Biomedical subjects

Geoffrey A Pietersz

Publications and source records attributed to Geoffrey A Pietersz.

15 recordsLinked to original sources

PubMed2007

Receptor-mediated delivery of antigens to dendritic cells: anticancer applications.

Recently, there has been a surge of interest in the use of ex vivo antigen-pulsed dendritic cells (DCs) in the immunotherapy for cancer. DCs are powerful adjuvants with the ability to prime naive CD4+ and CD8+ T cells. As antigen sources, various preparations, including peptides, proteins, crude tumor lysates, and DCs transfected or transformed with various viruses, have been used. These procedures that involve the isolation of patient DCs and reintroduction after in vitro manipulation are time-consuming and expensive. The DC populations used frequently in ex vivo clinical studies are IL-4 and GM-CSF cultured DCs that may not represent the in vivo DC populations. An attractive method of targeting in vivo DCs is to utilize various ligands or antibodies that bind discrete populations of DCs. These cell surface receptors will direct the antigen to different antigen processing pathways depending on the targeted receptor to induce cytotoxic T cell or T helper responses. This review will discuss the various approaches and receptors that have been used for antigen targeting that may eventually be translated to alternative DC-based immunotherapies.

Animals↗

PubMed2006

Poly-L-lysine-coated nanoparticles: a potent delivery system to enhance DNA vaccine efficacy.

DNA formulations provide the basis for safe and cost efficient vaccines. However, naked plasmid DNA is only poorly immunogenic and new effective delivery strategies are needed to enhance the potency of DNA vaccines. In this study, we present a novel approach for the delivery of DNA vaccines using inert poly-L-lysine (PLL) coated polystyrene particles, which greatly enhance DNA immunogenicity. Intradermal injection of plasmid DNA encoding for chicken egg ovalbumin (OVA) complexed with PLL-coated polystyrene nanoparticles induced high levels of CD8 T cells as well as OVA-specific antibodies in C57BL/6 mice and furthermore inhibited tumour growth after challenge with the OVA expressing EG7 tumour cell line. Importantly, vaccine efficacy depended critically on the size of the particles used as well as on the presence of the PLL linker. Our data show that PLL-coated polystyrene nanoparticles of 0.05 microm but not 0.02 microm or 1.0 microm in diameter are highly effective for the delivery of DNA vaccines.

Adjuvants, Immunologic↗

PubMed2006

Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1 [ISRCTN71711835].

INTRODUCTION: Mucin 1 (MUC1) is a high molecular weight glycoprotein overexpressed on adenocarcinoma cells and is a target for immunotherapy protocols. To date, clinical trials against MUC1 have included advanced cancer patients. Herein, we report a trial using early stage breast cancer patients and injection of oxidized mannan-MUC1. METHOD: In a randomized, double-blind study, 31 patients with stage II breast cancer and with no evidence of disease received subcutaneous injections of either placebo or oxidized mannan-MUC1, to immunize against MUC1 and prevent cancer reoccurrence/metastases. Twenty-eight patients received the full course of injections of either oxidized mannan-MUC1 or placebo. Survival and immunological assays were assessed. RESULTS: After more than 5.5 years had elapsed since the last patient began treatment (8.5 years from the start of treatment of the first patient), the recurrence rate in patients receiving the placebo was 27% (4/15; the expected rate of recurrence in stage II breast cancer); those receiving immunotherapy had no recurrences (0/16), and this finding was statistically significant (P = 0.0292). Of the patients receiving oxidized mannan-MUC1, nine out of 13 had measurable antibodies to MUC1 and four out of 10 had MUC1-specific T cell responses; none of the placebo-treated patients exhibited an immune response to MUC1. CONCLUSION: The results suggest that, in early breast cancer, MUC1 immunotherapy is beneficial, and that a larger phase III study should be undertaken.

Adenocarcinoma↗

PubMed2006

Mannan-MUC1-pulsed dendritic cell immunotherapy: a phase I trial in patients with adenocarcinoma.

PURPOSE: Tumor antigen-loaded dendritic cells show promise for cancer immunotherapy. This phase I study evaluated immunization with autologous dendritic cells pulsed with mannan-MUC1 fusion protein (MFP) to treat patients with advanced malignancy. EXPERIMENTAL DESIGN: Eligible patients had adenocarcinoma expressing MUC1, were of performance status 0 to 1, with no autoimmune disease. Patients underwent leukapheresis to generate dendritic cells by culture ex vivo with granulocyte macrophage colony-stimulating factor and interleukin 4 for 5 days. Dendritic cells were then pulsed overnight with MFP and harvested for reinjection. Patients underwent three cycles of leukapheresis and reinjection at monthly intervals. Patients with clinical benefit were able to continue with dendritic cell-MFP immunotherapy. RESULTS: Ten patients with a range of tumor types were enrolled, with median age of 60 years (range, 33-70 years); eight patients were of performance status 0 and two of performance status 1. Dendritic cell-MFP therapy led to strong T-cell IFNgamma Elispot responses to the vaccine and delayed-type hypersensitivity responses at injection sites in nine patients who completed treatments. Immune responses were sustained at 1 year in monitored patients. Antibody responses were seen in three patients only and were of low titer. Side effects were grade 1 only. Two patients with clearly progressive disease (ovarian and renal carcinoma) at entry were stable after initial therapy and went on to further leukapheresis and dendritic cell-MFP immunotherapy. These two patients have now each completed over 3 years of treatment. CONCLUSIONS: Immunization produced T-cell responses in all patients with evidence of tumor stabilization in 2 of the 10 advanced cancer patients treated. These data support further clinical evaluation of this dendritic cell-MFP immunotherapy.

Adenocarcinoma↗

PubMed2006

Delivery of tumor associated antigens to antigen presenting cells using penetratin induces potent immune responses.

Cytoplasmic delivery of proteins or CTL epitopes is crucial for the presentation of antigen for the generation of CTL. We previously described the use of the 16-amino acid peptide penetratin from the Drosophila Antennapedia domain (Int) to transport CTL epitopes into cells. Here we show that, Int, incorporating MUC1 CTL epitopes in tandem is able to facilitate their rapid uptake by macrophages and dendritic cells (DC) in an energy-dependent endocytic pathway. We also demonstrate for the first time that Int conjugated proteins are also able to be efficiently taken up by DC. Furthermore, C57BL/6 and HLA-A2 transgenic mice immunized with the Int-peptides or Int-proteins induce strong IFN-gamma secreting T cells and weak IgG1 antibodies. Immunized C57BL/6 mice were protected against the growth of a MUC1(+) tumor cell line.

Animals↗

PubMed2006

Methods for nano-particle based vaccine formulation and evaluation of their immunogenicity.

Nano- and microparticles have long been used for the delivery of drugs and are currently being evaluated as vaccine delivery systems. Particulates can elicit potent immune responses, either by direct immuno-stimulation of antigen presenting cells (APC) or/and by delivering antigen to specific cellular compartments and promoting antigen uptake by appropriate stimulatory cell types. Herein, we describe a detailed method for the preparation of a novel nanoparticle-based antigen delivery system which induces strong cellular and humoral immune responses in mice and sheep. This simple system is based on the use of 40 nanometer (nm) inert solid carrier beads to which antigen is covalently coupled before injection. Covalent conjugation of antigen to the nanobeads, assessment of conjugation efficiency, characterisation and measurement of in vivo immunogenicity by cytokine ELISPOT (to measure antigen-specific T-cell responses) and ELISA (to measure antibody titers), are described. Emphasis is placed on providing trouble-shooting advice to enable the reproducible production of soluble nano-size formulations that do not suffer from common problems such as aggregation, as well as understanding the causes and thus avoiding a range of prevalent technical problems that occur when using immune response detection assays, such as the cytokine ELISPOT assay and ELISA.

Animals↗

PubMed2006

Penetratin tandemly linked to a CTL peptide induces anti-tumour T-cell responses via a cross-presentation pathway.

Recently there has been increasing evidence to suggest that membrane translocating peptides enter cells by a receptor-dependent pathway. There have been some studies on the mechanism of major histocompatibility complex (MHC) class I presentation of membrane translocating peptides incorporating cytotoxic T lymphocyte epitopes. However, these have been on different cell lines and only a limited number of inhibitors of the antigen presentation pathway were used. Herein, we demonstrate a comprehensive study utilizing a full spectrum of inhibitors to various pathways of MHC class I to elucidate the mechanism of the membrane translocating peptide, penetratin from Antennapedia (Int). It is clear that Int, RQIKIWFQNRRMKWKK when tandemly linked to a cytotoxic T lymphocyte peptide of ovalbumin, SIINFEKL (IntSIIN) is endocytosed via phagocytosis or macropinocytosis by dendritic cells in an ATP-dependent manner and is processed by a proteasome- and tapasin-independent pathway for presentation and loading to MHC class I molecules. In addition, the majority of antigen is taken up by negatively charged receptors. IntSIIN activates T cells in vitro and in vivo and protects mice against challenge with an ovalbumin-expressing tumour.

Animals↗

PubMed2006

Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells.

Mannan, a polysaccharide isolated from yeast binds to C-type lectins of the mannose receptor family, expressed by antigen-presenting cells (APCs) including dendritic cells (DCs) and macrophages. As these receptors mediate endocytosis, they have been targeted with ligands to deliver antigens into APCs to initiate immune responses. Immunization with tumour antigen MUC1 conjugated to oxidized mannan (OM) or reduced mannan (RM) induced differential immune responses in mice, and only mice immunized with OM-MUC1 elicited strong MUC1-specific cytotoxic T lymphocyte responses and protected mice from a MUC1 tumour challenge. In this study, the adjuvant effect of mannan and its derivatives including OM and RM, in comparison to lipopolysaccharide, on DCs were investigated. Mannan, OM and RM were capable of stimulating mouse bone marrow-derived DC in vitro, eliciting enhanced allogeneic T-cell proliferation and enhancing OTI/OTII peptide-specific T-cell responses. Injection of mice with mannan, OM and RM induced a mature phenotype of lymph node and splenic DCs. Analysis by reverse transcription-polymerase chain reaction indicated that Manna, OM and RM also stimulated up-regulation of inflammatory cytokines including interleukin-1beta and tumour necrosis factor-alpha, and differential T helper 1 (Th1)/Th2 cytokines. Subsequent experiments demonstrated that activation of DCs was Toll-like receptor-4-dependent. The data presented here, together with evidence reported previously on OM and RM in induction of immune responses in vivo, suggest that OM and RM exert a dual capacity to target antigen to APCs as well as mature DCs.

Adjuvants, Immunologic↗

PubMed2006

Enhanced major histocompatibility complex class I binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide.

Designing peptide-based vaccines for therapeutic applications in cancer immunotherapy requires detailed knowledge of the interactions between the antigenic peptide and major histocompatibility complex (MHC) in addition to that between the peptide-MHC complex and the T-cell receptor. Past efforts to immunize with high-affinity tumour-associated antigenic peptides have not been very immunogenic, which may be attributed to the lack of T cells to these peptides, having been deleted during thymic development. For this reason, low-to-medium affinity non-canonical peptides represent more suitable candidates. However, in addition to the difficulty in identifying such antigens, peptide binding to MHC, and hence its ability to induce a strong immune response, is limited. Therefore, to enhance binding to MHC and improve immune responses, anchor modifications of non-canonical tumour-associated peptides would be advantageous. In this study, the non-canonical tumour-associated peptide from MUC1, MUC1-8 (SAPDTRPA), was modified at the MHC anchor residues to SAPDFRPL (MUC1-8-5F8L) and showed enhanced binding to H-2Kb and improved immune responses. Furthermore, the crystal structure of MUC1-8-5F8L in complex with H-2Kb was determined and it revealed that binding of the peptide to MHC is similar to that of the canonical peptide OVA8 (SIINFEKL).

Animals↗

PubMed2006

Structure and design of polycationic carriers for gene delivery.

The development of safe and effective gene delivery methods is a major challenge to enable gene therapy or DNA vaccines to become a reality. Currently there are two major approaches for delivery of genetic material, viral and non-viral. The majority of on-going clinical trials in gene therapy or DNA vaccines use retroviruses and adenoviruses for delivering genetic materials. Viral delivery systems are far more effective than non-viral delivery however there are concerns regarding toxicity, immunogenicity and possible integration of viral genetic material into the human genome. Given the negative charge of the phosphate backbone of DNA, polycationic molecules have been the major focus as carriers of DNA. There are several physiological barriers to overcome for effective systemic delivery of DNA. The ideal vector must be stable in the systemic circulation, escape the reticuloendothelial system, able to extravasate tissues, enter the target cell, escape lysosomal degradation and transport DNA to the nucleus to be transcribed. With increasing understanding of the physicochemical properties essential to overcome the various barriers, it is possible to apply rational design to the cationic carriers. A number of poly-amino acids, cationic block co-polymers, dendrimers and cyclodextrins have been rationally designed to optimize gene delivery. This review will discuss approaches that have been used to design various synthetic polycations with enhanced DNA condensing ability, serum stability and endosomolytic capability for efficient gene transfer in vitro and in vivo.

Cations↗

PubMed2005

Synthesis and study of the electrophoretic behavior of mannan conjugates with cyclic peptide analogue of myelin basic protein using lysine-glycine linker.

New approaches for the treatment of multiple sclerosis involve the design and synthesis of peptide or nonpeptide analogues of myelin sheath, which could alter the immune response of patients. For this purpose, the cyclo(75-82) myelin basic protein (MBP)(74-85) analogue was conjugated to mannan (a polymannose) via (Lys-Gly)(5) linker. Monitoring of synthesis of the (Lys-Gly)(5)-containing cyclic analogue of MBP, mannan oxidation, and the conjugation reaction of this analogue to oxidized mannan was performed with capillary electrophoresis (CE) in operating buffers of different pH values. The (Lys-Gly)(5)cyclo(75-82)MBP(74-85) was efficiently synthesized by solid-phase synthesis and purified to a high degree, as confirmed by CE analysis in a low-pH (3.0) phosphate buffer and normal polarity. Oxidation of mannan was monitored using a high-pH (9.3) borate buffer, and the generation of heterogeneous products and even UV-absorbing peaks was shown by CE. CE analysis in a pH 5.1 phosphate buffer offers high resolution of oxidized mannan and the conjugation product and can be used for screening of the reaction products. Mannan-peptide conjugates of varying degrees of substitution and unreacted mannan were observed. The developed CE analysis presents distinct advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis such as high versatility, high separation efficiency, short analysis time, low cost, and low solvent consumption.

Electrophoresis, Capillary↗

PubMed2004

Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors.

Infection can protect against subsequent disease by induction of both humoral and cellular immunity, but inert protein-based vaccines are not as effective. In this study, we present a new vaccine design, with Ag covalently conjugated to solid core nano-beads of narrowly defined size (0.04-0.05 microm) that localize to dendritic cells (DEC205(+) CD40(+), CD86(+)) in draining lymph nodes, inducing high levels of IFN-gamma production (CD8 T cells: precursor frequencies 1/5000 to 1/1000) and high Ab titers in mice. Conjugation of Ag to these nano-beads induced responses that were significantly higher (2- to 10-fold) than those elicited by other bead sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A). Responses were comparable to CFA/IFA immunization for Abs and ex vivo peptide-pulsed dendritic cell immunization for CD8 T cells. A single dose of Ag-conjugated beads protected mice from tumors in two different model challenges and caused rapid clearance of established tumors in mice. Thus, a range of Ags conjugated to nano-beads was effective as immunogens in both therapeutic and prophylactic scenarios.

Adjuvants, Immunologic↗

PubMed2004

Cripto: a novel target for antibody-based cancer immunotherapy.

Cripto, a member of the epidermal growth factor-Cripto-FRL-Criptic (EGF-CFC) family, has been described recently as a potential target for immunotherapy (Adkins et al., J Clin Invest 2003;112:575-87). We have produced rat monoclonal antibodies (mAbs) to a Cripto 17-mer peptide, corresponding to the "EGF-like" motif of Cripto. The mAbs react with most cancers of the breast, colon, lung, stomach, and pancreas but do not react or react weakly with normal tissues. The mAbs inhibit cancer cell growth in vitro, and this effect was greater with cytotoxic drugs such as 5-fluorouracil, epirubicin, and cisplatin. The anti-Cripto mAbs prevent tumor development in vivo and inhibit the growth of established tumors of LS174T colon xenografts in Scid mice. The growth inhibitory effects with these mAbs may be greater than those described elsewhere, possibly because of IgM giving more effective cross-linking or binding to a different epitope (EGF-like region versus CFC region). The mechanism of inhibitory effects of the Cripto mAbs includes both cancer cell apoptosis, activation of c-Jun-NH(2)-terminal kinase and p38 kinase signaling pathways and blocking of Akt phosphorylation. Thus, Cripto is a unique target, and mAbs to Cripto could be of therapeutic value for human cancers.

Amino Acid Sequence↗

PubMed2003

Aspects of cancer immunotherapy.

Cancer immunotherapy has traditionally undergone a 'revolution' every decade, from the use of Bacille Calmette-Guérin by scarification in the 1970s, to interleukin-2 therapies in the 1980s, and monoclonal antibody treatments in the early 1990s. Usually the early reports on the use of such agents were encouraging, but when more patients were studied in multiple centres, the initial promising results could not be confirmed. Now in a new century, we have more reagents and methods available than ever before - indeed, with such a plethora of reagents it is difficult to envisage them being fully and appropriately tested within the next decade, by which time there will be even more reagents to test. However, there have been three major advances which should lead to substantial progress in cancer immunotherapy: (1) the widespread use of genetic engineering, enabling identification of candidate vaccine proteins and manipulation of their sequences; (2) the production of antigens, antibodies and cytokines in large amounts by recombinant technologies, and (3) an understanding of the mode of presentation of peptides by major histocompatibility complex Class I and Class II molecules and their recognition by T cells. Despite these advances, there are major problems facing cancer immunotherapy, such as the ability of tumours to mutate and evade the immune system and the difficulty of precisely defining the interactions of effector cells in mediating 'rejection' or destruction of a tumour. There are clearly immunological similarities with diseases such as malaria and schistosomiasis, where the invading foreign organisms can use a variety of strategies to resist an elicited immune response. The failure to find a suitable vaccine for these diseases must lead to some pessimism for the development of immunotherapy for an autologous tumour. However, there are promising studies now in progress which should give an indication of the most important directions to follow. This review provides a commentary on aspects of cancer immunotherapy and in particular will deal with: (1) the selection of antigens as vaccine components; (2) the modes of presentation of antigens, particularly by major histocompatibility complex Class I molecules; and (3) new modes of delivery of vaccine immunogens.

Antigen Presentation↗

PubMed2002

Crystal structure of a non-canonical low-affinity peptide complexed with MHC class I: a new approach for vaccine design.

Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide-binding groove. Peptides that do not contain these canonical anchor residues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6 A resolution of an immunogenic, low-affinity peptide from the tumor-associated antigen MUC1, bound to H-2Kb. Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular interactions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines.

Animals↗