ABSTRACT
The agonistic action of several immunomodulatory monoclonal antibodies (mAbs) requires both target antigen binding and clustering of this mAb:target complex by the Fcs interacting with Fcγ receptors (FcγRs), in particular FcγRIIb, on neighboring bystander cells. Fc mutations were made in the immunoglobulin G4 (IgG4)-based TGN1412 anti-CD28 mAb to define the role of FcγR interactions in its "super-agonist" activity. The dual mutation, IgG4-ED269,270 AA, ablated interaction with all human FcγRs and agonistic action was consequentially lost, confirming the FcγR dependence on the action of TGN1412. The IgG4 lower hinge region (F234 L235 G236 G237 ) was modified by L235 E mutation (F234 E235 G236 G237 ), a mutation commonly used to ablate FcγR binding, including in approved therapeutic mAbs. However, rather than ablating all FcγR binding, IgG4-L235 E conferred specific binding to FcγRIIb, the inhibitory Fc receptor. Furthermore, in combination with the core hinge-stabilizing mutation (IgG4-S228 P, L235 E), this mutation increased affinity for FcγRIIb compared with wild-type IgG4. In addition to having FcγRIIb specificity, these engineered TGN1412 antibodies retained their super-agonistic ability, demonstrating that CD28- and FcγRIIb-specific binding are together sufficient for agonistic function. The FcγRIIb-specific nature of IgG4-L235 E has utility for mAb-mediated immune agonism therapies that are dependent on FcγRIIb interaction and of anti-inflammatory mAbs in allergy and autoimmunity that harness FcγRIIb inhibitory signaling.
Subject(s)
Immunoglobulin G , Receptors, IgG , Humans , Receptors, IgG/metabolism , Antibodies, Monoclonal/therapeutic use , Mutation/geneticsABSTRACT
Ab-dependent cellular cytotoxicity, phagocytosis, and Ag presentation are key mechanisms of action of Abs arising in vaccine or naturally acquired immunity, as well of therapeutic mAbs. Cells expressing the low-affinity FcγRs (FcγRII or CD32 and FcγRIII or CD16) are activated for these functions when receptors are aggregated following the binding of IgG-opsonized targets. Despite the diversity of the Fc receptor proteins, IgG ligands, and potential responding cell types, the induction of all FcγR-mediated responses by opsonized targets requires the presentation of multiple Fc regions in close proximity to each other. We demonstrated that such "near-neighbor" Fc regions can be detected using defined recombinant soluble (rs) dimeric low-affinity ectodomains (rsFcγR) that have an absolute binding requirement for the simultaneous engagement of two IgG Fc regions. Like cell surface-expressed FcγRs, the binding of dimeric rsFcγR ectodomains to Ab immune complexes was affected by Ab subclass, presentation, opsonization density, Fc fucosylation, or mutation. The activation of an NK cell line and primary NK cells by human IgG-opsonized influenza A hemagglutinin correlated with dimeric rsFcγRIIIa binding activity but not with Ab titer. Furthermore, the dimeric rsFcγR binding assay sensitively detected greater Fc receptor activity to pandemic H1N1 hemagglutinin after the swine influenza pandemic of 2009 in pooled human polyclonal IgG. Thus these dimeric rsFcγR ectodomains are validated, defined probes that should prove valuable in measuring the immune-activating capacity of IgG Abs elicited by infection or vaccination or experimentally derived IgG and its variants.
Subject(s)
Antibodies, Viral/analysis , Immunoglobulin G/analysis , Immunologic Techniques/methods , Receptors, Fc/immunology , Receptors, IgG/immunology , Antibodies, Viral/immunology , Antigen-Antibody Complex/immunology , Humans , Immunoglobulin G/immunology , Influenza A Virus, H1N1 SubtypeABSTRACT
Peptide-based vaccines for cancer have many advantages however, for optimization these immunogens should incorporate peptide epitopes that induce CD8, as well as CD4 responses, antibody and long term immunity. Cell penetrating peptides (CPP) with a capacity of cytosolic delivery have been used to deliver antigenic peptides and proteins to antigen presenting cells to induce cytotoxic T cell, helper T cell and humoral responses in mice. For this study, a tripartite CPP including a mucin 1 (MUC1) variable number of tandem repeat (VNTR) containing multiple T cell epitopes and tetanus toxoid universal T helper epitope peptide (tetCD4) was synthesised (AntpMAPMUC1tet) and immune responses investigated in mice. Mice vaccinated with AntpMAPMUC1tet + CpG show enhanced antigen-specific interferon-gamma (IFN-γ) and IL-4 T cell responses compared with AntpMAPMUC1tet vaccination alone and induced a Th1 response, characterised by a higher ratio of IgG2a antibody/IgG1 antibodies. Furthermore, vaccination generated long term MUC1-specific antibody and T cell responses and delayed growth of MUC1+ve tumours in mice. This data demonstrates the efficient delivery of branched multiple antigen peptides incorporating CPP and that the addition of CpG augments immune responses.
Subject(s)
Cell-Penetrating Peptides/immunology , Epitopes, T-Lymphocyte/immunology , Minisatellite Repeats/genetics , Mucin-1/genetics , T-Lymphocytes, Helper-Inducer/immunology , Amino Acid Sequence , Animals , Antibody Specificity/drug effects , Antigen-Presenting Cells/metabolism , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Differentiation , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Cytotoxicity, Immunologic/drug effects , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Endocytosis , Epitopes , Epitopes, T-Lymphocyte/chemistry , Female , HLA-A2 Antigen/metabolism , Immunity, Cellular/drug effects , Immunization , Interferon-gamma/metabolism , Interleukin-4/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms/immunology , Neoplasms/pathology , Oligodeoxyribonucleotides/metabolism , Tetanus Toxin/chemistryABSTRACT
BACKGROUND: H1N1 influenza viruses mutate rapidly, rendering vaccines developed in any given year relatively ineffective in subsequent years. Thus it is necessary to generate new vaccines every year, but this is time-consuming and resource-intensive. Should a highly virulent influenza strain capable of human-to-human transmission emerge, these factors will severely limit the number of people that can be effectively immunised against that strain in time to prevent a pandemic. An adjuvant and mode of administration capable of rendering ordinarily unprotective vaccine doses protective would thus be highly advantageous. METHODS: The carbohydrate mannan was conjugated to whole inactivated H1N1 influenza virus at a range of ratios, and mixed with it at a range of ratios, and various doses of the resulting preparations were administered to mice via the intranasal (IN) route. Serum immunity was assessed via antigen-specific IgG ELISA and the haemagglutination-inhibition (HI) assay, and mucosal immunity was assessed via IgA ELISA of bronchio-alveolar lavages. RESULTS: IN-administered inactivated H1N1 mixed with mannan induced higher serum IgG and respiratory-tract IgA than inactivated H1N1 conjugated to mannan, and HIN1 alone. Adjuvantation was mannan-dose-dependent, with 100 µg of mannan adjuvanting 1 µg of H1N1 more effectively than 10 or 50 µg of mannan. Serum samples from mice immunised with 1 µg H1N1 adjuvanted with 10 µg mannan did not inhibit agglutination of red blood cells (RBCs) at a dilution factor of 10 in the HI assay, but samples resulting from adjuvantation with 50 and 100 µg mannan inhibited agglutination at dilution factors of ≥ 40. Both serum IgG1 and IgG2a were induced by IN mannan-adjuvanted H1N1 vaccination, suggesting the induction of humoral and cellular immunity. CONCLUSIONS: Mixing 100 µg of mannan with 1 µg of inactivated H1N1 adjuvanted the vaccine in mice, such that IN immunisation induced higher serum IgG and respiratory tract IgA than immunisation with virus alone. The serum from mice thus immunised inhibited H1N1-mediated RBC agglutination strongly in vitro. If mannan similarly adjuvants low doses of influenza vaccine in humans, it could potentially be used for vaccine 'dose-sparing' in the event that a vaccine shortage arises from an epidemic involving a highly virulent human-to-human transmissable influenza strain.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/administration & dosage , Lung/immunology , Mannans/administration & dosage , Orthomyxoviridae Infections/prevention & control , Adjuvants, Immunologic/pharmacology , Administration, Intranasal , Animals , Antibodies, Viral/blood , Antibody Formation/drug effects , Female , Hemagglutination Inhibition Tests , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Lung/metabolism , Mannans/immunology , Mannans/pharmacology , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology , Vaccination/methodsABSTRACT
Cell penetrating peptides (CPP), including the TAT peptide from the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein and penetratin from Drosophila Antennapedia homeodomain protein, translocate various cargos including peptides and proteins across cellular barriers. This mode of delivery has been harnessed by our group and others to deliver antigenic proteins or peptides into the cytoplasm of antigen processing cells (APC) such as monocyte-derived dendritic cells (MoDC). Antigens or T cell epitopes delivered by CPP into APC in vivo generate antigen-specific cytotoxic T cell and helper T cell responses in mice. Furthermore, mice immunised with these peptides or proteins are protected from a tumour challenge. The functional properties of CPP are dependent on the various cargos being delivered and the target cell type. Despite several studies demonstrating superior immunogenicity of TAT and Antp-based immunogens, none has compared the immunogenicity of antigens delivered by TAT and Antp CPP. In the current study we demonstrate that a cytotoxic T cell epitope from the mucin 1 (MUC1) tumour associated antigen, when delivered by TAT or Antp, generates identical immune responses in mice resulting in specific MUC1 T cell responses as measured by in vivo CTL assays, IFNγ ELISpot assays and prophylactic tumour protection.
Subject(s)
Carrier Proteins/pharmacology , Cell-Penetrating Peptides/pharmacology , Cytotoxicity, Immunologic/drug effects , Epitopes, T-Lymphocyte/immunology , T-Lymphocytes, Cytotoxic/drug effects , tat Gene Products, Human Immunodeficiency Virus/pharmacology , Amino Acid Sequence , Animals , Bone Marrow Cells/cytology , Carrier Proteins/chemistry , Cell Proliferation/drug effects , Cell-Penetrating Peptides/chemistry , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Endocytosis/drug effects , Immunity, Cellular/drug effects , Immunization , Interferon-gamma/metabolism , Lymphocyte Activation/drug effects , Mice, Inbred C57BL , Molecular Sequence Data , Mucin-1/metabolism , Neoplasms/pathology , Ovalbumin/immunology , tat Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.
Subject(s)
Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/immunology , Immunoglobulin G/immunology , Receptors, IgG/chemistry , Receptors, IgG/immunology , Animals , Antigen-Antibody Complex/genetics , Crystallography, X-Ray , Epitope Mapping , Humans , Immunoglobulin G/chemistry , Mice , Models, Molecular , Polymorphism, Single Nucleotide , Protein Structure, Quaternary , Receptors, IgG/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Signal Transduction/immunology , Surface Plasmon ResonanceABSTRACT
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ABSTRACT
Joining a function-enhanced Fc-portion of human IgG to the SARS-CoV-2 entry receptor ACE2 produces an antiviral decoy with strain transcending virus neutralizing activity. SARS-CoV-2 neutralization and Fc-effector functions of ACE2-Fc decoy proteins, formatted with or without the ACE2 collectrin domain, were optimized by Fc-modification. The different Fc-modifications resulted in distinct effects on neutralization and effector functions. H429Y, a point mutation outside the binding sites for FcγRs or complement caused non-covalent oligomerization of the ACE2-Fc decoy proteins, abrogated FcγR interaction and enhanced SARS-CoV-2 neutralization. Another Fc mutation, H429F did not improve virus neutralization but resulted in increased C5b-C9 fixation and transformed ACE2-Fc to a potent mediator of complement-dependent cytotoxicity (CDC) against SARS-CoV-2 spike (S) expressing cells. Furthermore, modification of the Fc-glycan enhanced cell activation via FcγRIIIa. These different immune profiles demonstrate the capacity of Fc-based agents to be engineered to optimize different mechanisms of protection for SARS-CoV-2 and potentially other viral pathogens.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Humans , Peptidyl-Dipeptidase A/metabolism , RNA, Viral , SARS-CoV-2ABSTRACT
Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.
Subject(s)
Antigen Presentation , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class I/immunology , Neoplasms/immunology , Animals , Antennapedia Homeodomain Protein/immunology , Antennapedia Homeodomain Protein/metabolism , Antigens, Neoplasm/immunology , CD4 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8 Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Carrier Proteins , Cell-Penetrating Peptides , Drosophila , Drosophila Proteins/immunology , Interferon-gamma/biosynthesis , Interferon-gamma/metabolism , Mice , Mice, Inbred C57BL , Mucin-1/immunology , Mucin-1/metabolism , Ovalbumin/immunology , Ovalbumin/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR; FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab')2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibody-Dependent Cell Cytotoxicity/immunology , Immunoglobulin Fc Fragments/metabolism , Receptors, IgG/agonists , Receptors, IgG/metabolism , Binding Sites , Epitopes/genetics , Epitopes/immunology , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fc Fragments/immunology , Mutation , Phosphorylation , Platelet Activation , Protein Binding , Receptors, Fc , Receptors, IgG/geneticsABSTRACT
Receptor mediated gene delivery is an attractive non-viral method for targeting genetic material to specific cell types. We have previously utilized oxidized (OMPLL) and reduced mannan poly-L-lysine (RMPLL) to target DNA vaccines to antigen presenting cells and demonstrated that it could induce far stronger immune responses in mice compared to naked DNA immunization. In this study, we describe the immune enhancing attributes of mannan-PLL mediated DNA vaccination at the molecular level. Several attributes observed in similar gene delivery conjugates, such as entry via the endocytic pathway, low toxicity, protection from nucleases and compaction of particle size, were also evident here. In addition, OMPLL and RMPLL conjugates had profound effects on the antigen presentation functions of dendritic cells and macrophages, through the stimulation of cytokine production and maturation of dendritic cells. Interestingly, we demonstrate that OMPLL-DNA and RMPLL-DNA are able to mediate dendritic cell activation via toll-like receptor 2 as opposed to mannan alone which mediates via toll-like receptor 4. Overall, this report leads to greater understanding of how oxidized and reduced mannan mediated gene delivery could augment immune responses to DNA vaccination and provide insights into ways of further improving its immunogenicity.
Subject(s)
Drug Carriers , Gene Transfer Techniques , Mannans , Vaccines, DNA , Animals , B7-2 Antigen/immunology , Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Cytokines/immunology , Dendritic Cells/immunology , Dendritic Cells/physiology , Drug Carriers/chemistry , Humans , Mannans/chemistry , Mannans/immunology , Materials Testing , Mice , Mice, Inbred Strains , Mice, Knockout , Particle Size , Vaccines, DNA/chemistry , Vaccines, DNA/immunologyABSTRACT
The interaction of immune complexes with the human Fc receptor, FcgammaRIIa, initiates the release of inflammatory mediators and is implicated in the pathogenesis of human autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, so this FcR is a potential target for therapy. We have used the three-dimensional structure of an FcgammaRIIa dimer to design small molecule inhibitors, modeled on a distinct groove and pocket created by receptor dimerization, adjacent to the ligand-binding sites. These small chemical entities (SCEs) blocked immune complex-induced platelet activation and aggregation and tumor necrosis factor secretion from macrophages in a human cell line and transgenic mouse macrophages. The SCE appeared specific for FcgammaRIIa, as they inhibited only immune complex-induced responses and had no effect on responses to stimuli unrelated to FcR, for example platelet stimulation with arachidonic acid. In vivo testing of the SCE in FcgammaRIIa transgenic mice showed that they inhibited the development and stopped the progression of collagen-induced arthritis (CIA). The SCEs were more potent than methotrexate and anti-CD3 in sustained suppression of CIA. Thus, in vitro and in vivo activity of these SCE FcgammaRIIa receptor antagonists demonstrated their potential as anti-inflammatory agents for autoimmune diseases involving immune complexes.
Subject(s)
Antirheumatic Agents/chemistry , Antirheumatic Agents/therapeutic use , Arthritis, Experimental/drug therapy , Drug Design , Receptors, IgG/antagonists & inhibitors , Animals , Antirheumatic Agents/chemical synthesis , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Blood Platelets/drug effects , Blood Platelets/immunology , Blood Platelets/metabolism , Disease Models, Animal , Humans , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Transgenic , Platelet Activation/drug effects , Platelet Activation/immunology , Protein Conformation , Receptors, IgG/chemistry , Receptors, IgG/genetics , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunology , U937 CellsABSTRACT
Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4(+)/CD8(+) T cell and antibody responses. MDO also targeted lymph node DC to cross-present OVA, leading to OTI CD8(+) T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Antigens/immunology , Dendrimers/administration & dosage , Dendrimers/chemistry , Drug Delivery Systems , Mannose/immunology , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/therapeutic use , Amino Acid Sequence , Animals , Antigen Presentation/immunology , Antigens/administration & dosage , Antigens/chemistry , Cells, Cultured , Dendritic Cells/immunology , Egg Proteins/administration & dosage , Egg Proteins/immunology , Mannose/chemistry , Mannose/metabolism , Melanoma, Experimental/immunology , Melanoma, Experimental/prevention & control , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Ovalbumin/administration & dosage , Ovalbumin/immunology , Peptide Fragments , T-Lymphocytes/immunologyABSTRACT
DNA immunization is an attractive form of vaccination, which has shown promising results only in small animal models. There is a need to develop efficient gene delivery systems. We previously demonstrated that oxidized (OM) and reduced mannan (RM) complexed to ovalbumin DNA via poly-l-lysine (PLL), were able to generate potent immune responses in mice. Herein, we further investigated the suitability of OMPLL and RMPLL as carriers for mucin 1 (MUC1) DNA vaccination for cancer immunotherapy. Studies presented here showed that immune responses in C57BL/6 mice induced by OMPLL-MUC1 DNA and RMPLL-MUC1 DNA immunization were more immunogenic compared to MUC1 DNA alone. Moreover, tumor protection was evident at a dose as low as 0.5 microg. In addition, strong T cell responses were induced in HLA-A2 transgenic and human MUC1 transgenic mice. These results demonstrate the potential of OM and RM as efficient non-viral gene delivery carriers for DNA vaccines for use in cancer immunotherapy.