Biomedical subjects

David Boczkowski

Publications and source records attributed to David Boczkowski.

15 recordsLinked to original sources

PubMed2007

Vaccination against the forkhead family transcription factor Foxp3 enhances tumor immunity.

Depletion of CD4+CD25+ regulatory T cells (Treg) by treatment with alphaCD25 antibody synergizes with vaccination protocols to engender protective immunity in mice. The effectiveness of targeting CD25 to eliminate Treg is limited by the fact that CD25, the low-affinity interleukin-2 receptor, is up-regulated on conventional T cells. At present, foxp3 is the only product known to be exclusively expressed in Treg of mice. However, foxp3 is not expressed on the cell surface and hence cannot be targeted with antibodies. In this study, we tested the hypothesis that vaccination of mice against foxp3, a self-antigen expressed also in the thymus, is capable of stimulating foxp3-specific CTL that will cause the depletion of Treg and enhanced antitumor immunity. Vaccination of mice with foxp3 mRNA-transfected dendritic cells elicited a robust foxp3-specific CTL response and potentiated vaccine-induced protective immunity comparably with that of alphaCD25 antibody administration. In contrast to alphaCD25 antibody treatment, repeated foxp3 vaccination did not interfere with vaccine-induced protective immunity. Importantly, foxp3 vaccination led to the preferential depletion of foxp3-expressing Treg in the tumor but not in the periphery, whereas alphaCD25 antibody treatment led to depletion of Treg in both the tumor and the periphery. Targeting foxp3 by vaccination offers a specific and simpler protocol for the prolonged control of Treg that may be associated with reduced risk of autoimmunity, introducing an approach whereby specific depletion of cells is not limited to targeting products expressed on the cell surface.

Animals↗

PubMed2005

Tumor immunotherapy targeting fibroblast activation protein, a product expressed in tumor-associated fibroblasts.

Murine studies have shown that immunologic targeting of the tumor vasculature, a key element of the tumor stroma, can lead to protective immunity in the absence of significant pathology. In the current study, we expand the scope of stroma-targeted immunotherapy to antigens expressed in tumor-associated fibroblasts, the predominant component of the stroma in most types of cancer. Mice were immunized against fibroblast activation protein (FAP), a product up-regulated in tumor-associated fibroblasts, using dendritic cells transfected with FAP mRNA. Using melanoma, carcinoma, and lymphoma models, we show that tumor growth was inhibited in tumor-bearing mice vaccinated against FAP and that the magnitude of the antitumor response was comparable to that of vaccination against tumor cell-expressed antigens. Both s.c. implanted tumors and lung metastases were susceptible to anti-FAP immunotherapy. The antitumor response could be further enhanced by augmenting the CD4+ T-cell arm of the anti-FAP immune response, achieved by using a lysosomal targeting sequence to redirect the translated FAP product into the class II presentation pathway, or by covaccination against FAP and a tumor cell-expressed antigen, tyrosinase-related protein 2. No morbidity or mortality was associated with anti-FAP vaccination except for a small delay in wound healing. The study suggests that FAP, a product which is preferentially expressed in tumor-associated fibroblasts, could function as a tumor rejection antigen in a broad range of cancers.

Animals↗

PubMed2005

Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef.

Transfection with synthetic mRNA is a safe and efficient method of delivering antigens to dendritic cells for immunotherapy. Targeting antigens to the lysosome can sometimes enhance the CD4+ T-cell response. We transfected antigen-presenting cells (APCs) with mRNA encoding Gag-p24 and cytoplasmic, lysosomal, and secreted forms of Nef. Antigen-specific cytotoxic T cells were able to lyse the majority of transfected targets, indicating that transfection was efficient. Transfection of APCs with a Nef construct bearing lysosomal targeting signals produced rapid and prolonged antigen presentation to CD4+ and CD8+ T cells. Polyclonal CD4+ and CD8+ T-cell lines recognizing multiple distinct epitopes were expanded by coculture of transfected dendritic cells with peripheral blood mononuclear cells from viremic and aviremic HIV-infected subjects. Importantly, lysosome-targeted antigen drove a significantly greater expansion of Nef-specific CD4+ T cells than cytoplasmic antigen. The frequency of recognition of CD8 but not CD4 epitopes by mRNA-expanded T cells was inversely proportional to sequence entropy and was similar to ex vivo responses from a large chronic cohort. Thus human dendritic cells transfected with mRNA encoding lysosome-targeted HIV antigen can expand a broad, polyclonal repertoire of antiviral T cells, offering a promising approach to HIV immunotherapy.

CD4-Positive T-Lymphocytes↗

PubMed2005

Vaccination with mRNAs encoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efficiently primes CTL responses, but is insufficient to overcome tolerance to a model tumor/self antigen.

Immunization of mice with dendritic cells transfected ex vivo with tumor-associated antigen (TAA)-encoding mRNA primes cytotoxic T lymphocytes (CTL) that mediate tumor rejection. Here we investigated whether direct injection of TAA mRNA, encapsulated in cationic liposomes, could function similarly as cancer immunotherapy. Intradermal and intravenous injection of ovalbumin (OVA) mRNA generated specific CTL activity and inhibited the growth of OVA-expressing tumors. Vaccination studies with DNA have demonstrated that co-administration of antigen (Ag)- and cytokine-encoding plasmids potentiate the T cell response; in analogous fashion, the inclusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA enhanced OVA-specific cytotoxicity. The ability of this GM-CSF-augmented mRNA vaccine to treat an established spontaneous tumor was evaluated in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse, using the SV40 large T Ag (TAg) as a model tumor/self Ag. Repeated vaccination elicited vigorous TAg-specific CTL activity in nontransgenic mice, but tumor-bearing TRAMP mice remained tolerant. Adoptive transfer of naïve splenocytes into TRAMP mice prior to the first vaccination restored TAg reactivity, and slowed tumor progression. The data from this study suggests that vaccination with TAA mRNA is a simple and effective means of priming antitumor CTL, and that immunogenicity of the vaccine can be augmented by co-delivery of GM-CSF mRNA. Nonetheless, limitations of such vaccines in overcoming tolerance to tumor/self Ag may mandate prior or simultaneous reconstitution of the autoreactive T cell repertoire for this form of immunization to be effective.

Animals↗

PubMed2005

Induction of CD4(+) and CD8(+) T-cell responses to the human stromal antigen, fibroblast activation protein: implication for cancer immunotherapy.

PURPOSE: The propensity of tumor cells to escape immune elimination could limit, if not defeat, the long-term benefits of effective immunotherapeutic protocols. Immunologic targeting of tumor stroma could significantly reduce the ability of tumors to evade immune elimination. Murine studies have shown that inducing immunity against angiogenesis-associated products engenders potent antitumor immunity without significant pathology. It is, however, not known whether T cells corresponding to stromal products are present in humans. In this study, we describe a method to screen for human stromal products that have not triggered significant tolerance and could therefore serve as candidate antigens for cancer immunotherapy. EXPERIMENTAL DESIGN: To identify candidates for human stromal antigens, we used an in vitro-screening method to determine whether dendritic cells transfected with mRNA encoding products, which are overexpressed in the tumor stroma, are capable of stimulating cytotoxic CD8(+) (CTL) responses from human peripheral blood mononuclear cells. RESULTS: CTL responses could be consistently generated against fibroblast activation protein (FAP) but not against matrix metalloproteinase-9 (MMP-9) or MMP-14. To enhance the immunogenicity of the mRNA-translated FAP product, a lysosomal targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1) was fused to the COOH terminus of FAP to redirect the translated product into the class II presentation pathway. Dendritic cells transfected with mRNA encoding the FAP-LAMP fusion product stimulated enhanced CD4(+) and CD8(+) T-cell responses. CONCLUSION: This study identifies FAP, a protease preferentially expressed in tumor-associated fibroblasts, as a candidate human stromal antigen to target in the setting of cancer immunotherapy, and shows that differential expression of stromal products is not a sufficient criteria to indicate its immunogenicity in a vaccination setting.

Animals↗

PubMed2005

Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer.

Telomerase reverse transcriptase (hTERT) represents an attractive target for cancer immunotherapy because hTERT is reactivated in most human tumors. A clinical trial was initiated in which hTERT mRNA-transfected dendritic cells (DC) were administered to 20 patients with metastatic prostate cancer. Nine of these subjects received DC transfected with mRNA encoding a chimeric lysosome-associated membrane protein-1 (LAMP) hTERT protein, allowing for concomitant induction of hTERT-specific CD8+ and CD4+ T cell responses. Treatment was well tolerated. Intense infiltrates of hTERT-specific T cells were noted at intradermal injection sites after repeated vaccination. In 19 of 20 subjects, expansion of hTERT-specific CD8+ T cells was measured in the peripheral blood of study subjects, with 0.9-1.8% of CD8+ T cells exhibiting Ag specificity. Patients immunized with the chimeric LAMP hTERT vaccine developed significantly higher frequencies of hTERT-specific CD4+ T cells than subjects receiving DC transfected with the unmodified hTERT template. Moreover, CTL-mediated killing of hTERT targets was enhanced in the LAMP hTERT group, suggesting that an improved CD4+ response could augment a CTL response. Vaccination was further associated with a reduction of prostate-specific Ag velocity and molecular clearance of circulating micrometastases. Our findings provide a rationale for further development of hTERT-transfected DC vaccines in the treatment of prostate and other cancers.

Aged↗

PubMed2004

Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand.

The objective of this study was to investigate whether the immunostimulatory properties of human monocyte-derived dendritic cells (DCs) could be enhanced by triggering OX40/OX40L signaling. Since monocyte-derived DCs possess only low-cell surface levels of OX40L in the absence of CD40 signaling, OX40L was expressed by transfection of DCs with the corresponding mRNA. We show that OX40L mRNA transfection effectively enhanced the immunostimulatory function of DCs at multiple levels: OX40L mRNA transfection augmented allogeneic and HLA class II epitope-specific CD4+ T-cell responses, improved the stimulation of antigen-specific cytotoxic T lymphocytes (CTLs) in vitro without interfering with the prostaglandin E2 (PGE2)-mediated migratory function of the DCs, and facilitated interleukin 12 p70 (IL-12p70)-independent T helper type 1 (Th1) polarization of naive CD4+ T-helper cells. Furthermore, vaccination of tumor-bearing mice using OX40L mRNA-cotransfected DCs resulted in significant enhancement of therapeutic antitumor immunity due to in vivo priming of Th1-type T-cell responses. Our data suggest that transfection of DCs with OX40L mRNA may represent a promising strategy that could be applied in clinical immunotherapy protocols, while circumventing the current unavailability of reagents facilitating OX40 ligation.

Animals↗

PubMed2003

Injection of immature dendritic cells into adjuvant-treated skin obviates the need for ex vivo maturation.

A key and limiting step in the process of generating human monocyte-derived dendritic cells (DC) for clinical applications is maturation. In the setting of immunotherapy, DC are matured ex vivo by culturing them with various agents that mimic the conditions encountered at a site of inflammation. This study examined whether the ex vivo DC maturation step could be replaced by maturing DC in situ by injecting immature DC into sites pre-exposed to agents that induce a microenvironment conducive to in situ maturation of the injected DC. The hypothesis was that recapitulation of the physiological conditions occurring during pathogen infection would lead to optimal conditions for DC maturation, migration, and function. Murine immature DC injected into adjuvant (Adjuprime, poly-arginine, or Imiquimod)-pretreated skin exhibited lymph node migratory capacity comparable to and immunostimulatory capacity equal to or exceeding that of ex vivo matured DC. Acquisition of migratory capacity did not always correlate with enhanced immunostimulatory capacity. Immunostimulatory capacity was not enhanced when mature DC were injected into adjuvant-pretreated sites and remained below that seen with immature DC matured in situ. Immature DC injected into adjuvant-pretreated sites were more effective than mature DC in stimulating antitumor immunity in mice. (111)Indium-labeled human monocyte-derived immature DC injected into adjuvant (Imiquimod)-pretreated sites in cancer patients acquired lymph node migratory capacity comparable to ex vivo matured DC. This study shows that in situ maturation offers a simpler and potentially superior method to generate potent immunostimulatory DC for clinical immunotherapy.

Adjuvants, Immunologic↗

PubMed2003

Inhibition of invariant chain expression in dendritic cells presenting endogenous antigens stimulates CD4+ T-cell responses and tumor immunity.

Induction of potent and sustained antiviral or antitumor immunity is dependent on the efficient activation of CD8+ and CD4+ T cells. While dendritic cells constitute a powerful platform for stimulating cellular immunity, presentation of endogenous antigens by dendritic cells transfected with nucleic acid-encoded antigens favors the stimulation of CD8+ T cells over that of CD4+ T cells. A short incubation of mRNA-transfected dendritic cells with antisense oligonucleotides directed against the invariant chain enhances the presentation of mRNA-encoded class II epitopes and activation of CD4+ T-cell responses in vitro and in vivo. Immunization of mice with the antisense oligonucleotide-treated dendritic cells stimulates a more potent and longer lasting CD8+ cytotoxic T-cell (CTL) response and enhances the antitumor efficacy of dendritic cell-based tumor vaccination protocols. Transient inhibition of invariant chain expression represents a simple and general method to enhance the stimulation of CD4+ T-cell responses from endogenous antigens.

Animals↗

PubMed2003

Synergy between tumor immunotherapy and antiangiogenic therapy.

This study tested the hypothesis that combination of antiangiogenic therapy and tumor immunotherapy of cancer is synergistic. To inhibit angiogenesis, mice were immunized with dendritic cells (DCs) transfected with mRNA that encode products that are preferentially expressed during neoangiogenesis: vascular endothelial growth factor receptor-2 (VEGFR-2) and Tie2 expressed in proliferating endothelial cells, and vascular endothelial growth factor (VEGF) expressed in the angiogenic stroma as well as the tumor cells used in this study. Immunization of mice against VEGF or VEGFR-2 stimulated cytotoxic T lymphocyte (CTL) responses and led to partial inhibition of angiogenesis. Antiangiogenic immunity was not associated with morbidity or mortality except for a transient impact on fertility seen in mice immunized against VEGFR-2, but not VEGF. Tumor growth was significantly inhibited in mice immunized against VEGF, VEGFR-2, and Tie2, either before tumor challenge or in the setting of pre-existing disease in murine B16/F10.9 melanoma and MBT-2 bladder tumor models. Coimmunization of mice against VEGFR-2 or Tie2 and total tumor RNA exhibited a synergistic antitumor effect. Synergism was also observed when mice were coimmunized with various combinations of defined tumor-expressed antigens, telomerase reverse transcriptase (TERT) or TRP-2, and VEGF or VEGFR-2. This study shows that coimmunizing mice against angiogenesis-associated and tumor-expressed antigens can deliver 2 compatible and synergistic cancer treatment modalities via a common treatment, namely immunization.

Animals↗

PubMed2003

Immunotherapy with autologous, human dendritic cells transfected with carcinoembryonic antigen mRNA.

Immunizations with dendritic cells (DC) transfected with RNA encoding tumor antigens induce potent tumor antigen-specific immune responses in vitro and in murine models. We performed a phase I study of patients with advanced carcinoembryonic antigen (CEA)-expressing malignancies followed by a phase II study of patients with resected hepatic metastases of colon cancer to assess safety and feasibility of administering autologous DC loaded with CEA mRNA. The immunizations were well tolerated. Of the 24 evaluable patients in the dose-escalation phase, there was 1 complete response (by tumor marker), 2 minor responses, 3 with stable disease, and 18 with progressive disease. In the phase II study, 9 of 13 patients have relapsed at a median of 122 days. Evidence of an immunologic response was demonstrated in biopsies of DC injection sites and peripheral blood of selected patients. We conclude that it is feasible and safe to administer mRNA-loaded DC to patients with advanced malignancies.

Adult↗

PubMed2002

Enhanced induction of telomerase-specific CD4(+) T cells using dendritic cells transfected with RNA encoding a chimeric gene product.

Dendritic cells (DCs) transfected with mRNA encoding human telomerase reverse transcriptase (hTERT) have been shown to represent potent inducers of CTLs and antitumor immunity. However, it has become widely accepted that not only CTLs but also CD4(+) T helper cells are critical to the generation, as well as to the maintenance, of potent antitumor responses in vivo. In this study, we sought to determine whether human DCs transfected with mRNA encoding a chimeric hTERT/lysosome-associated membrane protein (LAMP-1) protein, carrying the endosomal/lysosomal sorting signal of the LAMP-1, are capable of stimulating concomitant hTERT-specific CD8(+) and CD4(+) T-cell responses in vitro. We show that processing of hTERT/LAMP-1 transcripts leads to enhanced stimulation of hTERT-specific CD4(+) T cells and does not negatively affect intracellular generation and subsequent presentation of MHC class I epitopes, hence, generating a CTL response. These findings provide a preclinical rationale of using DCs transfected with the chimeric hTERT/LAMP-1 RNA in vaccine trials to facilitate generation of antigen-specific CD4(+) T-cell responses that may be required to stimulate and maintain an optimal CD8(+) CTL response in vivo.

Antigens, CD↗

PubMed2002

Induction of tumor-specific cytotoxic T lymphocytes in cancer patients by autologous tumor RNA-transfected dendritic cells.

OBJECTIVE: To demonstrate the feasibility of inducing tumor antigen-specific immune responses in patients with metastatic cancer using total tumor RNA-loaded dendritic cells (DCs). SUMMARY BACKGROUND DATA: The authors have shown that DCs transfected with mRNA encoding defined tumor antigens induce tumor antigen-specific T-cell responses in vitro and in vivo. There may be significant advantages to inducing immune responses against the entire repertoire of antigens expressed by a patient's autologous tumor. METHODS: RNA was extracted from a metastatic colon cancer and used to load autologous DCs. The DCs were coincubated with autologous T cells and the cytolytic activity of the T cells was assessed by the ability to lyse the autologous tumor cells. RNA was then extracted from a metastatic lung cancer and used to load autologous DCs, followed by four injections of the DC vaccine given every 4 weeks. Tumor antigen-specific cytotoxic T lymphocyte activity was then evaluated by testing peripheral blood mononuclear cells for their ability to lyse an antigen-expressing target. RESULTS: DCs transfected with the total RNA content of autologous tumor cells stimulated antigen-specific T-cell responses that are capable of recognizing and lysing autologous, primary tumor cells in vitro. Tumor-specific immune responses were induced in a patient with a carcinoembryonic antigen-expressing adenocarcinoma after immunization with autologous DCs transfected with total tumor RNA. CONCLUSIONS: DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of tumor antigens of surgically resected malignancies.

Adenocarcinoma↗

PubMed2002

The feasibility and safety of immunotherapy with dendritic cells loaded with CEA mRNA following neoadjuvant chemoradiotherapy and resection of pancreatic cancer.

BACKGROUND: Resected pancreatic cancer has a high risk of recurrence and mortality despite the the use of chemoradiotherapy. Because pancreatic cancers express tumor antigens such as carcinoembryonic antigen (CEA), it may be possible to immunize patients to induce tumor antigen-specific immune responses. We hypothesize that high-frequency tumor antigen-specific immune responses will reduce recurrence and increase survival. Autologous dendritic cells (DCs) loaded with tumor antigens are particularly potent at inducing tumor antigen-specific immune responses. METHODS: Three patients with resected pancreatic adenocarcinoma following neoadjuvant chemoradiotherapy received autologous, monocyte-derived DCs loaded with the mRNA encoding CEA monthly for 6 mo. RESULTS: It was feasible to generate an adequate number of DC from these patients and to cryopreserve them for repeated use. The DC demonstrated the typical immature phenotype. The immunizations were well-tolerated without evidence of adverse events. All three developed injection site reactivity. All three are alive without evidence of disease at more than 2 1/2 yr from the original diagnosis. CONCLUSION: The postoperative period following neoadjuvant chemoradiotherapy and pancreaticoduodenectomy for pancreatic cancer is an ideal environment to test novel immune-based therapies. DC-based immunotherapy in this setting is safe and feasible and may lead to prolonged survival.

Adenocarcinoma↗

PubMed2002

RNA-transfected dendritic cells.

Based on their unique ability to stimulate primary immune responses, dendritic cells are the most potent antigen-presenting cells known. This ability stems from the fact that they are very efficient at the uptake and processing of antigen and they express high levels of major histocompatibility complex class I and class II, as well as costimulatory molecules, which are required to prime naive cytotoxic T-cells. Many groups of investigators have tried to take advantage of these features by developing dendritic cell-based vaccines against tumors and infectious diseases. While the basic principle in these studies is the same--dendritic cells pulsed with antigen are used to elicit cytotoxic T-cell responses--the methods used are varied. This is particularly true with respect to the nature of the antigen used and the method of antigen delivery. In this article, we will focus on the use of RNA as a form of antigen with which to load dendritic cells. We will discuss the rationale behind using RNA as an antigen source and will review recent studies in both murine and human settings that use RNA-pulsed dendritic cells as vaccines.

Animals↗