Ivo Minarik 1,2, Ivan Kawaciuk 1, Jirina Bartunkova 2
- 1 Department Of Urology, 2nd Medical School, Charles University, Prague
2 Department of Immunology, 2nd Medical School, Charles University, Prague
dendritic cells, immunotherapy, prostatic neoplasms, vaccines
- Introduction. Metastatic disease represents the incurable stage for prostate cancer patients. New treatment modalities have been invented and tested. They will supplement or even replace contemporary treatment procedures. Dendritic cell based vaccines will play an important role in the treatment of prostate cancer patients.
- The aim of the study. Summarize existing clinical studies utilizing dendritic cell vaccines in prostate cancer patients.
- Material and methods. Analysis of published studies in medical literature and internet databases.
- Conclusion. Dendritic cell vaccine based immunotherapy is a promising treatment alternative to prostate cancer patients, which needs further clinical evaluation. The future lies in the combination of dendritic cell vaccines and methods that lead to depletion of regulatory T-lymphocytes or change in tumor microenvironment.
Prostate carcinoma is one of the most frequent neoplasms in men in the Czech Republic. Its incidence was 68,3/100 000 men and mortality was 28.2/100 000 men in 2002. The incidence of this disease has increased over past years. The explanation for this phenomenon may be ageing of the population and initiation of PSA screening for prostate carcinoma. With the screening we manage to reveal earlier stages of prostate cancer and so more patients are indicated for radical prostatectomy or radiotherapy. About 40% of patients evolve biochemical relapse that is the sign of local recurrence or micrometastatic disease. There is no curable treatment we can offer to patients with metastases. We resort to hormonal therapy, brachytherapy or sometimes chemotherapy. In the past few years, the research engaged in new treatment modalities has advanced. Antitumor vaccines based on the encouragement of immune system against the tumor turned out to be the future trend in the treatment of malignant neoplasms.
Concept of immunotherapy is based on the fact that the development of malignant disease is caused by the failure of immune system to recognize and destroy malignant cells. Possibilities of immune system struggle against tumor are the production of antibodies by plasma cells or mainly cell immunity that comprises many different types of cells such as CD8+ and CD4+ T lymphocytes, NK cells, monocytes, dendritic cells.
Both elements of specific immunity are based on the principle of recognition of foreign antigen and initiation of reaction against it. Identification of such antigen in the case of prostate carcinoma is not simple. So far, nobody has discovered specific tumor antigen that would not be expressed by the healthy prostatic tissue. On the other hand, there are few molecules that are produced only in the prostate and are highly expressed by the tumor such as PSA - prostate specific antigen, PSMA - prostate specific membrane antigen, PSCA - prostate stem cell antigen, or PAP - prostate acid phosphatase. The existence of these molecules provides scientists with a substrate for development of antitumor vaccines.
Specific immunity, especially cell-mediated immunity, plays the key role in the fight of immune system against tumor cells. The main elements involved are T-lymphocytes. CD8+ T-lymphocytes, i. e. cytotoxic T-lymphocytes, eliminate the cells which present such peptides in the complex with MHC I that are not normally expressed in the healthy tissue. These could be peptides produced in the early embryonic stage or new peptides created by tumor cells after mutations of DNA that occur frequently in neoplastic tissue. In order to initiate the cascade of tumor elimination, cytotoxic T lymphocyte needs to be activated at first. The process involves CD 4+ T-lymphocytes (T-helper) and antigen presenting cells (APC). APCs engulf antigen and present them to T-lymphocytes, which are thus activated. Another element of cellular immunity are NK cells that destroy cells that suppress their expression of MHC I molecules on the surface (the mechanism that enables a tumor cell to escape T-lymphocyte elimination).
Recently, there was a great boom of vaccines based on dendritic cells. Dendritic cells are the most efficient antigen-presenting cells in the human body. They are used to initiate immune response against the specific antigen (e. g. PSA). The process of delivery of antigen is called pulsing. We can pulse mature dendritic cells only with the peptides that are able to bind directly to MHC I molecules, i. e. they do not require endocytosis. On the other hand, for their high phagocytic activity immature dendritic cells can engulf even larger molecules, e. g. proteins, whose fragments are then exposed on the surface in conjunction with both MHC I and MHC II molecules and so offered to T-lymphocytes. In the following text, we will deal with the types of antigens used in the development of vaccines and the efficiency of these vaccines.
DC pulsed with peptides
Peptides bind directly to MHC I molecules on the surface of dendritic cells. Therefore, we have to present them to mature dendritic cells. The drawback is their bond to specific MHC I molecule, which differs individually. A few studies have been carried out so far using peptides as a substrate for pulsing dendritic cells of patients with prostate carcinoma.
In 1999 Murphy, Tjoa et al. published results of the clinical study phase I/II, where dendritic cells were pulsed with HLA-A2-specific PSMA peptides. Thirty-three patients, who advanced from the phase I, participated in the study. All of them had locally advanced tumor T4 or metastatic disease, and had experienced hormonal therapy before. Patients were followed before, during and after the vaccination therapy. Authors monitored the level of serum PSA (total PSA, free PSA), PSMA (Western blot), blood count, bone ALP (alkaline phosphatase), bone scintigraphy, ProstaScint(R) and initial chest X-ray. Pulsed immature dendritic cells were then administered to peripheral blood in 6-week intervals, total 6×. As an immunoadjuvant they applied GM-CSF subcutaneously. Partial response (PR) was observed in 9 patients [based on NPCP criteria (National Prostate Cancer Project) and 50% decrease of PSA], 11 patients had stable disease without progression and 13 patients developed progression. In the PR group, they registered the gradual PSA decline of patients with initially low, but even high PSA. On the other hand, PSA gradually rose in the group with progression. There were no significant differences of the level of PSMA, hematocrit, and white blood cells. In certain patients, the concentration of ALP and bone scintigraphy correlated with the stage of the disease. ALP gradually decreased between the days 30-480 after the vaccine administration, in some cases even to the normal level. However, around the day 500 the growth of bone ALP was observed. It corresponded to clinical progression. The average time to progression during the phase I and II was about 280 days in the group with PR and stable disease. The average survival in the stage T4 N1-3 M1 was about 600 days. This is significantly longer survival then in the group of patients who missed vaccination (6 months).
In 2006 Waeckerle-Men et al. published the study of phase I/II that involved 6 patients with androgen-independent prostate carcinoma. Dendritic cells were pulsed with peptides PSA, PSMA, PSCA and PAP. Vaccines were administered intradermally, at first in 2-week intervals up to the total count 6, then in month intervals. Besides immunological parameters (ELISPOT, cytotoxic tests) they monitored serum PSA concentrations. Four patients completed the study; three of them developed specific cytotoxic reaction against the given antigens. PSA decreased in one patient. After 34 weeks PSA started to rise steeply and the study with this patient had to be disrupted. The other 2 patients who continued with the study prolonged PSA doubling time (PSAdt). The study was brought to an end after the development of new distant metastases. The clinical progression correlated with the loss of ability to lyse cells presenting given antigen.
DC pulsed with proteins
So far, only a few studies using proteins for pulsing dendritic cells have been carried out in the world. Barrow et al from Paris, France utilized recombinant PSA protein (Dendritophage - rPSA) for pulsing dendritic cells gained by differentiation of monocytes of peripheral blood. The study comprised 24 patients with biochemical relapse after radical prostatectomy. The initial PSA value was between 1-10 ng/ml, the clinical stage T1-3 N0 M0 and no additional treatment was applied. They administered vaccines to these patients in 9 series by various modes of injection - subcutaneously, intradermally and intravenously. They determined the PSA concentration, number of circulating tumor cells (real-time quantitative PCR = RQ-PCR), number of circulating specific T-lymphocytes reacting against PSA (Enzyme-linked immunospot = ELISpot) and observed the development of delayed-type hypersensitivity reaction against the toxin of Clostridium tetani. Circulating tumor cells were detected in 6 patients, but after 6 and 12 months they were undetectable. No patient demonstrated the decrease of the PSA concentration by 50% of its initial value. The PSA decline was 6-39% in 11 patients, but this phenomenon was temporary and lasted maximum 6 months. Specific T-lymphocytes were detected in 11 patients after the first vaccination. No specific antibodies were produced. There was no correlation between the PSA decline and Gleason score after prostatectomy. No correlation was found between the immunological reaction against PSA and PSA decline, Gleason score, or delayed-type hypersensitivity skin test.
Another protein used in vaccination trials was prostate acid phosphatase (PAP). The Dendreon Company in the USA developed a vaccine connecting GM-CSF and autologous dendritic cells pulsed with PAP (Provenge(R)). Several clinical studies of phase I and II were carried out involving patients with androgen-independent prostate carcinoma (AIPC) or biochemical relapse. The first results were promising. In 2000, EJ Small published the results of phase II clinical study that comprised 19 patients with AIPC. Vaccination induced the development of specific cellular immune reaction in all patients; however, the clinical outcome was not as convincing. The PSA concentration was reduced by >50% in 3 patients and between 25-49% in other 3 patients. No improvement was detected by diagnostic imaging. Median to progression was 29 weeks, but 7 patients had no progress at the end of the study (lasted 1 year).
In 2005 Schellhammer published the results of randomized placebo-controlled study of 127 patients with AIPC, who were divided into 2 groups. The first received Provenge(R), the other placebo. They monitored the time to radiological progression, the pain level and survival over the three years during which the study occurred. The disease advanced in 115 men. The average time to progression was 11.1 weeks in the Provenge(R) group, unlike 10 weeks in the placebo group. However, when subdivided according to Gleason score (GS) the difference was even more enhanced. The men with Provenge(R) and GSŁ7 progressed after 16 weeks, had higher probability of longer painless period, whereas the progression was noted in the group with placebo after 9 weeks. The average survival of patients with Provenge(R) and GSŁ7 was 30.7 months, which is 8.4 months more than in the placebo group. No clinical therapeutic effect was recorded in patients with GS>7.
In 2005, Beinart published the results of phase II study that comprised men after the definitive primary treatment of prostate carcinoma (prostatectomy, radiotherapy) with biochemical relapse without demonstrable metastatic disease. They received Provenge(R). The PSA doubling time extended (from 4.9 to 7.9 months), but the PSA decline by >50% was not registered in any patient.
DC pulsed with nucleic acids
There have been a few trials utilizing genetic information extracted from autologous tumor cells, mainly in the form of mRNA, e. g. mRNA coding PSA or telomerase, for pulsing dendritic cells. Messenger RNA (mRNA) has several advantages in comparison with cDNA and peptides - 1) preparation and characterization is much more simple than in the case of protein, 2) the mRNA template can be further modified to extend the elimination halftime of this molecule, 3) no risk of incorporation into the genome, 4) antigens coded by mRNA can be presented with various haplotypes of MHC I. The prepared mRNA is then transfected into dendritic cells either by electroporation or coincubation. In the case of prostate carcinoma Axel Heiser and colleagues best elaborated the methodology of vaccine preparation. They first published the clinical studies based on PSA-mRNA. The phase I comprised 13 patients in the stage D1 - D3 with PSA >4 ng/ml who had no radiotherapy, chemotherapy or immunotherapy in 6 previous weeks. Vaccine was administered intravenously and intradermally. Vaccine was not toxic and without severe adverse reactions. All patients developed specific T-lymphocyte reaction against various epitopes of PSA protein. The intensity depended on the vaccine dose. They managed to follow the PSA velocity in 7 patients, it decreased in 6 patients. In one case even PSA decreased. They monitored the kinetics of tumor cells in the peripheral blood in 6 patients. The tumor cells disappeared from the blood in all cases after the first dose; however, they reappeared after a few weeks. This was just the clinical study of phase I so it is impossible to state whether this technique had any clinical effect.
The same authors dealt with in vitro pulsing of dendritic cells with mRNA extracted from a tumor cell. They anticipated proliferation of T-lymphocytes reacting against the wider spectrum of antigens and so the probability of eliminating tumor burden would be higher. The benefit of mRNA extraction lies in the possibility of mRNA amplification and so it is not necessary to isolate larger amount of tumor tissue; this is a great problem in the case of obtaining viable prostate cancer cells from any prostatectomy specimen. They determined the % lysis of dendritic cells by cytotoxic T-lymphocytes. Dendritic cells were pulsed with total tumor mRNA, PSA-mRNA and TERT (telomerase) - mRNA and PSA peptides. The results show that the cytotoxic reaction raised by pulsing with the total mRNA is aimed against the wider spectrum of antigens and the % lysis is the greatest compared to the reaction raised by pulsing by other molecules.
Norwegian group under the supervision of LJ Mu engaged in the immunotherapy with dendritic cells pulsed with mRNA extracted from the prostate cancer cell lines (LNCaP, DU145, PC-3). In 2005, they published the first results of the clinical study of phase I/II, which involved 19 patients. The inclusion criteria were the androgen-independent prostate carcinoma and verifiable progression of the disease while receiving androgen suppression. In the beginning 14 patients had bone metastases. They administered the vaccine to 10 patients intranodally, 9 patients intradermally. They utilized matured dendritic cells. The study lasted 3 months. They monitored the vaccine safety as well as the first clinical and immunological effects. Specific T-lymphocyte response developed in 12 of 19 patients, but during the study it gradually decreased. The specific response was augmented by repeated vaccine administration. For example, 46 T-lymphocyte clones were isolated from which two clones were CD8+, and the rest was CD4+. Neither patient had verifiable improvement of bone scan, however, the progression of bone scan halted in those who developed immune reaction. With regard to PSA 11 patients had stable disease (less than 50% decrease or increase of PSA concentration) with the maximum decline of 48%. The PSA velocity decreased in 13 patients. At the end of the study, 11 patients had stable disease, 10 of them developed specific immune response as well as two of the 8 remaining patients with clinical progression.
In 2000, Nair et al published the first in vitro studies and murine studies describing application of dendritic cells pulsed with RNA coding human telomerase (hTERT). The latter is a complex protein in the cell nucleus preventing shortening of telomere and the cell death. This protein is normally expressed only in stem cells and spermatogonia, but the level of production is incomparable to several types of tumor cells. The pulsed dendritic cells stimulated the cell division and rise of activity of cytotoxic T-lymphocytes reacting against hTERT; these lymphocytes were able to lyse several tumor cell lines of renal and prostate cancer. If the pulsed dendritic cells were administered to mice with melanoma or thymoma, then the induced immune response prevented the progression of the disease.
These results inspired Su et al who published the results of the clinical study of phase II involving patients with metastatic prostate carcinoma. Twelve patients completed the study. The clinical response was not demonstrable in any patient during the 2-month lasting study. The only indicator, which significantly differed in the beginning and the end of the study, was PSA doubling time (PSAdt) in 5 patients who had received 6 doses of vaccine. The median of PSAdt in this group was 2.9 months in the beginning of the study, but after 6 doses of vaccine the PSAdt prolonged to 100 months. However, after the completion of the study the PSAdt returned to its initial value. The PSAdt did not change significantly in the group of 7 patients who had received just 3 doses of vaccine.
DC pulsed with tumor cells
A tumor cell bears lots of various antigens on its surface; some of them are not expressed on normal cells. Identification of such antigens is complicated; therefore some researchers use whole tumor cells for induction of more intense immune reaction. Autologous tumor cells seem to be the best source. Their advantage is the expression of antigens, which are produced in the tumor of the patient. These cells are then used for pulsing dendritic cells in the form of either necrotic or apoptotic cells. Dendritic cells pulsed with apoptotic bodies produce significantly higher amount of IL-12 (induction of cellular immunity), but the difference was not confirmed in the murine in vivo studies that monitored the reduction of tumor mass.
It is quite impossible to obtain sufficient number of tumor cells in the case of several tumors, e. g. prostate carcinoma; therefore the researchers resort to utilizing tumor cell lines as the source of antigen. The advantage of using cell lines is the possibility of cultivating infinite number of tumor cells, on the other hand, the spectrum of antigens present on the surface may differ significantly from those expressed in the tumor. It leads to limited immune reaction.
So far the number of published studies regarding whole tumor cells for pulsing dendritic cells of patients with prostate cancer has been limited. In 2004, Pandha et al published results of the clinical study of phase I/II that comprised 16 patients with androgen-independent prostate carcinoma. The study lasted 3 months during which the patients received 6 doses of vaccine. Dendritic cells were pulsed with lysed (necrotic) cells of tumor cell lines LNCaP and DU145. Just 1 patient developed the PSA decline from 64 to 54 ng/ml, the PSA velocity was observed in 1 patient, and 6 patients prolonged PSA doubling time.
While testing dendritic cell vaccines we run across heterogeneity of material for pulsing. There had been no consensus so far on which type of antigen is the most suitable. The extent of immune reaction is limited when using a defined antigen, e. g. PSA. When pulsing with a tumor cell or total mRNA the extent of immune reaction is much more intense because of broader spectrum of antigens. This fact gives a tumor cell little chance to escape the elimination by immune cells. On the other hand, there are a lot of undefined antigens on the surface of tumor cells whose utilization raises controversies - will the immune reaction be directed only against tumor or could this method lead to development of severe autoimmune diseases? However, no severe autoimmune reactions were recorded in existing studies.
Immunotherapy of malignant neoplasms has advanced in the past several years. The first results of studies brought more controversies than satisfactory effect. The causes are still being identified. The important breakthrough was the elucidation of dendritic cell biology. The difference between mature and immature dendritic cells led to substantial improvement of preparation protocol of vaccines. Immature dendritic cells have a high capability of phagocytosis, but induce tolerance to antigens. On the other hand, mature dendritic cells are able to shift from the place of administration to regional lymph nodes where they initiate immune reaction. Majority of the studies were performed with immature dendritic cells [4-7, 9-18]. Another factor that plays a role in inducing effective immune reaction is the site of vaccination. Correct selection of location of vaccine administration is crucial for biological effect of the vaccine.
Still there is a lot to discover about dendritic cells. They cooperate with various types of cells, e. g. regulatory T-lymphocytes. These represent a group of lymphocytes whose role resides in the suppression of immune response. Their number is elevated mainly in the tumor of patients with malignant neoplasms, where they assist tumor cells in their survival and suppression of local immune response. The future lies in immunotherapeutic approaches combining dendritic cell vaccines and methods leading to reduction of immunosuppressive effect of tumor microenvironment, e. g. antibodies against certain cytokines (e. g. VEGF), or methods resulting in the depletion of regulatory T-lymphocytes. One of the possibilities is the application of the conjugate consisting of IL-2 and diphtheria toxin, which is preferentially trapped by regulatory T-lymphocytes (1000× higher affinity of IL-2 receptor on regulatory T-lymphocytes than other types of lymphocytes), and so the cell is destroyed. Another mechanism of elimination of regulatory T-lymphocytes is so called adoptive cell transfer, which is based on the principle of reduction of lymphocyte population with a chemotherapeutic drug in nonmyeloablative doses (e. g. cyclophosphamide, fludarabine), followed by vaccine administration containing ex vivo stimulated lymphocytes.
The important aspect of contemporary vaccines is their effect, which is limited to the period of vaccination. So the research has been heading towards the development of a method that would lead not only to the expansion of cytotoxic response, but also to the differentiation of memory cells.
Dendritic cell immunotherapy represents the alternative to well-established treatment protocols in prostate cancer patients. The results of existing studies have not impressed by their efficacy, however, better understanding of dendritic cell biology, accurate definition of patients profiting from DC-immunotherapy or combination with other immunotherapeutic approaches will undoubtedly contribute to longer survival of patients with prostate carcinoma, or even their complete cure.
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