The novel 2-Diethylaminoethyl-Dextran Methyl Methacrylate Copolymer Non-Viral Vector (Ryujyu Science Corporation) was conjugated with plasmid pSicop53 from the company Addgene for the first time. The aim of the study was to evaluate the safety and efficacy of targeted gene therapy in a Lewis lung cancer model. Indeed, although the pharmacokinetics of the different administration modalities differs, the intratumoral administration presented increased survival and decreased distant metastasis. Intratumoral gene therapy could be considered as an efficient local therapy for lung cancer.Gene Therapy advance online publication, 28 November 2013; doi:10.1038/gt.2013.68.
... In another phase I/II trial with an autologous tumor cell lysate-pulsed DC vaccine, Kim et al. showed five SD and one PR at a median follow up of 17.5 months in nine patients with mRCC. 52 Except for one patient, all patients showed an antigen-specific lymphocyte proliferation response after the first cycle, and patients with PR or SD had higher responses by day 42. Similar findings have been published suggesting the value of DC vaccines. ...
Renal cell carcinoma is the most common malignant tumor originating from the kidney. Compared with other solid tumors, it does not respond to traditional management modalities, such as chemotherapy and radiotherapy. However, it is well known that renal cell carcinoma represents one of the most immune-responsive cancers and several immunotherapeutic strategies have been investigated in the management of renal cell carcinoma with variable degrees of success. The development of immunotherapy with α-interferon or high-dose interleukin-2 is the best established treatment, and is associated with durable disease control. Although the lack of defined antigens in renal cell carcinoma has hindered more specific vaccine development, research regarding vaccination therapy has been of special interest for the treatment of renal cell carcinoma for more than 30 years. At present, there are three types of cell-based vaccines in renal cell carcinoma treatment: autologous tumor-cell vaccines, genetically modified tumor vaccines and dendritic cell-based vaccines. A further type is peptide-based vaccination with tumor-associated antigens as possible targets, such as carbonic anhydrase IX, survivin and telomerase that are overexpressed in renal cell carcinoma. In the present article, we review data from completed clinical trials of vaccine therapy, and discuss future trials to assess the current knowledge and future role of vaccine therapy for renal cell carcinoma in the era of recently developed targeted therapy.
... Different phase I/II trials using DC pulsed with tumor lysates (either from autologous tumors or allogeneic tumor cell lines) have demonstrated modest clinical responses. [16][17][18][19] More recently, DC-tumor cell hybrids were used as a vaccine, but they also showed only modest efficacy. 20,21 The use of allogeneic rather than autologous cells has been established since allogeneic HLA molecules pose a potent immunogenic signal and may lead to T cell responses through expression of immunostimulatory cytokines by allo-reactive T cells. ...
Multi-kinase inhibitors have been established for the treatment of advanced renal cell cancer (RCC), but long-term results are still disappointing and immunotherapeutic approaches remain an interesting experimental option particularly in patients with a low tumor burden. DC are crucial for antigen-specific MHC-restricted T cell immunity. Furthermore, allogeneic HLA-molecules pose a strong immunogenic signal and may help to induce tumor-specific T cell responses. In this phase I/II trial, 7 patients with histologically confirmed progressive metastatic RCC were immunized repetitively with 1 × 10 ( 7) allogeneic partially HLA-matched DC pulsed with autologous tumor lysate following a schedule of 8 vaccinations over 20 weeks. Patients also received 3 Mio IE IL-2 sec.c. once daily starting in week 4. Primary endpoints of the study were feasibility and safety. Secondary endpoints were immunological and clinical responses. Vaccination was feasible and safe with no severe toxicity being observed. No objective response could be documented. However, while all patients had documented progress at study entry, 29% of the patients showed SD throughout the study with a mean TTP of 24.6 weeks (range 5 to 96 weeks). In 3/7 patients, TH1-polarized immune responses against RCC-associated antigens were observed. In one patient showing a minimal clinical response and a TTP of 96 weeks, clonally proliferated T cells against yet undefined antigens were induced by the vaccine. Vaccination with tumor antigen loaded DC remains an interesting experimental approach, but should rather be applied in the situation of minimal residual disease after systemic therapy. Additional depletion of regulatory cells might be a promising strategy.
... DC-based vaccination trials in human solid cancers have aimed to activate the immune system to recognize and destroy tumor cells. The safety and the feasibility of anti-cancer DC vaccines have been confirmed in several clinical trials (5)(6)(7)(8)(9). Nevertheless, reports on DC therapy for lung cancer patients were limited (10)(11)(12). ...
The anti-tumor effect of monocyte-derived DC (MoDC) vaccine was studied in lung cancer model with feasible but weak Ag-specific immune response and incomplete blocking of tumor growth. To overcome this limitation, the hematopoietic stem cell-derived DC (SDC) was cultured and the anti-tumor effect of MoDC & SDC was compared in mouse lung cancer minimal residual model (MRD). Therapeutic DCs were cultured from either CD34(+) hematopoietic stem cells with GM-CSF, SCF and IL-4 for 14 days (SDC) or monocytes with GM-CSF and IL-4 for 7 days (MoDC). DCs were injected twice by one week interval into the peritoneum of mice that are inoculated with Lewis Lung Carcinoma cells (LLC) one day before the DC injection. Anti-tumor responses and the immune modulation were observed 3 weeks after the final DC injection. CD11c expression, IL-12 and TGF-β secretion were higher in SDC but CCR7 expression, IFN-γ and IL-10 secretion were higher in MoDC. The proportion of CD11c(+)CD8a(+) cells was similar in both DC cultures. Although both DC reduced the tumor burden, histological anti-tumor effect and the frequencies of IFN-γ secreting CD8(+) T cells were higher in SDC treated group than in MoDC. Conclusively, although both MoDC and SDC can induce the anti-tumor immunity, SDC may be better module as anti-tumor vaccine than MoDC in mouse lung cancer.
... Autologous DC generation. DCs were generated from blood monocytes, as reported previously (22), with modifications. White blood cells obtained from the HCC patients through leukapheresis. ...
Dendritic cells (DCs) are increasingly used as adjuvants for vaccination strategies; however, there has been very little development in DC vaccines for patients with hepatocellular carcinoma (HCC). In this study, we assessed the safety, feasibility and efficacy of a multiple tumor-associated antigen (TAA)-pulsed DC vaccine in 5 patients with advanced HCC. DCs were generated by culturing blood monocytes in the presence of granulocyte macrophage-colony stimulating factor and interleukin-4 for 5 days. The DC vaccine was prepared by pulsing DCs with cytoplasmic transduction peptide-attached α-fetoprotein, glypican-3 and MAGE-1 recombinant fusion proteins and cultivating them in the presence of maturation cocktail. DCs were injected subcutaneously near the inguinal lymph nodes, followed by topical application of toll-like receptor-7 agonist around the injection site. We showed that our DC vaccine was safe and well-tolerated over 6 vaccinations in 5 patients. All 5 patients showed T cell responses against TAAs. Clinical benefit was observed in one of the 5 patients. In conclusion, the feasibility, safety and immune activity of DCs pulsed with TAAs were confirmed in HCC patients. However, clinical response was detected only in one patient. Future trials may consider applying this therapy in a less advanced stage to obtain better clinical responses.
... SEREX is still a valued, commonly used tool for antigen identification (Wang et al. 2009; Kiyamova et al. 2010). Several modifications have been incoporated to overcome restrictions for instance regarding the prokaryotic expression system, which does not allow for posttranslational modifications (Kim et al. 2007). With the advent of the " omics " era a comparatively high throughput analysis of differences in the gene expression level could be done with relative ease. ...
Recent studies have revealed that tumor-associated macrophages are the most abundant stromal cells in the tumor microenvironment and play an important role in tumor initiation and progression. Furthermore, the proportion of macrophages in the tumor microenvironment is associated with the prognosis of patients with cancer. Tumor-associated macrophages can polarize into anti-tumorigenic phenotype (M1) and pro-tumorigenic phenotype (M2) by the stimulation of T-helper 1 and T-helper 2 cells respectively, and then exert opposite effects on tumor progression. Besides, there also is wide communication between tumor-associated macrophages and other immune compositions, such as cytotoxic T cells, regulatory T cells, cancer-associated fibroblasts, neutrophils and so on. Furthermore, the crosstalk between tumor-associated macrophages and other immune cells greatly influences tumor development and treatment outcomes. Notably, many functional molecules and signaling pathways have been found to participate in the interactions between tumor-associated macrophages and other immune cells and can be targeted to regulate tumor progression. Therefore, regulating these interactions and CAR-M therapy are considered to be novel immunotherapeutic pathways for the treatment of malignant tumors. In this review, we summarized the interactions between tumor-associated macrophages and other immune compositions in the tumor microenvironment and the underlying molecular mechanisms and analyzed the possibility to block or eradicate cancer by regulating tumor-associated macrophage-related tumor immune microenvironment.
Due to its size, shape, and inherent expression of pathogen-associated molecular patterns and invasion-assistant adhesion proteins, Burkholderia pseudomallei can easily attach to, and then be internalized by, dendritic cells (DCs), leading to more efficient antigen cross-presentation if modified as carrier. Herein, we engineered Burkholderia pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and adjuvant CpG (C) to be used as a tumor vaccine (SB-LC). We found that the adhesion proteins of Burkholderia pseudomallei promote internalization of the SB-LC vaccine by DCs, and result in enhanced DC maturation and antigen cross-presentation. SB-LC induces robust cellular and humoral antitumor responses that synergistically inhibit tumor growth with minimal adverse side effects in several tumor models. Moreover, SB-LC vaccination reverses the immunosuppressive tumor microenvironment, apparently as a result of CD8+-induced tumor ferroptosis. Thus, SB-LC is a potential model tumor vaccine for translating into a clinically viable treatment option.
Cancer immunotherapy has advanced rapidly over the past decade leading to clinical approval of immune checkpoint blockade and adoptive cell transfer therapies. Further efforts into development of therapeutic vaccines had generated promising results in pre-clinical and clinical studies. Here, we demonstrate novel methodology for preparation of cell membranes into nano-sized vesicles and the development of characterization methods via nanoparticle flow cytometry. Cancer cell membranes from murine melanoma cell line expressing model antigen, ovalbumin, were used for generation of PEGylated vehicles (PEG-NPs), which efficiently delivered endogenous membrane-associated cancer antigens to the draining lymph nodes after subcutaneous administration. PEG-NPs were efficiently taken up by dendritic cells and, when dosed with a potent adjuvant, led to antigen-specific T cell activation and proliferation approximately 4-fold greater than treatment with traditional freeze-thaw lysates. In combination with immune checkpoint blockade (anti-PD-1 treatment), our vaccination approach led to therapeutic cure of 63% of mice and persistent memory responses rejecting additional tumor rechallenge. We further utilized our nanoparticle platform by using adjuvant-matured dendritic cells (DCs) generating MPLA-activated dendritic cell membrane vesicles ((MPLA)DC-MVs). This preparation led to nanoparticles carrying T cell activation ligands (CD80 and CD86) and promoted their proliferation activation in vitro compared to antigen peptide alone, as demonstrated by 2-fold increase in proliferation and 5- to 8-fold increase in live cell numbers and expression of CD25 activation marker. In addition, (MPLA)DC-MVs, but not unstimulated DC-MVs, resulted in activation of immature dendritic cells in vitro, indicated by 2- and 1.3-fold greater expression of CD40 and CD80, respectively. Administration of this formulation in vivo together with OVA peptide epitope led to 2-fold enhanced expansion and maintenance of antigen-specific T cells compared to peptide alone in mice that received adoptive cell transfer or had established OVA-expressing tumors. These studies had demonstrated the use for cell membranes in immunotherapy as vaccine vehicles, but further characterization and optimization could allow for improved efficacy, prompting us to adopt flow cytometry methods aimed at nanoparticle analysis. The technique was established by analysis of lipid-based synthetic formulations focused on demonstrating effective fluorescence detection and separation of individual particle populations. Proof of concept studies were used to confirm presence of ovalbumin on membrane-derived vesicles with antibody staining. Finally, we had utilized this technique to examine antigen display on hepatitis virus C vaccine formulation in order to determine if broadly neutralizing antibodies can bind efficiently and thus if they can be raised in mice immunized with these formulations. Our studies demonstrate that similar levels of broadly neutralizing antibody binding to nanoparticles translate to similar level of protection against cross-strain viral challenge. Taken together, this work has generated a foundation for further research into the use of cell membranes as nanoparticles for immunotherapeutic approaches and techniques necessary for their characterization.
Mobilizing antitumour immunity through vaccination potentially constitutes a powerful anticancer strategy but has not yet provided robust clinical benefits in large patient populations. Although major hurdles still exist, we believe that currently available strategies for vaccines that target dendritic cells or use them to present antitumour antigens could be integrated into existing clinical practice using prime–boost approaches. In the priming phase, these approaches capitalize on either standard treatment modalities to trigger in situ vaccination and release tumour antigens or vaccination with dendritic cells loaded with tumour lysates or patient-specific neoantigens. In a second boost phase, personalized synthetic vaccines specifically boost T cells that were triggered during the priming phase. This immunotherapy approach has been enabled by the substantial recent improvements in dendritic cell vaccines. In this Perspective, we discuss these improvements, highlight how the prime–boost approach can be translated into clinical practice and provide solutions for various anticipated hurdles.
Autoimmune diseases affect 10% of the world's population, and 1 in 200 people worldwide suffer from either multiple sclerosis (MS) or type 1 diabetes (T1D). While the targeted organ systems are different, MS and T1D share similarities in terms of autoreactive immune cells playing a critical role in pathogenesis. Both diseases can be managed only symptomatically without curative remission, and treatment options are limited and non‐specific. Most current therapies cause some degree of systemic immune suppression, leaving the patients susceptible to opportunistic infections and other complications. Thus, there is considerable interest in the development of immunotherapies not associated with generalized immune suppression for these diseases. This review presents current and preclinical strategies for MS and T1D treatment, emphasizing those aimed to modulate the immune response, including the most recent strategies for tolerance induction. A central focus is on the emerging approaches using nano‐ and microparticle platforms, their evolution as immunotherapeutic carriers, including those incorporating specific antigens to induce tolerance and reduce unwanted generalized immune suppression.
Despite the promise and advantages of autologous cancer cell vaccination, it remains challenging to induce potent anti-tumor immune responses with traditional immunization strategies with whole tumor cell lysate. In this study, we sought to develop a simple and effective approach for therapeutic vaccination with autologous whole tumor cell lysate. Endogenous cell membranes harvested from cancer cells were formed into PEGylated nano-vesicles (PEG-NPs). PEG-NPs exhibited good serum stability in vitro and draining efficiency to local lymph nodes upon subcutaneous administration in vivo. Vaccination with PEG-NPs synthesized from murine melanoma cells elicited 3.7-fold greater antigen-specific cytotoxic CD8+ T lymphocyte responses, compared with standard vaccination with freeze-thawed lysate in tumor-bearing mice. Importantly, in combination with anti-programmed death-1 (αPD-1) IgG immunotherapy, PEG-NP vaccination induced 4.2-fold higher frequency of antigen-specific T cell responses (P < 0.0001) and mediated complete tumor regression in 63% of tumor-bearing animals (P < 0.01), compared with FT lysate + αPD-1 treatment that exhibited only 13% response rate. In addition, PEG-NPs + αPD-1 IgG combination immunotherapy protected all survivors against a subsequent tumor cell re-challenge. These results demonstrate a general strategy for eliciting anti-tumor immunity using endogenous cancer cell membranes formulated into stable vaccine nanoparticles.
Antigen-specific treatments are highly desirable for autoimmune diseases in contrast to treatments which induce systemic immunosuppression. A novel antigen-specific therapy has been developed which, when administered semi-therapeutically, is highly efficacious in the treatment of the mouse model for multiple sclerosis, namely experimental autoimmune encephalomyelitis (EAE). The treatment uses dual-sized, polymeric microparticles (dMPs) loaded with specific antigen and tolerizing factors for intra- and extra-cellular delivery, designed to recruit and modulate dendritic cells toward a tolerogenic phenotype without systemic release. This approach demonstrated robust efficacy and provided complete protection against disease. Therapeutic efficacy required encapsulation of the factors in controlled-release microparticles and was antigen-specific. Disease blocking was associated with a reduction of infiltrating CD4⁺ T cells, inflammatory cytokine-producing pathogenic CD4⁺ T cells, and activated macrophages and microglia in the central nervous system. Furthermore, CD4⁺ T cells isolated from dMP-treated mice were anergic in response to disease-specific, antigen-loaded splenocytes. Additionally, the frequency of CD86hiMHCIIhi dendritic cells in draining lymph nodes of EAE mice treated with Ag-specific dMPs was reduced. Our findings highlight the efficacy of microparticle-based drug delivery to mediate antigen-specific tolerance, and suggest that such a multi-factor combinatorial approach can act to block autoimmunity.
Cell-free and concentrated ascites reinfusion therapy (CART) is an effective treatment for patients with refractory ascites. Cellular components such as cancer cells and blood cells are removed and discarded. The aim of this study was to investigate the alteration of immune cells in lavage fluid and the generation of dendritic cells (DCs) from lavage fluid obtained by CART. Flow cytometry analysis showed a trend toward immunosuppression and impairment in innate immunity in lavage fluid. Immature DCs with downregulation of CD14 and increased antigen-uptake were generated by culturing monocytes obtained from lavage fluid with GM-CSF and IL4. Following the culture with proinflammatory mediators, mature DCs with upregulation of CD83 and potent ability of T cell activation were induced. There were no significant phenotypical or functional differences between these DCs and DCs derived from peripheral blood, indicating lavage fluid might be employed for an alternative cellular source for the generation of DCs. © 2017 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy
Dendritic cells (DCs) represent a complex network of antigen-presenting cells that play a crucial role in the initiation of primary immunity, as well as maintaining the balance between immune tolerance and reactivity [1]. The modern field of DC biology was initiated in 1973 by Steinman and Cohn, who identified a subpopulation of murine splenocytes that had distinctive morphologic and phenotypic characteristics and powerfully stimulated T cell responses [2]. DCs have subsequently been described as the most potent antigen-presenting cells, which demonstrate the unique capacity to induce primary immune responses. Stimulation of naive T cells requires antigen presentation in the context of co-stimulatory and adhesion molecules, which serve as secondary signals needed for the activation of primary immunity. Antigen-presenting cells, such as B cells and macrophages, are effective in maintaining immune responses, but are incapable of initiating primary responses to novel antigens. In contrast, DC richly express MHC class I, II, co-stimulatory and adhesion molecules and are uniquely potent in initiating cellular immunity (Fig. 45-1) [3-6]. DCs also mediate humoral responses through the activation of helper T cells and direct effects on B cells [7]. DC activation of innate immunity has been demonstrated through their effects on NK cells and NKT cells [8, 9]. DCs have emerged as an area of intense interest in the fields of tumor immunotherapy and transplant biology.
Objective: To investigate the clinical effect and safety of autologous dendritic cells (DCs) stimulated by cytokine-induced killer (CIK) cells in patients with advanced renal cell carcinoma (RCC). Methods: During July 2011 to June 2012, peripheral blood mononuclear cells (PBMCs) were collected from 22 patients (12 males and 10 females, median age 60. 8 years [21-79 years J]) with advanced renal carcinoma in the Department of Medical Oncology of Suzhou Hospital affiliated to Nanjing Medical University, and DC-CIK cells were individually prepared from these PBMCs. Flow cytometry was performed to analyz the proportion of CD3 + T, CD8 + T, CD4 + T, NK(CD3 CD56 +)and NKT(CD3 + CD56 + Jcells in the DC-CIK cells. The cytotoxicity of DC-CIK cells on leukemia K562 cells (sensitive to NK cells) and renal carcinoma 786-0 cells (insensitive to NK cells) was detected by MTT assay. DC-CIK cells therapy was started when the conventional therapy (surgery + chemotherapy + radiotherapy/ cytokine therapy) finished and continued for 4 weeks. Each time, about (5.0±0.5)×l08 DC-CIK cells were returned through intravenous transfusion, and each patient received 5 times (one cycle) of DC-CIK cells intravenous infusion. The immunological index such as lymphocyte subsets and cytokine concentration in the peripheral blood were detected before and after DC-CIK infusion. The adverse reactions during the treatment were closely observed and recorded. Results: In the total number of DC-CIK cells, the proportion of CD3 positive (CD3) lymphocytes was (86. 92 ±5. 32)%, while the proportions of CD3 CD56 NKT cells and CD3 CD56 NK cells were (52. 04 ± 7. 33)% and(7. 85 ± 3. 15)% respectively. DC-CIK cells showed similar in vitro killing activities on 786-0 cells and K562 cells in vitro, with a killing rate of (16. 5 ±1.7) % and (18.4±1.9)% respectively (P =0.014), when the ratio of effect cells and target cells was 3:1. After patients received DC-CIK cell therapy, the proportion of peripheral lymphocyte sub-group (CD3 T, CD4 T, CD8 T and CD3 CD56 NK cells) showed no significant change (P >0. 05); compared to the cytokine levels prior to DC-CIK cells infusion, the levels of interleukine 2 (IL-2), in-terleukin 12 (IL-12) and interferon gamma (IFN-r) increased significantly (P <0.05), while the levels of tumor necrosis factor-a (TNF-α) and interleukine 10 (IL-10) changed unremarkably (P >0. 05). Two patients experienced a transient fever lasting 4-6 hours and one felt a short time of fatigue after DC-CIK cells infusion. Conclusion: DC-CIK cells can kill 786-0 cells and K562 cells effectively. The DC-CIK cells infusion may improve immune response of some patients with a safe level, and it can be one of the assistant treatment methods for advanced RCC patients.
Objectives: The first total hip replacement (THR) in Nigeria was performed in 1974. But due to infrastructural decay in public institutions, arthroplasty outcome was poor. National Orthopedic Hospital, Enugu (NOHE) - a regional trauma and orthopedic centre took the initiative in 2008. This paper presents our preliminary results and lists our challenges in establishing this service in a resource- constrained economy. Design: Prospective Setting: NOHE, Nigeria Subjects: Fifty-two patients who had primary hip arthroplasty between November 2008 and November 2010. Method: Details of demographic data, joints affected, etiology, co-morbidities, anesthesia, postoperative treatment, complications, and follow-up were recorded, analyzed and challenges noted Intervention: Total hip replacement. Main Outcome Measures: Improvement in patient's function and re-operation rate Result: Fifty-four THRs were done in fifty-two patients. Twenty nine (53.7%) patients were male. The mean age was 52 ± 2.4 years. Two patients had staged bilateral hip replacement. Twenty five (48.1%) patients had primary osteoarthritis. The commonest complaint at presentation was incapacitating hip pain. Half of the patients 26 (49.9%) had this pain for over four year. Trauma related secondary arthritis was responsible for 21 cases and old unreduced hip dislocation in five (9.6%) patients. Six patients had previous hip surgeries. Implant dislocation occurred in three (5.5%) patients. The functional status improved in all patients as shown by Harris Hip scores. Conclusion: There is an absolute need to develop arthroplasty service in Nigeria. A good number of the cases were complex primary arthroplasties. Most of the patients were relatively young and will outlive their implant.
Objective: Therapeutic efficacy of dendritic cell-based vaccine for renal cell carcinoma remains limited. In this study, we investigated whether anti-CD137 monoclonal antibody is capable of potentiating anti-tumor effect of dendritic cell-based vaccine. Design: Experimental study Setting: Research laboratory Subjects: Balb/c mice (8-10 weeks old) Interventions: A renal cell carcinoma model was established by subcutaneous injection of Renca tumor cells into Balb/c mice on day 0. After three days, tumor-bearing mice were treated with Renca tumor lysate-pulsed dendritic cells (i.e., dendritic cell-based vaccine), anti-CD137 monoclonal antibody, or combination of Renca tumor lysate-pulsed dendritic cells with anti-CD137 monoclonal antibody. Mice were killed on day 20 after tumor cell inoculation, and spleens were harvested for analysis of anti-tumor immune responses. Main Outcome Measures: The anti-tumor immune responses were analyzed by measuring proliferation and activity of T cells, which have the ability to kill tumor cells. The anti-tumor effect was assessed by measuring tumor size. Results: The combination therapy with Renca tumor lysate-pulsed dendritic cells and anti-CD137 antibody significantly increased T-cell proliferation and activity, and significantly inhibited tumor growth, compared with a single treatment with Renca tumor lysate-pulsed dendritic cells or anti-CD137 antibody. Conclusion: These results suggest that combination therapy can enhance anti-tumor effect by increasing T-cell proliferation and activity.
Renal cell carcinoma (RCC) is a chemotherapy-resistant disease, and current molecularly targeted therapies offer limited clinical benefit but no cures. The observation that RCC is immunogenic led to immune-based strategies for the treatment of this disease. Immunotherapy with high-dose IL-2 can induce long-term, complete responses in a small percentage of patients. IFN has been used as an immune intervention as well but with much less success than IL-2. Currently IFN is used only in combination with the anti-VEGF monoclonal antibody bevacizumab. Further investigation of novel immune interventions in RCC is ongoing with promising results. Dendritic cell vaccines have been tested in single-arm clinical trials suggesting improved survival when added to standard anti-angiogenic therapy. Monoclonal antibodies against PD-1 and PD-L1, novel immune targets, have shown promising response in phase I trials. Peptide vaccines have shown efficacy in phase II trials as well. Phase III trials to test these immune interventions are currently ongoing and have the potential to bring new, effective treatment options for patients with advanced RCC.
Objective: To evaluate the sterilization methods for whole tumor antigen stimulated-dendritic cells (DCs). Methods: The extract of whole tumor antigens was added to DC culture media. The antigen-stumulated DCs were cultured with graduated concentrations of vancomycin hydrochloride and imipenem/cilastatin sodium. The bacteria and fungi in the co-culture media were examined after 24 and 48 h. Results: Fungi were not detected in all 39 specimens of control group, but bacteria were detected in 7 samples. All specimens in experimental group showed negative fungi and bacteria. Conclusion: Addition of vancomycin hydrochloride and imipenem/cilastatin sodium in DC culture media with whole fumor antigen extract can be used for sterile culture of antigen-specific DC cells.
Keywords: Tumor vaccine Macrophage inflammatory protein 3a Tumor suppressive microenvironment DC vaccine a b s t r a c t The combination of several potential strategies so as to develop new tumor vaccines is an attractive field of translational medicine. Pulsing tumor lysates with dendritic cells (DCs), in-vivo attraction of DCs by macrophage inflammatory protein 3a (MIP-3a), and reversion of the tumor suppressive microenviron-ment have been tested as strategies to develop tumor vaccines. In this study, we generated an alginate microsphere (named PaLtTcAdMIP3a) that encapsulated tumor lysates, live tumor cells engineering with a recombinant MIP-3a adenovirus and BCG. We used PaLtTcAdMIP3a as a model vaccine to test its antitumor activities. Our results showed that PaLtTcAdMIP3a expressed and excreted MIP-3a, which effectively attracted DCs ex vivo and in vivo. Injection of PaLtTcAdMIP3a into tumor-bearing mice effectively induced both therapeutic and prophylactic antitumor immunities in CT26, Meth A, B16-F10 and H22 models, but without any ensuing increase in adverse effects. Both tumor-specific cellular and humoral immune responses, especially the CD8 þ T cell-dependent cytotoxic T immunity, were found in the mice injected with PaLtTcAdMIP3a. The anti-tumor activity was abrogated completely by depletion of CD8 þ and partially by CD4 þ T lymphocytes. In addition, the number of IFN-g-producing CD8 þ T cells in spleen and tumor tissues was significantly increased; but the number of CD4 þ CD25 þ FOXP3 þ regulatory T cells (Treg) in tumor tissues was decreased. These data strongly suggest that a combination of multi-current-using strategies such as the novel approach of using our PaLtTcAdMIP3a microspheres could be an effective tumor model vaccine.
Cytokine induced killer (CIK) cells-based treatments have shown antitumor activity against renal cell carcinoma (RCC) in vitro and in vivo. But the therapeutic options and benefits of various CIK cells were unknown for different stages of RCC. In this random clinical trial, the 3-year disease free survival (DFS) of operable RCC patients treated with autologous tumor lysates-pulsed dendritic cells co-cultured with cytokine induced killer (Ag-DC-CIK) was 96.7% compared with 57.7% in control group. Ag-DC-CIK immunotherapy decreased the risk of post-operative disease progression and relapse (P=0.0418). In inoperable RCC patients treated with CIK, the 3-year overall survival (OS) and progression-free survival (PFS) were significantly longer than control group (P=0.0116 and P=0.0212). The CD4(+)/CD8(+) T cell ratio in peripheral blood increased after last cell infusion in CIK treatment group, and especially further increased in Ag-DC-CIK treatment group (P=0.002). No severe toxicity was observed after infusion of CIK cells. Therefore, tumor antigen-sensitized Ag-DC-CIK cells might be more efficient and personalized for the patients with tumor resection, and CIK cells could improve the prognosis for those inoperable patients. According to the stages of RCC patients, different CIK cells-based immunotherapies would help to achieve more beneficial effects.
Copyright © 2015. Published by Elsevier Ireland Ltd.
Since the mid-1990s, dendritic cells have been used in clinical trials as cellular mediators for therapeutic vaccination of patients with cancer. Dendritic cell-based immunotherapy is safe and can induce antitumour immunity, even in patients with advanced disease. However, clinical responses have been disappointing, with classic objective tumour response rates rarely exceeding 15%. Paradoxically, findings from emerging research indicate that dendritic cell-based vaccination might improve survival, advocating implementation of alternative endpoints to assess the true clinical potency of dendritic cell-based vaccination. We review the clinical effectiveness of dendritic cell-based vaccine therapy in melanoma, prostate cancer, malignant glioma, and renal cell carcinoma, and summarise the most important lessons from almost two decades of clinical studies of dendritic cell-based immunotherapy in these malignant disorders. We also address how the specialty is evolving, and which new therapeutic concepts are being translated into clinical trials to leverage the clinical effectiveness of dendritic cell-based cancer immunotherapy. Specifically, we discuss two main trends: the implementation of the next-generation dendritic cell vaccines that have improved immunogenicity, and the emerging paradigm of combination of dendritic cell vaccination with other cancer therapies.
Unlabelled:
Dendritic cells are heterogeneous population of the leukocytes and most potent APC in activation of naive T lymphocytes. Therefore the DCs generated in vitro are under research for their application in anti-tumor immunotherapy. The aim of the study was generation of the immature dendritic cells from peripheral blood monocytes collected from colorectal cancer patients and comparison of their ability to endocytosis, cytokine production and immunophenotype to DCs generated from healthy donors.
Material and methods:
16 adenocarcinoma stage II patients were included in the study. Dendritic cells were generated in the presence of rhGM-CSF and IL-4. PBMC were isolated from the blood of patients and 16 healthy donors - control group. Immunophenotype, ability of endocytosis of Dextran- FITC as well as intracellular IL-12 expression of the generated dendritic cells was measured using flow cytometry. The cytokines (IL-6, IL-10, IL-12p70, IFN-γ) concentration in the supernatants of DCs culture was measured by ELISA.
Results:
The percentage of the immature dendritic cells and expression of CD206 and CD209 antigens was significantly higher in patients group (p <0.05 and p <0.001 respectively). Significantly (p <0.001) higher expression of the antigens which initiate the Th2 immune response (CD80-/CD86 + and B7-H2 + / CD209 +) was in the patients group. There were no differences in endocytosis ability and the cytokines (IL-6, IL-10, IL-12p70, IFN-γ) concentration between investigated groups.
Conclusions:
High immature markers expression on the generated dendritic cells together with identical endocytosis ability in patients group is advantageous in antitumor autologous cells immunotherapy planning. However there is one troubling fact--high expression of markers, which may induce tolerance to particular antigen. It seems to be more reasonable to use the autologous DCs in the antitumor immunotherapy, especially due to the incompatibility in allogenic cells in the context of HLA complex.
Die Prognose von fortgeschrittenen Nierenzellkarzinomen ist nach wie vor infaust. Die mediane Überlebenszeit für das metastasierte Stadium liegt unter einem Jahr. Da bei Nierenzellkarzinomen im Vergleich zu anderen Tumoren häufiger Spontanremissionen beobachtet werden, liegt die Vermutung nahe, dass das Nierenzellkarzinom prinzipiell auf eine immunologische Therapie anspricht. Dabei zeigten sich die Standardtherapieverfahren der unspezifischen Immuntherapie mit Interferonen und Interleukin-2 als nicht besonders erfolgversprechend hinsichtlich der Verlängerung der Gesamtüberlebenszeit bei gleichzeitig nicht zu unterschätzenden Nebenwirkungen. Daher ist die Entwicklung innovativer Strategien auf der Basis zellulärer spezifischer Immuntherapien, insbesondere für das fortgeschrittene Stadium, von eminent wichtiger Bedeutung. Dies gilt insbesondere unter Berücksichtigung der Verbesserung oder wenigstens Beibehaltung der Lebensqualität. Mittlerweile liegen für das Nierenzellkarzinom eindeutige Hinweise vor, dass es in besonderem Maße durch eine T-Zell-vermittelte Immunantwort behandelbar ist. In mehreren klinischen Phase-I/II-Studien sowie Einzelfallbeschreibungen konnte gezeigt werden, dass die Behandlung von Nierenzellkarzinomen mit einer spezifischen, zellbasierten Immuntherapie erfolgreich sein kann. Dabei wurden autologe Tumorvakzinen, Peptidvakzinen sowie autologe dendritische Zellen zur Behandlung eingesetzt. Die Therapien wurden allgemein gut vertragen, schwerwiegende Nebenwirkungen wurden für keine der Vakzinierungsstrategien beschrieben.
Summary
Advanced renal cell carcinomas belong to the tumors with up to now unfavorable prognosis. Once metastasized the median survivals are less than one year. Because of observed spontaneous remissions of metastases it is likely that an immunotherapy can be a successful treatment option. However, the treatment with an unspecific immunotherapy with Interferon or Interleukine-2 failed to have a significant impact on the survival and the side effects are high. Thus the development of a specific immunotherapy is required. Meanwhile, several groups have shown that a specific immunotherapy based on cellular vaccines can induce a clinical antitumor response in patients with renal cell carcinoma, especially in advanced metastatic disease stages.
After decades of research, dendritic cell (DC)-based vaccines for renal cell carcinoma have progressed from preclinical rodent models and safety assessments to Phase I/II clinical trials. DC vaccines represent a promising therapy that has produced measurable immunological responses and prolonged survival rates. However, there is still much room to improve in terms of therapeutic efficacy. The key issues that affect the efficiency and reliability of DC therapy include the selection of patients who will respond best to treatment, the proper preparation and administration of DC vaccines, and a combination of DC vaccination with other immune-enhancing therapies (e.g., removal of Tregs, CTLA-4 blockade and lymphodepletion). Additional antiangiogenic agents will hopefully lead to greater survival benefits for patients in early disease stages. This review focuses on the different approaches of DC-based vaccination against renal cell carcinoma and potential strategies to enhance the efficacy of DC vaccination.
It remains a challenge to inhibit the local recurrence or distant metastasis of localized or locally advanced renal cell carcinoma (RCC) after surgical resection. We investigated the feasibility, safety and efficacy of immunotherapy using autologous tumor lysate (TL)-pulsed dendritic cells (DCs) and cytokine-induced killer (CIK) cells in patients with localized or locally advanced RCC.
From January 2001 to July 2009, we collected 137 patients that met the selection criteria and randomly divided them into three groups. After surgery, immunotherapy with TL-pulsed DCs-CIK cells (DC-CIK group) and interferon (IFN)-α (IFN-α group) was performed in 46 patients, respectively. The other 45 patients received no postoperative adjuvant therapy (the control group). The changes in the numbers of T lymphocyte subsets, including CD4(+)CD25(high) regulatory T cells (Treg), were determined before the operation and after immunotherapy. The overall survival was compared among the three groups.
An increase of the CD4(+)/CD8(+) ratio and a decrease of CD4(+)CD25(high) cells were observed after TL-pulsed DC-CIK cells or IFN-a immunotherapy. All patients tolerated the TL-pulsed DC-CIK cells immunotherapy very well, and side effects in the DC-CIK group were less than in the IFN-α group. The metastasis and recurrence rates were significantly decreased after TL-pulsed DC-CIK cells or IFN-α immunotherapy compared with the control group (P < 0.01). The Log-rank test showed that the overall survival rates were significantly higher in the DC-CIK group and IFN-α group than that in the control group (P < 0.01), but there was no difference between the DC-CIK group and IFN-α group (P > 0.05).
Postoperative immunotherapy with TL-pulsed DC-CIK cells may prevent recurrence/metastasis and increase the overall survival rate after surgery in localized or locally advanced RCC.
We performed continuous dendritic cells (DCs) vaccination to treat a patient with chemotherapy-resistant recurrent rectal carcinoma and lung and bone metastases. A patient has received a total of 66 DC vaccinations at 14-day intervals for 3 years until his death. Necrotic whole tumor cells (WTC) were selected as the tumor-associated antigen source because they showed a greater capacity for DC maturation and interleukin-12 secretion than both necrotic tumor lysate alone and necrotic tumor cell fragment alone. After the sixth vaccination, both skin test and interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) response by peripheral blood T-cells to WTC-pulsed DCs were positive. Importantly, T-cell responses were positive towards DCs pulsed with several synthetic peptides including Carcinoembryonic antigen (CEA), Melanoma associated antigen (MAGE)1 and MAGE3. A biopsy specimen collected from the pelvic bone metastasis after the 6th vaccination showed marked necrotic change of the carcinoma cells, with many infiltrating mononuclear cells. The patient did not show any particular adverse reactions to vaccination such as autoimmune phenomena. Our experience of this case suggests that continuous long-term vaccination with autologous WTC-pulsed DCs can elicit in vivo T-cell response against multiple tumor-associated antigens and induce tumor regression in disease that has proven resistant to intensive chemo- or radiation therapy.
Dendritic cells (DCs) represent the bridging cell compartment between a variety of nonself antigens (i.e., microbial, cancer and vaccine antigens) and adaptive immunity, orchestrating the quality and potency of downstream immune responses. Because of the central role of DCs in the generation and regulation of immunity, the modulation of DC function in order to shape immune responses is gaining momentum. In this respect, recent advances in understanding DC biology, as well as the required molecular signals for induction of T-cell immunity, have spurred many experimental strategies to use DCs for therapeutic immunological approaches for infections and cancer. However, when DCs lose control over such 'protective' responses - by alterations in their number, phenotype and/or function - undesired effects leading to allergy and autoimmune clinical manifestations may occur. Novel therapeutic approaches have been designed and currently evaluated in order to address DCs and silence these immunopathological processes. In this article we present recent concepts of DC biology and some medical implications in view of therapeutic opportunities.
CD34+ cells in human cord blood and marrow are known to give rise to dendritic cells (DC), as well as to other myeloid lineages. CD34+ cells are rare in adult blood, however, making it difficult to use CD34+ cells to ascertain if DC progenitors are present in the circulation and if blood can be a starting point to obtain large numbers of these immunostimulatory antigen-presenting cells for clinical studies. A systematic search for DC progenitors was therefore carried out in several contexts. In each case, we looked initially for the distinctive proliferating aggregates that were described previously in mice. In cord blood, it was only necessary to deplete erythroid progenitors, and add granulocyte/macrophage colony-stimulating factor (GM-CSF) together with tumor necrosis factor (TNF), to observe many aggregates and the production of typical DC progeny. In adult blood from patients receiving CSFs after chemotherapy for malignancy, GM-CSF and TNF likewise generated characteristic DCs from HLA-DR negative precursors. However, in adult blood from healthy donors, the above approaches only generated small DC aggregates which then seemed to become monocytes. When interleukin 4 was used to suppress monocyte development (Jansen, J. H., G.-J. H. M. Wientjens, W. E. Fibbe, R. Willemze, and H. C. Kluin-Nelemans. 1989. J. Exp. Med. 170:577.), the addition of GM-CSF led to the formation of large proliferating DC aggregates and within 5-7 d, many nonproliferating progeny, about 3-8 million cells per 40 ml of blood. The progeny had a characteristic morphology and surface composition (e.g., abundant HLA-DR and accessory molecules for cell-mediated immunity) and were potent stimulators of quiescent T cells. Therefore, large numbers of DCs can be mobilized by specific cytokines from progenitors in the blood stream. These relatively large numbers of DC progeny should facilitate future studies of their Fc epsilon RI and CD4 receptors, and their use in stimulating T cell-mediated resistance to viruses and tumors.
Recombinant human interleukin-2 (aldesleukin) and recombinant human interferon alfa can induce notable tumor regression in a limited number of patients with metastatic renal-cell carcinoma. We conducted a multicenter, randomized trial to determine the effect of each cytokine independently and in combination, and to identify patients who are best suited for this treatment.
Four hundred twenty-five patients with metastatic renal-cell carcinoma were randomly assigned to receive either a continuous intravenous infusion of interleukin-2, subcutaneous injections of interferon alfa-2a, or both. The main outcome measure was the response rate; secondary outcomes were the rates of event-free and overall survival. Predictive factors for response and rapid progression were identified by multivariate analysis.
Response rates were 6.5 percent, 7.5 percent, and 18.6 percent (P<0.01) for the groups receiving interleukin-2, interferon alfa-2a, and interleukin-2 plus interferon alfa-2a, respectively. At one year, the event-free survival rates were 15 percent, 12 percent, and 20 percent, respectively (P=0.01). There was no significant difference in overall survival among the three groups. Toxic effects of therapy were more common in patients receiving interleukin-2 than in those receiving interferon alfa-2a. Response to treatment was associated with having metastasis to a single organ and with receiving the combined treatment. The probability of rapid progression of disease was at least 70 percent for patients with at least two metastatic sites, liver metastases, and a period of less than one year between the diagnosis of the primary tumor and the appearance of metastases.
Cytokines are active in a few patients with metastatic renal-cell carcinoma. The higher response rate and longer event-free survival obtained with a combination of cytokines must be balanced against the toxicity of such treatment.
Vascular endothelial growth factor (VEGF) is known to play crucial roles in tumor angiogenesis. We have investigated the circulating level of VEGF in sera from cancer patients as well as from healthy normal controls using a sensitive enzymatic immunoassay. Immunoreactive VEGF proteins were detectable in normal sera, and the cutoff level was determined to be 180 pg/ml. In examined patients with all types of cancer, including breast, gastrointestinal, hepatobiliary, and lung cancer, an aberrant increase in the circulating level of VEGF was detected. For example, in 137 primary breast cancer patients, 12 (8.8%) showed an aberrant increase in VEGF levels. This aberrant expression of VEGF in sera was significantly associated with the progression of the disease, and with VEGF protein expression in tumor tissues. In addition, a Western blot analysis confirmed the presence of the VEGF165 form in sera from a patient with recurrent breast cancer. It was concluded that VEGF was detectable in normal sera, and its level was increased in some populations of cancer patients. A positive angiogenesis regulator, VEGF might function as an endocrine growth factor, particularly for solid tumors.
Angiogenesis and immunosuppression are the main biological mechanisms responsible for cancer progression. Moreover, recent observations suggesting a negative influence of angiogenesis on anticancer immunity have shown that some angiogenic factors, such as VEGF, may induce immunosuppression. In addition, the evidence of abnormally high blood levels of VEGF has been proven to be associated with resistance to IL-2 immunotherapy. The present study was performed to establish a possible relation ship between the efficacy of IL-2 cancer immunotherapy and changes in circulating levels of VEGF, IL-12, mature and immature dendritic cells (DC). The study included 25 metastatic renal cell cancer patients who underwent subcutaneous low-dose IL-2 immunotherapy (6 MIU/day for 6 days/week for 4 weeks). Immature and mature DCs were identified as CD123+ and CD11c+ cells, respectively. The clinical response consisted of partial response (PR) in five, stable disease (SD) in 11 and progressive disease (PD) in the remaining nine patients. The mean IL-12 levels observed during IL-2 immunotherapy were significantly higher in patients with PR or SD than in those with PD, whereas the mean VEGF concentrations were significantly higher in patients who had PD than in those with PR or SD. Finally, a significant increase in the mean number of circulating mature DCs occurred only in patients with PR or SD, whereas no significant change was seen in patients with PD. By contrast, no significant change was observed in the mean number of immature DCs. This study shows that the efficacy of IL-2 immunotherapy is associated with a significant increase in circulating mature DCs and IL-12, without any concomitant increase in VEGF concentrations. Further studies will be required to better define the relationship between activation of anticancer immunity and control of angiogenesis-related mechanisms.
Polyvalent cancer vaccines targeting the entire antigenic spectrum on tumor cells may represent a superior therapeutic strategy for cancer patients than vaccines solely directed against single Ags. In this study, we show that autologous dendritic cells (DC) transfected with RNA amplified from microdissected tumor cells are capable of stimulating CTL against a broad set of unidentified and critical prostate-specific Ags. Although the polyclonal CTL responses generated with amplified tumor RNA-transfected DC encompassed as a subcomponent a response against prostate-specific Ag (PSA) as well as against telomerase reverse transcriptase, the tumor-specific CTL were consistently more effective than PSA or telomerase reverse transcriptase CTL to lyse tumor targets, suggesting the superiority of the polyclonal response. Although tumor RNA-transfected DC stimulated CTL, which recognized not only tumor but also self-Ags expressed by benign prostate tissue, these cross-reactive CTL were exclusively specific for the PSA, indicating an immunodominant role of PSA in the prostate cancer-specific immune response. Our data suggest that tumor RNA-transfected DC may represent a broadly applicable, potentially clinically effective vaccine strategy for prostate cancer patients, which is not limited by tumor tissue availability for Ag preparation and may minimize the risk of clonal tumor escape.
We wanted to evaluate feasibility and safety of dendritic cell-based immunotherapy in patients with metastatic renal cell carcinoma (RCC).
Patients with metastatic RCC (n = 35) received vaccinations (i.v. or i.d.) of CD83+ autologous monocyte-derived dendritic cells (moDCs). MoDCs were loaded with lysate of cultured autologous or allogeneic permanent tumor cells (A-498) as well as keyhole limpet hemocyanin as control and helper antigen. Maturation of moDCs was induced by a combination of tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, and prostaglandin E2.
Treatment was associated with transient flu-like symptoms. In 2 of 27 evaluable patients, any evidence of disease disappeared (complete response). In both cases, metastatic tissue had been the source of tumor antigen. One patient had an objective partial response. Seven patients had stable disease, the remaining 17 patients had progressive disease. In 11 of 11 patients evaluated, moDCs induced strong immune responses against keyhole limpet hemocyanin. In 5 of 6 patients tested, enhanced immune responses against oncofetal antigen (immature laminin receptor; OFA/LRP) could also be detected. The strongest responses against OFA/LRP were detectable in 2 patients with complete response and partial response, respectively. At the time of submission, mean follow up is 32 months and 8 patients are currently alive.
Our data indicate that moDC-based vaccination is well tolerated and has immunological as well as clinical effects in patients with metastatic RCC. OFA/LRP might be an attractive candidate antigen for DC-based immunotherapy of RCC.
The molecular characterisation of human tumour antigens recognised by T cells has provided new impetus for immunisation of patients bearing tumours expressing well-defined antigens. After evaluating the immunogenicity of the new, molecularly characterised antigens in vitro, several clinical studies were conducted to assess the in vivo immunogenicity and the clinical efficacy of vaccines including these antigens. The findings generated by trials based on the administration of peptides or DNA-encoding antigens are discussed to highlight the limits of this therapeutic approach; however, this approach has resulted in some complete and durable regressions, although still in a unsatisfactory small number of cases (5-25%). The recent use of dendritic cells loaded ex vivo with tumour antigens suggests that a high frequency of tumour-specific immune responses can be achieved. Possible means of overcoming the clinical limits and improving the outcome of previous studies are also discussed.
Tumour immunotherapy using dendritic cells (DCs) is a new therapeutic approach, which has been applied to a variety of different cancers over the last 5 years. Here we discuss the clinical results of these trials in relation to the different protocols used to generate DCs, and in particular the effect that DC maturation state has had on clinical responses. In ten different melanoma trials a total of 167 patients have been treated, resulting in 9 complete tumour regressions, 24 partial regressions, 26 patients with stable disease, and 108 with progressive disease. Favourable response, defined as any outcome other than progressive disease, was not associated with previous chemotherapy, but was significantly correlated ( p<0.001) with the addition of TNF-alpha for the maturation of DCs in vitro. Hence DC maturation state has had an impact on clinical responses to therapy. However, TNF-alpha is not the only molecule capable of inducing DC maturation, and strategies for improving clinical responses by optimizing DC maturation are discussed.
B and T lymphocytes are the mediators of immunity, but their function is under the control of dendritic cells. Dendritic cells in the periphery capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. They not only activate lymphocytes, they also tolerize T cells to antigens that are innate to the body (self-antigens), thereby minimizing autoimmune reactions. Once a neglected cell type, dendritic cells can now be readily obtained in sufficient quantities to allow molecular and cell biological analysis. With knowledge comes the realization that these cells are a powerful tool for manipulating the immune system.
We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo.
Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients).
All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. In a second patient, DTH-derived T cells showed specific lysis of tumor cells expressing the antigens used for DC pulsing. Only patients vaccinated with mature DCs showed objective clinical responses. Interestingly, both patients with long-term progression-free survival (22 and >40 months) were both vaccinated with mature DCs and demonstrated antigen-specific T-cell reactivity of DTH-derived T cells.
We conclude that mature DC are superior to immature DC in the induction of immunological responses in melanoma patients, which may translate into improved clinical results.
Autologous dendritic cells transfected with total renal tumor RNA have been shown to be potent stimulators of CTLs and antitumor immunity in vitro. A Phase I trial was conducted to evaluate this strategy for feasibility, safety, and efficacy to induce tumor-specific T-cell responses in subjects with metastatic renal cell carcinoma. Renal tumor RNA-transfected dendritic cells were administered to 10 evaluable study patients with no evidence of dose-limiting toxicity or vaccine-related adverse effects including autoimmunity. In six of seven evaluable subjects, expansion of tumor-specific T cells was detected after immunization. The vaccine-induced T-cell reactivities were directed against a broad set of renal tumor-associated antigens, including telomerase reverse transcriptase, G250, and oncofetal antigen, but not against self-antigens expressed by normal renal tissues. Although most patients underwent secondary therapies after vaccination, tumor-related mortality of the study subjects was unexpectedly low with only 3 of 10 patients dying from disease after a mean follow-up of 19.8 months. These data provide a scientific rationale for continued clinical investigation of this polyvalent vaccine strategy in the treatment of metastatic renal cell carcinoma and, potentially, other cancers.
A phase I trial was conducted to evaluate the feasibility, safety, and efficacy of a dendritic cell-based vaccination in patients with metastatic renal cell carcinoma (RCC). Autologous mature dendritic cells derived from peripheral blood monocytes were pulsed with the HLA-A2-binding MUC1 peptides (M1.1 and M1.2). For the activation of CD4(+) T-helper lymphocytes, dendritic cells were further incubated with the PAN-DR-binding peptide PADRE. Dendritic cell vaccinations were done s.c. every 2 weeks for four times and repeated monthly until tumor progression. After five dendritic cell injections, patients additionally received three injections weekly of low-dose interleukin-2 (1 million IE/m(2)). The induction of vaccine-induced T-cell responses was monitored using enzyme-linked immunospot and Cr release assays. Twenty patients were included. The treatment was well tolerated with no severe side effects. In six patients, regression of the metastatic sites was induced after vaccinations with three patients achieving an objective response (one complete response, two partial responses, two mixed responses, and one stable disease). Additional four patients were stable during the treatment for up to 14 months. MUC1 peptide-specific T-cell responses in vivo were detected in the peripheral blood mononuclear cells of the six patients with objective responses. Interestingly, in patients responding to the treatment, T-cell responses to antigens not used for vaccinations, such as adipophilin, telomerase, or oncofetal antigen, could be detected, indicating that epitope spreading might occur. This study shows that MUC1 peptide-pulsed dendritic cells can induce clinical and immunologic responses in patients with metastatic RCC.
Immune surveillance of cancer requires antigen-presenting cells which activate T cells specific for tumor-associated antigens. We show here that substantial numbers of dendritic cells, which are the most potent antigen-presenting cells, emigrate from renal-tumor explants in organ culture. Tumor-derived dendritic cells presented with all characteristics of mature dendritic cells. Dendritic cells could be identified by typical cytoplasmic projections (=veils). They expressed high levels of MHC products and of the co-stimulator CD86 (B7-2). Dendritic cells expressed the CD45RO isoform but not CD45RA. The most important point was that up to 9% of the emigrating leukocytes expressed the CD83 antigen, a specific marker for mature dendritic cells. CD83⁺ cells were approximately 40-fold enriched in the tumor tissue as compared to the peripheral blood. In contrast to cultured blood dendritic cells, tumor-emigrant dendritic cells had a reduced potential to capture soluble antigen, as shown by the exclusion of fluoresceinated Dextran molecules. Finally, in mixed leukocyte reactions, tumor-derived dendritic cells were able to stimulate naive T cells from cord blood, which is a unique feature of dendritic cells. This study demonstrates that genuine dendritic cells reside in or infiltrate renal-cell carcinoma tissue. The failure of patients with renal-cell carcinoma to mount an anti-tumor immune response despite the presence of professional antigen-presenting cells in the tumor tissue suggests that tumor-associated dendritic cells are suppressed in situ, in a similar way to that described for tumor-infiltrating lymphocytes. © 1996 Wiley-Liss, Inc.
Background. Treatment with interleukin-2 (IL-2) and lymphokine-activated killer cells (LAK) resulted in responses in some patients with advanced renal cell carcinoma (RCC). However, the relative therapeutic benefit of the addition of LAK to IL-2 was unknown.
PURPOSEThe objective of this study was to determine the tolerance and effect of moderate-dose recombinant human interleukin-2 (rHu IL-2) and tumor-infiltrating lymphocytes (TIL) in patients with metastatic melanoma (MM) or renal cell carcinoma (RCC) refractory to standard therapy.PATIENTS AND METHODS
Twenty-six patients (18 MM and eight RCC) were entered onto this pilot study. TIL were isolated from fresh biopsy material and activated with anti-CD3 antibody, OKT3, for 48 hours and expanded in 100 IU/mL r-methionyl Hu IL-2 alanine 125 (r-met Hu IL-2 [ala-125]). At least 10(10) TIL were reinfused intravenously in three divided injections on days 2, 4, and 6 of the protocol. A maximum dose of 30,000 U/kg of IL-2 per injection was administered every 8 hours from day 2 through day 11 for a total of 28 doses.RESULTSSixteen melanoma patients completed the study. Of these, three (19%) showed a durable complete response (CR), nine (56%) had no response (NR), and four (25%) had progressive disease (PD). One nonrespo...
CD34+ cells in human cord blood and marrow are known to give rise to dendritic cells (DC), as well as to other myeloid lineages. CD34+ cells are rare in adult blood, however, making it difficult to use CD34+ cells to ascertain if DC progenitors are present in the circulation and if blood can be a starting point to obtain large numbers of these immunostimulatory antigen-presenting cells for clinical studies. A systematic search for DC progenitors was therefore carried out in several contexts. In each case, we looked initially for the distinctive proliferating aggregates that were described previously in mice. In cord blood, it was only necessary to deplete erythroid progenitors, and add granulocyte/macrophage colony-stimulating factor (GM-CSF) together with tumor necrosis factor (TNF), to observe many aggregates and the production of typical DC progeny. In adult blood from patients receiving CSFs after chemotherapy for malignancy, GM-CSF and TNF likewise generated characteristic DCs from HLA-DR negative precursors. However, in adult blood from healthy donors, the above approaches only generated small DC aggregates which then seemed to become monocytes. When interleukin 4 was used to suppress monocyte development (Jansen, J. H., G.-J. H. M. Wientjens, W. E. Fibbe, R. Willemze, and H. C. Kluin-Nelemans. 1989. J. Exp. Med. 170:577.), the addition of GM-CSF led to the formation of large proliferating DC aggregates and within 5-7 d, many nonproliferating progeny, about 3-8 million cells per 40 ml of blood. The progeny had a characteristic morphology and surface composition (e.g., abundant HLA-DR and accessory molecules for cell-mediated immunity) and were potent stimulators of quiescent T cells. Therefore, large numbers of DCs can be mobilized by specific cytokines from progenitors in the blood stream. These relatively large numbers of DC progeny should facilitate future studies of their Fc epsilon RI and CD4 receptors, and their use in stimulating T cell-mediated resistance to viruses and tumors.
Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered i.v. due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by i.v., intradermal (i.d.), or intralymphatic (i.l.) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-γ production, however, was seen only with i.d. and i.l. routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the i.v. route developed Ag-specific Abs compared with one of six for i.d. and two of six for i.l. routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.
. Up to now, clinical tumor-markers for renal cell carcinoma (RCC) have been lacking. Increased plasma levels of transforming growth factor-ş (TGF-ş) were described as a tumor-marker and prognostic factor in RCC. The aim of this study was to test the clinical suitability of plasma TGF-ş as a tumor-marker for RCC. The concentrations of active and latent TGF-ş were determined in plasma of patients with localized (n=39) and metastasised (n=17) RCC. A newly developed, highly sensitive ELISA, which is specific for the isoform ş, was used. Active TGF was directly measured in the EDTA plasma. To determine the amount of latent TGF-ş, which is bound predominantly at Ō-macroglobulin, an optimized activation procedure was applied. Patients with localized RCC showed median concentrations of 16,700 ng/l (6,200-54,800 ng/l) for latent TGF-ş. A total of 94 patients with various non-malignant urological diseases were recruited as a control group. In comparison, this group had median concentrations of 19,900 ng/l (2,640-52,300 ng/l) for latent TGF-ş. There was no significant difference (nonparametric Kruskal-Wallis ANOVA) between these groups. Patients with metastatic RCC showed median concentrations of 34,500 ng/l (6,800-48,960 ng/l) for latent TGF-ş. In comparison to the localized RCC group, a statistically significant difference was found. Plasma levels after operative therapy (days 1, 5 and 10) and during follow-up without evidence of disease (2-6 months) showed no significant differences. Contrary to other study groups, our results suggest that TGF-ş is not a suitable tumor-marker for the diagnosis of localized RCC. In the face of higher TGF-ş plasma levels in metastatic disease, TGF-ş may be useful in the early detection of RCC recurrence or to control the success of immunochemotherapy.
Dendritic cells (DCs) elicit potent anti-tumoral T-cell responses in vitro and in vivo. However, different types of DC have yet to be compared for their capacity to induce anti-tumor responses in vivo at different developmental stages. Herein, we correlated the efficiencies of different types of monocyte-derived DC as vaccines on the resulting anti-tumor immune responses in vivo. Immature and mature DCs were separately pulsed with a peptide derived from tyrosinase, MelanA/MART-1 or MAGE-1 and a recall antigen. Both DC populations were injected every 2 weeks in different lymph nodes of the same patient. Immune responses were monitored before, during and after vaccination. Mature DCs induced increased recall antigen-specific CD4+ T-cell responses in 7/8 patients, while immature DCs did so in only 3/8. Expansion of peptide-specific IFN-γ–producing CD8+ T cells was observed in 5/7 patients vaccinated with mature DCs but in only 1/7 using immature DCs. However, these functional data did not correlate with the tetramer staining. Herein, immature DCs also showed expansion of peptide-specific T cells. In 2/4 patients vaccinated with mature DCs, we observed induction of peptide-specific cytotoxic T cells, as monitored by chromium-release assays, whereas immature DCs failed to induce peptide-specific cytotoxic T cells in the same patients. Instead, FCS-cultured immature DCs induced FCS-specific IgE responses in 1 patient. Our data demonstrate that this novel vaccination protocol is an efficient approach to compare different immunization strategies within the same patient. Thus, our data define FCS-free cultured mature DCs as superior inducers of T-cell responses in melanoma patients. © 2001 Wiley-Liss, Inc.
Background:
Treatment with interleukin-2 (IL-2) and lymphokine-activated killer cells (LAK) resulted in responses in some patients with advanced renal cell carcinoma (RCC). However, the relative therapeutic benefit of the addition of LAK to IL-2 was unknown.
Methods:
A randomized Phase III trial was conducted in patients with RCC comparing continuous intravenous infusion (CI) IL-2 alone with CI IL-2 plus LAK. Interleukin-2 was administered at 3 x 10(6) U/m2/day on days 1-5, 13-17, 21-24, and 28-31. Patients on the LAK treatment arm underwent leukapheresis on days 8-10 and LAK cell reinfusion on days 13-15. The results are reported with long-term follow-up. The published experience with IL-2 alone or with the addition of LAK was investigated in a quantitative literature survey. The response proportions were studied by schedule (high dose bolus, moderate dose, low dose) and by concomitant administration of LAK.
Results:
Seventy-one patients were treated, 36 on the IL-2 arm and 35 on the IL-2 plus LAK arm. Four patients (6%) had major responses (two complete, two partial). The median survival of all patients was 13 months (95% confidence interval [CI], 9-18 months). There were no differences between treatment arms with regard to response (P = 0.61) and survival (P = 0.67). More patients on the LAK arm experienced pulmonary toxicity (P = 0.008). The overall weighted response proportion was 16% (95% CI, 8%-24%) for the 39 published series of 1291 patients treated with IL-2. The 95% confidence intervals for response proportion overlapped when compared by schedule and by administration of LAK.
Conclusions:
The dose and schedule of IL-2 used in this study resulted in a low level of antitumor activity and the addition of LAK did not improve the response rate against RCC. Given the infrequent, but reproducible, responses with IL-2 and interferon-based regimens, continued investigation of these agents is warranted as is the study of new cytokines. Alternative treatment strategies should be studied in RCC and new agents and treatment regimens that appear promising in Phase II studies must be studied in randomized trials.
Dendritic cells (DCs) are predicted to participate in natural tumor immunity by migrating into tumors, where they acquire antigen, undergo activation, and migrate to lymph nodes to initiate a T-lymphocyte response against tumor-associated antigens. The presence of DCs using defined lineage markers and their function in human tumors has not been assessed previously. The monoclonal antibodies against CMRF-44 and CD83, which are differentiation/activation antigens on DCs, were used in immunohistological and flow cytometry studies to analyze the DC subtypes infiltrating 14 cases of human renal cell carcinoma (RCC). The functional immunocompetence of the DCs isolated from RCC was assessed by testing their ability to stimulate an allogeneic mixed leukocyte reaction. The majority of leukocytes present within the RCC were macrophages (62% +/- 14.7) or T lymphocytes (19% +/- 9.5), with CD45+ HLA-DR+ lineage-negative putative DCs accounting for less than 10% of the leukocytes present. Of these, a subset, comprising less than 1% of total leukocytes, had an activated CMRF-44+ or CD83+ DC phenotype. Activated CMRF-44+ and CD83+ DCs were more evident outside the tumor in association with T-lymphocyte clusters. The number of CMRF-44+ DCs correlated closely with the number of S-100-positive DCs. Isolation of DCs from eight RCCs was achieved, and flow cytometry studies confirmed the small proportion of activated CMRF-44+ DCs. The CMRF-44+ DCs stimulated an allogeneic mixed leukocyte reaction, but the CMRF-44- DCs (normal tissue DC precursors and other cells) failed to do so. These results suggest that RCCs recruit few DCs into the tumor substance, and the tumor environment fails to initiate the expected protective activation of DCs. These two mechanisms, amongst others, may contribute to tumor escape from immunosurveillance. In vitro loading of DCs with tumor-associated antigens may be a useful therapeutic maneuver.
The objective of this study was to determine the tolerance and effect of moderate-dose recombinant human interleukin-2 (rHu IL-2) and tumor-infiltrating lymphocytes (TIL) in patients with metastatic melanoma (MM) or renal cell carcinoma (RCC) refractory to standard therapy.
Twenty-six patients (18 MM and eight RCC) were entered onto this pilot study. TIL were isolated from fresh biopsy material and activated with anti-CD3 antibody, OKT3, for 48 hours and expanded in 100 IU/mL r-methionyl Hu IL-2 alanine 125 (r-met Hu IL-2 [ala-125]). At least 10(10) TIL were reinfused intravenously in three divided injections on days 2, 4, and 6 of the protocol. A maximum dose of 30,000 U/kg of IL-2 per injection was administered every 8 hours from day 2 through day 11 for a total of 28 doses.
Sixteen melanoma patients completed the study. Of these, three (19%) showed a durable complete response (CR), nine (56%) had no response (NR), and four (25%) had progressive disease (PD). One nonresponder demonstrated complete tumor regression within 1 year of treatment. Of four assessable RCC patients, two experienced a minor response (MR) and two showed NR. All TIL cultures showed comparably high cytotoxic activity as determined by antibody-redirected lysis (ARL). More importantly, melanoma TIL from responders possessed significantly higher cytotoxicity against autologous tumor cells than TIL from nonresponders (P < .05). Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-gamma), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and IL-4 was similar for TIL from melanoma responders and nonresponders, or TIL from RCC patients.
Immunotherapy with polyclonally activated TIL and moderate-dose IL-2 could be successfully used for the treatment of immunogenic tumors with less toxicity and lower costs as compared with high-dose IL-2 protocols.
GM-CSF has been shown to be important for the survival and function of cells of dendritic cell/Langerhans cell (LC) lineage in vitro. Since these cells have been demonstrated to infiltrate human lung and some lung carcinomas, we hypothesized that the production of GM-CSF in the lung could be important in their recruitment and differentiation. Using both immunohistochemistry and in situ hybridization, we have shown that: (a) GM-CSF was produced by normal bronchiolar epithelium, the only site were CD1a+ LC are observed in the normal lung, whereas neither GM-CSF production nor LC were identified in normal alveolar epithelium. (b) In inflamed pulmonary tissue, hyperplastic alveolar cells produced GM-CSF, and CD1a+ LC accumulated adjacent to these cells. (c) Some, but not all, lung carcinomas produced this cytokine, and a close correlation was found between the production of GM-CSF and the number of CD1a+ LC infiltrating these tumors. Since GM-CSF was produced at all sites where CD1a+ LC are known to accumulate, but not at other locations within the lung, these data suggest that the local production of GM-CSF by certain lung cells may play an important role in determining the distribution and differentiated state of dendritic cell/LC in the human lung.
Dendritic cells (DC) are essential for the initiation of T- and T-dependent immune responses by virtue of their capacities to acquire, process and present antigens and to deliver activation signals to mature resting T cells. In contrast, antigen presentation by other types of antigen-presenting cells (APC) may lead to peripheral T cell tolerance. This paper reviews the immunobiology of DC, and considers the possibility that immune unresponsiveness of tumour-bearing hosts may be due to clonal anergy of T cells. An important issue is whether it is possible to use DC to vaccinate tumour-free individuals, to overcome the immunological unresponsiveness of tumour-bearing hosts, and ultimately to cause rejection of primary and metastatic cancers.
Immune surveillance of cancer requires antigen-presenting cells which activate T cells specific for tumor-associated antigens. We show here that substantial numbers of dendritic cells, which are the most potent antigen-presenting cells, emigrate from renal-tumor explants in organ culture. Tumor-derived dendritic cells presented with all characteristics of mature dendritic cells. Dendritic cells could be identified by typical cytoplasmic projections (=veils). They expressed high levels of MHC products and of the co-stimulator CD86 (B7-2). Dendritic cells expressed the CD45RO isoform but not CD45RA. The most important point was that up to 9% of the emigrating leukocytes expressed the CD83 antigen, a specific marker for mature dendritic cells. CD83+ cells were approximately 40-fold enriched in the tumor tissue as compared to the peripheral blood. In contrast to cultured blood dendritic cells, tumor-emigrant dendritic cells had a reduced potential to capture soluble antigen, as shown by the exclusion of fluoresceinated Dextran molecules. Finally, in mixed leukocyte reactions, tumor-derived dendritic cells were able to stimulate naive T cells from cord blood, which is a unique feature of dendritic cells. This study demonstrates that genuine dendritic cells reside in or infiltrate renal-cell carcinoma tissue. The failure of patients with renal-cell carcinoma to mount an anti-tumor immune response despite the presence of professional antigen-presenting cells in the tumor tissue suggests that tumor-associated dendritic cells are suppressed in situ, in a similar way to that described for tumor-infiltrating lymphocytes.
Tumors are tolerated by the immune system notwithstanding the expression of tumor-associated antigens. PROb tumor cells, derived from a rat colon carcinoma, are rejected by tumor-immune hosts but give rise to progressive tumors in naive hosts. Paradoxically, these tumors are heavily infiltrated by dendritic cells that express MHC class II and ICAM-1. These tumor-infiltrating dendritic cells (TiDCs) could be expected to process and present to T cells the antigens released by the adjacent tumor cells. Indeed, we report here that TiDCs, compared with splenic dendritic cells, are poor stimulators of primary allogeneic T-cell proliferation and cytokine [interleukin-2 (IL-2) and interferon-gamma] production. Most of them (89-97%) do not express B7, an essential co-stimulatory signal for T cells, even after a culture period allowing B7 up-regulation on epidermal Langerhans cells. GM-CSF in association with tumor necrosis factor-alpha or IL-4, or cell-associated CD40-ligand, all known to be potent stimulators of B7 expression on other dendritic cells, did not restore B7 expression by TiDCs. After a first exposure to TiDCs, allogeneic T-cell response to a second challenge to splenic dendritic cells was decreased. The failure of most dendritic cells infiltrating PROb tumors to express B7, even after stimulation, may contribute to their poor capacity to stimulate T cells and could play a role in the immune tolerance allowing tumor growth.
T cell defined antigens have now been characterized in a large variety of tumor types, in both mice and humans. An increasing number of these antigens appear to result from tumor-specific mutations, and some of these mutations may be implicated in oncogenesis. The priority is now to demonstrate that immunization against some of these antigens is clinically valuable for antitumor therapy, and the first results of clinical pilot studies are now emerging.
Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-alpha/IL-1/IL-6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)-gamma and no IL-4 or IL-10. Production of IFN-gamma was significantly induced by addition of PGE2, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN-gamma(low), IL-4(neg) cells, TNF-alpha/IL-1/IL-6 promoted growth of IFN-gamma(intermediate), IL-4(neg) CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN-gamma production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-alpha/IL-1/IL-6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF-alpha/IL-1, IL-6 + PGE2-cultured DC seem to be optimal for generation of IFN-gamma-producing CD4/CD8+ T cells.
Developments in methods for identifying antigens from infectious agents
and cancers has provided exciting new opportunities in prevention and treatment
through vaccination. In many of these situations, however, traditional immunization
techniques do not stimulate protective immunity because they fail to fully
mobilize the appropriate immune responses. This limitation, together with
new insights into the underlying mechanism of immune responses, has spurred
development of several new approaches for vaccine delivery. We discuss some
of the current efforts being developed to provide effective vaccine delivery
systems.
Dendritic cells are the most potent stimulators of immune responses including antitumor responses. We performed a pilot study of cultured antigen loaded dendritic cells in patients with metastatic renal cell carcinoma.
Dendritic cells were obtained by culturing plastic adherent mononuclear cells from peripheral blood for 5 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. Day 5 dendritic cells were loaded with cell lysate from cultured autologous tumor cells and with the immunogenic protein keyhole-limpet hemocyanin (KLH) which serves as a helper antigen and as a tracer molecule. During the antigen pulse dendritic cells were activated with a combination of tumor necrosis factor-alpha and prostaglandin E2. Dendritic cells were administered by 3 intravenous infusions at monthly intervals. Cellular and humoral immune responses to KLH and cell lysate were measured in vitro before and after the vaccinations.
Preparation of 12 dendritic cell vaccines from patients with advanced renal cell carcinoma was successful. Treatment with fully activated CD83+ dendritic cells was well tolerated with moderate fever as the only side effect. Potent immunological responses to KLH and, most importantly, against cell lysate could be measured in vitro after the vaccinations.
Our data demonstrate that a dendritic cell based vaccine can induce antigen specific immunity in patients with metastatic renal cell carcinoma. Dendritic cell based immunotherapy represents a feasible, well tolerated and promising new approach for the treatment of advanced renal cell carcinoma.
The potential to harness the potency and specificity of the immune system underlies the growing interest in cancer immunotherapy. One such approach uses bone marrow-derived dendritic cells, phenotypically distinct and extremely potent antigen-presenting cells, to present tumor-associated antigens and thereby generate tumor-specific immunity. Support for this strategy comes from animal studies that have demonstrated that dendritic cells, when loaded ex vivo with tumor antigens and administered to tumor-bearing hosts, can elicit T cell-mediated tumor destruction. These observations have led to clinical trials designed to investigate the immunologic and clinical effects of antigen-loaded dendritic cells administered as a therapeutic vaccine to patients with cancer. In the design and conduct of such trials, important considerations include antigen selection, methods for introducing the antigen into MHC class I and II processing pathways, methods for isolating and activating dendritic cells, and route of administration. Although current dendritic cell-based vaccination methods are cumbersome, promising results from clinical trials in patients with malignant lymphoma, melanoma, and prostate cancer suggest that immunotherapeutic strategies that take advantage of the antigen presenting properties of dendritic cells may ultimately prove both efficacious and widely applicable to human tumors.
We have evaluated whether the addition of a foreign helper protein, keyhole limpet hemocyanin (KLH), can augment the efficacy of tumor lysate-pulsed dendritic cells and peptide-pulsed DC immunizations in vivo. Besides being used as a "surrogate antigen" in approaches to measure immunological response in cancer patients, KLH is also an immunogenic carrier protein to elicit T-cell help. Using the D5 subline of B16 melanoma, we demonstrate that DCs pulsed with both KLH and tumor lysate mediate enhanced immune priming and rejection of established metastases in vivo, which is dependent on host-derived T cells. Interleukin 2 augments the enhancement afforded by KLH, as measured by cure rates and overall survival, in the absence of autoimmune depigmentation. KLH added to DC immunizations markedly enhances tumor-specific T cell production of IFN-gamma. D5 melanoma exposed to similar levels of IFN-gamma results in substantial expression of MHC class I molecules. DCs pulsed with KLH and mouse tyrosinase-related protein-2 peptide results in enhanced reduction of B16 melanoma metastases; the effect is most pronounced in a setting where tyrosinase-related protein-2 peptide-pulsed DCs alone are completely ineffective. Collectively, these findings demonstrate that KLH addition to tumor antigen-pulsed DC immunizations can augment IFN-gamma production and enhance in vivo antitumor activity.
Approximately 31,000 new cases of renal cell carcinoma (RCC) are diagnosed in the United States each year and 30% to 40% of these will eventually become metastatic disease. The primary tumor often grows to considerable size before symptoms are apparent, which could explain the high rate of metastatic RCC (mRCC). The median survival of mRCC after diagnosis is 8 to 12 months and the 5-year survival is less than ideal. Traditionally, surgery has been the treatment of choice for mRCC. Chemotherapeutic agents tested so far have been disappointing, perhaps because of a high expression of the multidrug resistance gene or the high content of glutathione in RCC cells. However, the spontaneous regression of mRCC in some cases suggests that these tumor cells are responsive to immunologic mechanisms. Initial interest has focused on two cytokines, interferon-alpha (IFN-alpha) and interleukin-2 (IL-2), with response rates ranging from 15% to 20%. Both IL-2 and IFN-alpha are pleiotropic compounds with specific effects on many leukocyte subsets, in addition to directly affecting tumor proliferation, angiogenesis, and antigen expression. The mechanisms by which these immunoenhancing cytokines exert antitumor effects are still unknown. However, many agree that activation of T cells and natural killer cells is a pivotal part of the antitumor efficacy. For example, some investigators have found that pretreatment levels of natural killer cells and T cells predict a response to IL-2 and IFN-alpha in mRCC. Others report a relationship between activation of peripheral blood lymphocytes and response to cytokine therapy. Expansion of activated T cells in blood during treatment with these two cytokines seems to relate to clinical efficacy in patients with RCC.
Up to now, clinical tumor-markers for renal cell carcinoma (RCC) have been lacking. Increased plasma levels of transforming growth factor-beta1 (TGF-beta1) were described as a tumor-marker and prognostic factor in RCC. The aim of this study was to test the clinical suitability of plasma TGF-beta1 as a tumor-marker for RCC. The concentrations of active and latent TGF-beta1 were determined in plasma of patients with localized (n = 39) and metastasised (n = 17) RCC. A newly developed, highly sensitive ELISA, which is specific for the isoform beta1, was used. Active TGF was directly measured in the EDTA plasma. To determine the amount of latent TGF-beta1, which is bound predominantly at beta2-macroglobulin, an optimized activation procedure was applied. Patients with localized RCC showed median concentrations of 16,700 ng/l (6,200-54,800 ng/l) for latent TGF-beta1. A total of 94 patients with various nonmalignant urological diseases were recruited as a control group. In comparison, this group had median concentrations of 19,900 ng/l (2,640-52,300 ng/l) for latent TGF-beta1. There was no significant difference (nonparametric Kruskal-Wallis ANOVA) between these groups. Patients with metastatic RCC showed median concentrations of 34,500 ng/l (6,800-48,960 ng/l) for latent TGF-beta1. In comparison to the localized RCC group, a statistically significant difference was found. Plasma levels after operative therapy (days 1, 5 and 10) and during follow-up without evidence of disease (2-6 months) showed no significant differences. Contrary to other study groups, our results suggest that TGF-beta1 is not a suitable tumor-marker for the diagnosis of localized RCC. In the face of higher TGF-beta1 plasma levels in metastatic disease, TGF-beta1 may be useful in the early detection of RCC recurrence or to control the success of immunochemotherapy.
In this study we have presented in vitro data and results of a preliminary clinical trial using dendritic cells (DC) in patients with progressive metastatic renal cell carcinoma. DC precursor cells were obtained from peripheral blood mononuclear cells (PBMC). DC were pulsed with autologous tumor cell lysate if available. In total, 15 patients were treated with a median of 3.95 x 10(6) DC administered and ultrasound-guided into a lymph node or into adjacent tissue. Seven patients remained with progressive disease (PD), 7 patients showed stable disease (SD), and one patient displayed a partial response (PR). Most interestingly, the patient who was treated with the highest number of DC (14.4 x 10(6) DC/vaccine) displayed a PR. Delayed-type hypersensitivity (DTH) reaction using autologous tumor lysate was positive in 3 out of 13 patients, including the patient with PR. Two out of 3 patients receiving additional treatment with keyhole limpet hemocyanin (KLH) showed reactivity to KLH after vaccination. CD3+CD4+ and CD3+CD28+ cells as well as the proliferation rate of peripheral blood lymphocytes (PBL) increased significantly in the blood of patients during therapy. In conclusion, our observations confirm the capability of tumor-lysate pulsed autologous DC vaccines to stimulate an immune response in patients with metastatic renal cell carcinoma even in the presence of a large tumor burden. The lack of adverse effects together with immunologic effects support further investigation of this novel therapeutic approach. Further studies are necessary to demonstrate clinical effectiveness in cancer patients, in particular in patients with less advanced disease.
A novel approach to vaccination against cancer is to exploit dendritic cells (DCs) as "nature's adjuvants" and actively immunize cancer patients with a sample of their own DCs primed with tumor antigens. DC vaccination is, however, still at an early stage, slowed in part by the need to carry out research in humans. Nevertheless, valuable proofs of concept have been obtained with respect to the capacity of DCs to expand cancer-directed immune responses. The methods for preparing DCs are being improved continuously, and there are many opportunities to improve efficacy at the level of DC biology. An increased number of Phase I, II and III studies will drive this new area of human research.
Dendritic cells are the most potent antigen-presenting cells, and the possibility of their use for cancer vaccination has renewed the interest in this therapeutic modality. Nevertheless, the ideal immunization protocol with these cells has not been described yet. In this paper we describe the preliminary results of a protocol using autologous tumor and allogeneic dendritic hybrid cell vaccination every 6 weeks, for metastatic melanoma and renal cell carcinoma (RCC) patients. Thirty-five patients were enrolled between March 2001 and March 2003. Though all patients included presented with large tumor burdens and progressive diseases, 71% of them experienced stability after vaccination, with durations up to 19 months. Among RCC patients 3/22 (14%) presented objective responses. The median time to progression was 4 months for melanoma and 5.7 months for RCC patients; no significant untoward effects were noted. Furthermore, immune function, as evaluated by cutaneous delayed-type hypersensitivity reactions to recall antigens and by peripheral blood proliferative responses to tumor-specific and nonspecific stimuli, presented a clear tendency to recover in vaccinated patients. These data indicate that dendritic cell-tumor cell hybrid vaccination affects the natural history of advanced cancer and provide support for its study in less advanced patients, who should, more likely, benefit even more from this approach.
Cultured tumor lysate-loaded dendritic cells (TuLy-DC) have been demonstrated in vitro to stimulate potent immune modulations and generate significant antitumor response. We report the results of a pilot trial of TuLy-DC vaccine for patients with metastatic renal cell carcinoma (mRCC). Fourteen mRCC patients underwent nephrectomy to obtain autologous TuLy prepared by subjecting tumor cells to 3 freeze/thaw cycles. Dendritic cells were generated from peripheral blood CD14+ precursors cultured in the presence of GM-CSF, IL-4, and 10% autologous serum. Patients received one vaccination of TuLy alone as an immunologic control, followed by 3 weekly vaccinations of DC-TuLy injected intradermally in the midaxillary region. Peripheral blood lymphocytes were collected before and after weekly vaccines and were assessed for changes in phenotype, cytotoxicity, and cytokine profile. The TuLy-DC vaccine was successfully prepared and administered to 12 patients, whereas 2 patients did not receive vaccine treatment due to declines in postoperative performance status. The vaccines were well tolerated, with only grade 1 toxicities noted. One patient had a partial response to treatment that did not correspond to any significant change in immunologic profile. This pilot trial demonstrated both the safety and feasibility of reliably preparing a DC-based vaccine for mRCC patients. Our data suggest that autologous TuLy-DC vaccines generate only limited clinical response. Further clinical studies are needed to identify the most potent treatment regimen that can consistently mediate an antitumor immune response in vivo.
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- Banchereau
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Dendritic cells in cancer immunotherapy
- Fong
The use of dendritic cells in cancer immunotherapy
- Schuler