ANTIGENIC COMPOSITIONS AND USE OF SAME IN THE TARGETED DELIVERY OF NUCLEIC ACIDS
Methods and compositions are provided for delivery of therapeutic nucleic acids to a target cell. A chimeric antigen is provided to encapsulate, bind, or otherwise carry a nucleic acid molecule to a target cell where the chimeric antigen and nucleic acid are internalized, for example by receptor-mediated endocytosis. The chimeric antigen has a nucleic acid interaction domain, a target binding domain, and an immune response domain that may include a target antigen. Targeting is generally provided by the specificity of the target binding domain for a particular target cell receptor, but may also be provided by inclusion of a targeting antigen within the immune response domain. The combined delivery of chimeric antigen and nucleic acid, which may be a siRNA, may be synergistic in certain applications, for example in breaking host tolerance to a virus or in providing immunostimulation.
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This application is a divisional application of U.S. patent application Ser. No. 12/675,560, filed Jan. 28, 2011, which is national stage application under 35 U.S.C. 371 of PCT Application No. PCT/CA2008/01547, having an international filing date of Aug. 29, 2008, which designated the United States, which PCT application claims the benefit of priority under 35 U.S.C. §119(e) from U.S. Provisional Application No. 60/968,978, filed Aug. 30, 2007. The entire disclosure of each is incorporated herein by reference.
FIELD OF THE INVENTIONThe present invention relates to compositions for use in the delivery of nucleic acids. More particularly, chimeric antigens are provided for encapsulating, binding, or otherwise carrying and delivering nucleic acids to a target cell.
BACKGROUND OF THE INVENTIONDespite recent advances in the identification and refinement of nucleic acid therapeutics, finding suitable delivery means for these molecules in various applications has proved challenging. Moreover, while it is desirable to minimize the dosage of these expensive molecules, by localizing or targeting nucleic acid therapies to tissues/cells of interest, many technologies have been investigated, with few promising results.
RNAi [Fire A., et al (1998) Nature 391:801-11] has emerged as a means for sequence specific, posT transcriptional gene silencing, mediated by short interfering RNAs (siRNAs) homologous to the gene targeted for silencing. However, to be effectively used as drugs, the siRNAs (or their larger RNA precursors) must be delivered directly into the target cell. Targeted delivery of siRNA into specific cells of interest has been the main obstacle to achieving in vivo gene silencing by RNAi technologies. Specific delivery, dosage reduction, and minimizing toxicity are all important unmet objectives in this field.
Potential targeted siRNA delivery systems have emerged, such as antibody-mediated delivery, and liposomal delivery. Antibody mediated siRNA delivery may allow preferential accumulation of siRNA in target cells with less effect on normal tissues, and it has been suggested that such ligands can further be conjugated to delivery agents, such as liposomes, to promote uptake into target cells by receptor mediated endocytosis.
A further potential method for in vivo delivery of siRNA to specific target cells employs the nucleic acid binding properties of protamine, combined with the specificity of antibody-mediated delivery. Injection of siRNAs complexed with an antibody fragmenT protamine fusion protein have been used to selectively deliver siRNAs into target cells expressing the cell surface receptor recognized by the antibody [reviewed in Dykxhoorn, D. M., et al (2006) Gene Therapy 13-541-552; Song E. et al (2005) Nature Biotech. 23 (6):709-717].
The specific cell type or targeted organ will generally vary with the type of therapeutic being delivered. For example, dendritic cells may be a key focus in cancer immunotherapy applications, as these potent antigen presenting cells are uniquely capable of inducing immunity to break tolerance to cancer antigens. It has been suggested that RNAi can be used for immune modulation by targeting gene expression in dendritic cells [Hill, J. A., et al (2003) J. Immunol. 171:691-696].
SOCS-1 has been shown to control the tolerogenic and immunogenic state of the dendritic cell, as well as the extent of antigen presentation and hence the magnitude of adaptive immunity [reviewed in Yoshimura, A., et al (2007) Nature Rev. Immunol. 7:454-465]. Silencing of SOCS-1 by siRNA enhances both antigen presentation by dendritic cells and antigen-specific anti-tumour immunity and may offer a selective means of breaking in host tolerance, of enhancing antigen-specific anti-tumour and anti-viral immunity, and of increasing the efficiency of dendritic cell-based cancer vaccines. Silencing SOCS-1 in dendritic cells may reduce the threshold of the cell's responsiveness to endogenous stimuli, permit persistent activation of antigen-specific T cells in vivo, and boost the anti-cancer activity of T cells.
In an ex vivo study, dendritic cells showed enhanced antigen-specific anti-tumour immunity when SOCS-1 was silenced in the dendritic cells before their vaccination with a cancer antigen [Shen, T. (2004) Nature Biotech 22(12): 1546-1553]. In an in vivo study in mice, silencing of SOCS-1 induced an anti-HIV-1 CD8+ and CD4+ T cell response as well as antibody responses [Song, X-T. et al (2006) PLoS Med 3:1-18].
The use of siRNA in the treatment of viral disease has also been suggested. In particular, the manifestation of chronic viral diseases relies on avoidance of the host immune system. It has been speculated that viral gene expression may be silenced by administration of virus-specific siRNA to the infected host.
In subjects with chronic viral or parasitic infections (where the organism is resident inside a host cell at some point during its life cycle), antigens are produced by and expressed in the host cell, and secreted antigens are present in the circulation. As an example, in the case of a chronic human hepatitis B virus (HBV) infected carrier, virions, HBV surface antigens, and a surrogate of the core antigens (in the form of the e-antigen) can be detected in the blood but are apparently tolerated by the host immune system.
Similarly, in cancer, tumour escape from immune surveillance and attack is a major determinant for tumour survival in the host. A need exists for new, therapeutically effective compounds, compositions and methods for eliciting or enhancing immune responses against infectious diseases or cancer, or to break tolerance to infectious diseases or cancer.
SUMMARYIn accordance with a first aspect of the invention, there is provided a method for inhibiting expression of a target gene within a target cell, the method comprising the steps of: providing a nucleic acid molecule suitable for effecting RNAi of a target gene; providing a chimeric antigen comprising: a nucleic acid binding domain comprising an amino acid sequence corresponding to HBV core protein or a fragment thereof, and a target binding domain comprising a ligand for binding to a receptor on a target cell; and administering the nucleic acid molecule and the chimeric antigen to the target cell.
In an embodiment, the step of administering the nucleic acid molecule and chimeric antigen to the target cell comprises mixing the nucleic acid molecule with a suitable amount of the chimeric antigen to create a nucleic acid delivery complex, and then administering the nucleic acid delivery complex to the target cell.
In various embodiments, the HBV Core protein fragment may be the assembly domain of HBV Core protein, the protamine domain of HBV Core protein, or any other suitable HBV Core protein fragment.
In certain embodiments, the nucleic acid binding domain may be operatively attached to the N-terminus or to the C-terminus of the target binding domain.
In an embodiment, the target cell is a mammalian host cell, and the step of administering the nucleic acid and chimeric antigen to the target cell comprises administering said nucleic acid and chimeric antigen to the mammalian host. in such embodiment, the target binding domain may comprise a xenotypic antibody fragment.
In accordance with a second aspect of the invention, there is provided a method for eliciting an immune response to a target antigen, the method comprising the steps of: providing a nucleic acid molecule suitable for effecting RNAi of a target gene; providing a chimeric antigen comprising: a nucleic acid binding domain comprising an amino acid sequence corresponding to HBV core protein or a fragment thereof, a target binding domain comprising a ligand for binding a receptor on an antigen presenting cell, and an immune response domain comprising a target antigen; and administering the nucleic acid molecule and the chimeric antigen to the target cell.
In an embodiment, the step of administering the nucleic acid molecule and chimeric antigen to the target cell comprises first mixing the nucleic acid molecule with a suitable amount of the chimeric antigen to create a nucleic acid delivery complex, and then administering the nucleic acid delivery complex to the target cell.
In various embodiments, the HBV Core protein fragment may be the assembly domain of HBV Core protein, the protamine domain of HBV Core protein, or any other suitable HBV Core protein fragment.
In certain embodiments, the nucleic acid binding domain may be operatively attached to the N-terminus or to the C-terminus of the target binding domain.
In an embodiment, the target binding domain comprises a xenotypic antibody fragment.
In specific embodiments, the target gene is an immunomodulatory gene or a viral gene, and the target antigen may be a cancer antigen, a viral antigen, or any other suitable antigen.
In accordance with another aspect of the invention, there is provided a composition for use in silencing the expression of a target gene within a target cell, the composition comprising: a nucleic acid sequence corresponding to the target gene; and a chimeric antigen comprising a nucleic acid interaction domain corresponding to HBV core protein or a fragment thereof, and a target binding domain operatively attached to the nucleic acid interaction domain, the target binding domain comprising a ligand for binding to a receptor on the surface of the target cell.
In certain embodiments, the target gene may be an immunomodulatory gene or a viral gene.
In an embodiment, the target binding domain is a xenotypic Fc fragment.
In various embodiments, the nucleic acid sequence is a siRNA, shRNA, antisense DNA, or plasmid for inhibiting expression of the target gene.
In suitable embodiments, the HBV core protein fragment may be the assembly domain, the protamine domain, or another fragment of HBV core protein.
In an embodiment, the binding of the ligand to receptor initiates internalization, for example by receptor-mediated endocytosis, of the chimeric antigen and nucleic acid.
In accordance with a further aspect of the invention, there is provided a chimeric antigen for use in delivering a nucleic acid to a target cell, the chimeric antigen comprising: a nucleic acid interaction domain corresponding to HBV core protein or a fragment thereof; and a target binding domain operatively attached to the nucleic acid interaction domain, the target binding domain comprising a ligand for binding to a receptor on the surface of the target cell.
In suitable embodiments, the HBV core protein fragment is the protamine domain of HBV core protein or the assembly domain of HBV Core protein.
In an embodiment, the nucleic acid interaction domain is attached to the C-terminus of the target binding domain.
In an embodiment, the chimeric antigen further comprises a second nucleic acid interaction domain operatively attached to the target binding domain, the second nucleic acid interaction domain corresponding to HBV core protein or a fragment thereof.
In further embodiments, the chimeric antigen further comprises an immune response domain comprising an antigenic amino acid sequence, operatively attached to the nucleic acid interaction domain, or to the target binding domain. The immune response domain may provide targeting to a secondary target cell.
Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.
Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures, wherein:
Generally, the present description, with reference to the Figures, provides chimeric antigen compositions for use in the delivery of nucleic acids to a target cell. The compositions include a Nucleic Acid Interaction Domain (NAID), and may be used to encapsulate, tether, or otherwise carry a nucleic acid. The compositions may be particularly useful in the delivery of immunotherapies, as delivery directly to antigen presenting cells, such as dendritic cells, is possible. Targeting to other cell types, for example hepatocytes, is also possible.
Chimigen® VaccinesThe Applicant has previously described chimeric antigens and methods for making same, for example in US 2004/0001853; US 2005/0013828; PCT/CA2004/001469; and US2005/0031628 to George et al, which are each incorporated herein by reference in their entirety. These prior patent applications describe chimeric antigens for use in targeting and activating antigen presenting cells (such as dendritic cells), inducing cellular and/or humoral immune responses, and in breaking host tolerance to chronic and/or viral infections. Also described are chimeric antigens in which an antigen of interest is combined with a xenotypic antibody fragment to improve immunogenicity, broadening the immune response. Chimeric antigens containing Hepatitis B virus (HBV) and Hepatitis C virus (HCV) proteins are also described.
With reference to
Moreover, when the Chimigen® Molecule is produced in insect cells, non-mammalian glycosylation is imparted to the molecule, which facilitates uptake of the Vaccine through host lectin receptors, and increases immunogenicity in the host. The structure of the basic Chimigen® Molecule is able to incorporate any antigen, and may be used to target multiple specific receptors on APC's or other cells of interest.
Various Chimigen® Vaccines have been described by the Applicant for prophylactic and/or therapeutic use, including vaccines directed to Hepatitis C, Hepatitis B, Western Equine Encephalitis, and Influenza.
With reference to US 20050013828, which is incorporated herein by reference, methods for incorporation of HBV proteins into the Chimigen® Vaccine structure are described. Specifically, HBV core protein was placed within the immune response portion 10 of the Chimigen® Molecule.
HBV Core ProteinWith reference to
With reference again to
The immune response domain 10 of the chimeric antigen provides the desired antigenic properties. Typically, the immune response domain includes one or more antigens or antigenic fragments, or one or more recombinant antigens. Specifically, the immune response domain may include an antigen 11, which has been previously recognized as “self” by the host immune system. Further, the immune response domain may include a series of antigens to which immunity is desired.
The immune response domain may include, for example, an antigenic portion of an infectious agent, such as a virus or an obligate intracellular parasite, or of a cancer antigen. Examples of infectious viruses, obligate intracellular parasites and cancer antigens include those described in the published patent application PCT/CA2004/001469. The immune response domain of the chimeric antigen may further include a 6× His tag 12, fused to the one or more antigenic portions.
In certain embodiments, it may be desirable to include an antigen within the immune response domain that may provide some degree of binding to a target cell, to improve the specificity of delivery. For example, HBV S1/S2 binds to liver-derived HepG2 cells, and may be useful in targeting the chimeric antigen to hepatocytes.
Target Binding DomainThe target binding domain 20 binds to or otherwise directs the chimeric antigen to a target cell. Typically, the target binding domain is an antibody fragment capable of binding to a receptor on an antigen presenting cell, such as a dendritic cell, and which enables subsequent transport of the chimeric antigen into the antigen presenting cell by receptor mediated uptake. In addition, the glycosylation of the target binding domain facilitates the receptor-specific binding of the chimeric antigen to C-type lectin receptors on various cell types including antigen presenting cells.
The target binding domain 20 is formed from a xenotypic Fc fragment, which may extend from the C-terminal end to the immune response domain, and is typically recognized by the host as foreign, thereby increasing immunogenicity of the chimeric antigen. The target binding domain may provide customized delivery to a particular receptor on a specific cell type, for example FcγRI, FcγRII and FcγRIII (CD64, 32 and 16), on antigen presenting cells (such as dendritic cells) to bind, internalize, process, and present antigenic epitopes through MHC class I and MHC class II pathways to T and B cells and elicit a broad immune response. In this case, the epitopes ultimately presented by the antigen presenting cell may be epitopes from the immune response domain of the chimeric antigen.
When the chimeric antigen includes a NAID, the nucleic acid associated with the chimeric antigen may be similarly internalized within the antigen presenting cell. Further, the target binding domain may be designed as a ligand to provide selective binding with a specific receptor on a desired target cell type, leading to internalization of the chimeric antigen and associated nucleic acid within the target cell. For example, a target binding domain may be designed to bind Fcγ receptors on dendritic cells, or other antigen presenting cells, or designed to target lectin receptors.
In suitable embodiments, the target binding domain 20 includes a Fc fragment 21, a hinge region 22, and a portion of a CH1 region 23. The chimeric antigen also includes a peptide linker 24 suitable for linking the target binding domain 20 to the immune response domain 10. The target binding domain may include an immunoglobulin heavy chain fragment, and may or may not include a hinge region. Details are provided in PCT/CA2004/001469, for example.
Testing to date has shown that Chimigen® Bionanoparticles bearing a xenotypic Fc domain and carrying siRNA directed to CD86 are able to deliver nucleic acid to dendritic cells and to effect RNAi as evidenced by down-regulation of CD86 in these cells (see examples below). Chimigen® Bionanoparticles were able to effect RNAi and immunomodulation in T cells.
Nucleic Acid Interacting Domain (NAID)The Nucleic Acid Interacting Domain (NAID), is a portion of the presently described chimeric antigen that provides interaction with nucleic acids—encapsulating, sequestering, binding, or otherwise allowing the nucleic acid to be carried by the chimeric antigen to the target cell where it may be internalized upon meeting of the target binding domain with the target cell.
The NAID may be provided by incorporating a HBV core protein sequence within a chimeric antigen structure such as the Chimigen® Molecule. When a sequence corresponding to the assembly domain 31 of HBV core protein 30 is incorporated within the immune response domain 10 or within the target binding domain 20 of the chimeric antigen, the innate encapsulating ability of HBV core protein is retained within the chimeric antigen, enabling aggregation of chimeric antigen molecules. When the protamine domain 32 of the HBV core protein is incorporated within the chimeric antigen structure, the innate nucleic acid binding ability of the HBV core protein is retained within the chimeric antigen. When the entire HBV core protein 30 is present within the chimeric antigen, nucleic acid will be bound and a capsid will form about the nucleic acid. Each of the assembly domain 31 and the protamine domain 32 may be termed a NAID, as each interacts with nucleic acid for the purpose of delivery to a target cell. Specifically, the protamine domain 32 binds nucleic acid, while the assembly domain 31 encapsulates the bound nucleic acid.
Chimeric Antigens for Encapsulating Nucleic AcidHBV core protein 30 or a fragment thereof may be incorporated within the immune response domain of the Chimigen® Molecule structure to enable aggregation about a nucleic acid. When a HBV core fragment is incorporated within the Chimigen® Molecule structure for this purpose, it is preferable that the fragment includes the assembly domain (amino acids 1-143 of SEQ ID NO:2, approx.) of HBV Core. The protamine domain (amino acids 144 to 183 of SEQ ID NO:2, approx.) may also be included. The HBV core sequence or fragment is preferably inserted into the Chimigen® Molecule at the C-terminus or within the immune response domain.
Additional antigens may be added to the N-terminus or C-terminus of the HBV core protein or fragment, or at a suitable location within the HBV core protein or fragment, for example at the immunodominant site between amino acid residues 79 (proline) and 80 (alanine) of HBV core.
Similarly, HBV core or a fragment thereof may be incorporated within the target binding domain of the Chimigen® Molecule, which also enables aggregation of the Chimigen® Molecule about nucleic acids. When a HBV core fragment attached in this manner, for example to the C-terminus of the Chimigen® Molecule, it is preferable that the fragment include at least the assembly domain (amino acids 1-143 of SEQ ID NO:2, approx.). The protamine domain (amino acids 144 to 183 of SEQ ID NO:2, approx.) may also be included.
Aggregation of Chimigen® HBV Core Bionanoparticles about a nucleic acid is shown schematically in
HBV Core Protein 30 or a protamine-like fragment 33 thereof may be incorporated within a chimeric antigen structure to enable direct binding to nucleic acid molecules. When a HBV core protamine-like fragment is incorporated within the Chimigen® Molecule for this purpose, it is preferable that the fragment include a significant portion of the protamine-like domain (eg. amino acids 144-184 of SEQ ID NO:2: ETTVVRRRDRGRSPRRRTP SPRRRRSQSPRRRRSQSR ESQC and provided by SEQ ID NO:17) of HBV Core. The HBV core or protamine-like fragment is preferably included within the immune response domain of the chimeric antigen or at the C-terminus
With reference to
While this particular fusion protein was in fact able to bind nucleic acid, preliminary data indicates that this C-terminal protamine tail and nucleic acid binding location may impede interaction of the target cell receptors with the Fc portion of the chimeric antigen.
Introduction of Nucleic Acid to the Chimeric AntigenThe desired chimeric antigen may first be produced as a fusion protein, for example expressed in insect cells using the baculovirus expression system. The nucleic acid is synthesized separately, and mixed with chimeric antigen to form a chimeric antigen/nucleic acid complex.
When the chimeric antigen is intended to encapsulate nucleic acid, the purified fusion protein is produced, and nucleic acid is added under denaturing conditions. The denaturant is then removed by dialysis or gel filtration, and the chimeric antigen is renatured to form a Chimigen® Molecule/nucleic acid complex. The complex should be sufficiently stable so that, as the target binding domain binds to the receptors on the target cell, the nucleic acid, for example an siRNA, is delivered to the cytosol. The chimeric antigen is then processed through the antigen presentation pathways, while the siRNA interacts with RNA-induced silencing complex (RISC) in the cytosol, resulting in the annealing of the siRNA with the target mRNA and silencing the expression of the gene, via mRNA degradation.
When the chimeric antigen includes a C-terminal protamine tail, the nucleic acid may simply be prepared and added to the purified chimeric antigen.
Nucleic AcidsThe Chimigen® Molecule is designed to bind to and be internalized by a target cell, carrying the associated (encapsulated or bound) nucleic acid molecule into the cytosol of the target cell. Accordingly, based on the nucleic acid to be delivered, an appropriate target cell type is selected, and the target binding domain is designed to bind a specific receptor on the target cell. For most applications, it is desirable to choose a target receptor that is specific to the target cell of interest, and to which an appropriate target binding domain may be specifically bound. Such care in design will minimize the amount of chimeric antigen and nucleic acid required, will improve potency, and also may minimize non-specific off-target effects. It should be noted that targeting of the chimeric antigen molecule to a particular cell type may also be accomplished by specific design of the immune response domain. For example, when HBV S1/S2 protein is used as an antigen within the immune response domain, targeting to hepatocytes is observed.
Nucleic acids for use with the presently described NAIDs include siRNA, dsRNA, shRNA, and plasmids, for example plasmids encoding shRNAs, which form a desirable siRNA sequence in situ. To date, plasmid DNA sequence of up to 5.3 kb have been encapsulated in Chimigen® Molecules.
U.S. Pat. No. 7,078, 196 B2 and U.S. Pat. No. 7,056,704 B2 describe materials and methods for RNAi herein incorporated by reference. U.S. Pat. No. 7,056,704 B2, demonstrates, siRNA-mediated gene silencing in mammalian cells. U.S. Pat. No. 7,056,704 B2 also describes: a preferred structure of siRNA for efficient silencing; methods of preparing dsRNA for use in RNAi; and methods of mediating targeT specific nucleic acid modification, particularly RNAi and/or DNA methylation in a cell or an organism.
RNAi has been used for immune modulation by targeting gene expression in dendritic cells [Hill, J. A., et al (2003) J. Immunol. 171:691-696]. Silencing of SOCS-1 by siRNA was also shown to enhance both antigen presentation by dendritic cells and antigen-specific anti-tumour immunity. In an ex vivo study, dendritic cells showed enhanced antigen-specific anti-tumour immunity when SOCS-1 is silenced in the dendritic cells before their vaccination with a cancer antigen [Shen, T. (2004) Nature Biotech 22(12): 1546-1553]. In an in vivo study in mice, silencing of SOCS-1 induced an enhanced HIV-1 CD8+ and CD4+ T cell response as well as antibody responses [Song, X-T. et al (2006) PLoS Med 3:1-18].
Nucleic acid molecules for silencing SOCS-1 expression were delivered using the presently described Chimigen® Molecules, as described in the Examples section. Many other target genes would be suitable for delivery with the presently described chimeric antigens, as will be apparent to the reader upon reading of the present description combined with knowledge in the art.
Methods of Utilizing Chimeric Antigens and Nucleic AcidsAs discussed above, chimeric antigens incorporating a Nucleic Acid Interaction Domain may be administered to a cell, tissue, target, or host along with nucleic acids. Such co-administered chimeric antigens and nucleic acids may be used to break host tolerance to an antigen, enhance immune responses, or to generate other desirable effects in antigen presenting cells or other cell types of interest. It is also contemplated that a chimeric antigen composition without a nucleic acid interaction domain may simply be co-administered with a nucleic acid(s). While this may provide some degree of efficacy, the co-administration of nucleic acid with chimeric antigen incorporating a NAID will provide a more specific and desirable effect, as both the chimeric antigen and nucleic acid are provided to the same cell or group of cells simultaneously.
The chimeric antigen (which may be a Chimigen® Molecule) will allow generation of a cellular and/or humoral immune response to the target antigen, which may have previously been recognized as “self” during a chronic infection, and which will then become recognized as “foreign.” Accordingly, the host's immune system will mount a cytotoxic T lymphocyte response to eliminate the target antigen-infected cells. At the same time, antibodies produced by the host in response to the administered antigen will bind to the target antigen or infectious agent and remove it from the circulation or block binding of the target antigen or infectious agent to host cells. Accordingly, administration of a chimeric antigen along with an siRNA against a specific gene may induce a broad immune response in hosts who have chronic infections that are unrecognized or otherwise tolerated by the host immune system. Such administration may be used to break tolerance to a target antigen in a host who is chronically infected with an infectious agent, or who has a cancer or another immune disorder. For example, when the siRNA is designed to silence a viral gene product, such treatment may decrease viremia through RNAi, which may assist in clearing the infection. This will also be aided by the immune response generated by administration of the Chimigen® Vaccine.
While the Chimigen® Molecule is useful in eliciting or enhancing an immune response, the siRNA further silences a specific host gene. For example, the siRNA may further enhance the immune response to the Chimigen® Molecule by silencing the expression of a target gene, such as a gene encoding an inhibitor of cytokine signaling, for example silencing SOCS gene expression, and reducing SOCS-mediated inhibition of cytokine signaling.
The chimeric antigen and nucleic acid may be encapsulated together within a liposome. That is, the compositions of the present invention may be formulated for delivery either encapsulated in or attached to a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Alternatively, compositions of the present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles.
The Chimeric antigens and nucleic acids may be used for activating antigen presenting cells or enhancing antigen presentation in an antigen presenting cell (APC) in vivo or ex vivo. Antigen presenting cells contacted with the chimeric antigen and nucleic acid will result in binding and internalization of the chimeric antigen by APCs, activating the APCs and enhances antigen presentation of more than one epitope. This multi-epitopic response can include presentation of one or more epitopes of the immune response domain and/or presentation of one or more epitopes of the target binding domain.
An immune-treatable condition may be treated by co-administration, to a subject in need thereof, a therapeutically effective amount of a chimeric antigen and siRNA. Examples of immune-treatable conditions include viral infections such as HBV or HCV; parasitic infections; and cancers. For the treatment of HBV, suitable antigens for incorporation into the immune response domain of the Chimigen® Molecule may include at least one antigenic portion of a protein selected from the group consisting of a HBV Core protein, a HBV S protein, a HBV S1 protein, a HBV S2 protein, HBV Polymerase protein, HBV X protein, and/or combinations of same. For the treatment of HCV, the immune response domain may include at least one antigenic portion of a protein selected from the group consisting of HCV Core protein, E1 protein, E2 protein, NS2 protein, NS3 protein, NS4A protein, NS4B protein, NS5A protein, NS54B protein and/or combinations of same. HBV core protein or a fragment thereof may also be included within the immune response domain in order to provide encapsulation and/or binding of nucleic acid.
The amplitude of the immune response can be measured, for example, (i) by the amount of antigen-specific antibody present in the subject; (ii) by the amount of IFN-γ secreted by T cells in response to being exposed to APC loaded with the chimeric antigen or immune response domain alone; or (iii) by the amount of antigen-specific CD8+ T cells elicited in response to being exposed to APCs loaded with the chimeric antigen or immune response domain alone.
The chimeric antigen can be evaluated for its efficacy in generating an immune response by presenting the chimeric antigen to DCs ex vivo or in vivo. The DCs process and present the chimeric antigen to T cells, which are evaluated for proliferation of T cells and for the production of IFN-γ as markers of T cell response. Specifically, in the ex vivo situation, peripheral blood mononuclear cells (PBMCs) are isolated from naive donors, and are used for producing dendritic cells (DCs) and isolation of T cells. Activation of the T cells by the DCs is evaluated by measuring markers, e.g. IFN-γ levels, by a known procedure [See, e.g., Berlyn, et al., (2001) Clin. Immunol. 3:276-283]. A marked increase in the percentage of T cells induced to produce IFN-γ ex vivo will help predict efficacy in vivo. In the case of the in vivo situation, the chimeric antigen is directly introduced parenterally in the host where available DCs and other APCs have the capacity to interact with antigens and to process them accordingly.
The chimeric antigens and nucleic acids may also be used in prophylactically or therapeutically vaccinating a subject against an infection. The bifunctional nature of the molecule helps to target the antigen to APCs, e.g. DCs, making it a unique approach in the therapy of chronic infectious diseases by specifically targeting the APCs with the most effective stoichiometry of antigen to antibody. Such vaccines may be useful in the development of vaccines against infections caused by HBV, HCV, human immunodeficiency virus, human papilloma virus, herpes simplex virus, alphaviruses, influenza viruses, other types of viruses, obligate intracellular parasites and may also be applicable to all autologous antigens in diseases such as cancer and autoimmune disorders. The administration of these fusion proteins can elicit a broad immune response from the host, including both cellular and humoral responses. Thus, they can be used as therapeutic vaccines to treat subjects that are immune tolerant to an existing infection, in addition to being useful as prophylactic vaccines to immunize subjects at risk for developing a particular infection.
EXAMPLES Example 1 Cloning and Expression of a Chimeric Antigen with a C-Terminal Protamine TailStep 1. Cloning—DNA encoding a target binding domain (TBD) containing a 5′ Not I site and a 3′ Xba I site was produced by PCR using previously generated pFastBacHTa-TBD as template with unique primers that add the respective restriction enzyme sites. The primers used were;
The PCR fragment was isolated, digested with Not I and Xba I and cloned into a Not I/Xba I digested pFastBacHTa-gp64 plasmid.
For HBV Core protamine tail, the sequence was obtained by PCR of previously produced plasmid pFastBacHTa HBV Core-TBD as template using primers that add a unique Xba I site to the 5′ end and a unique Hind III site to the 3′ end. The primers used were:
The PCR product was isolated, digested with Xba I/Hind III and cloned into a Xba I/Hind III sites of the re-cloned TBD plasmid, described above, to create pFastBacHTa-64 TBD-HBV Core protamine.
A chimeric antigen containing HCV NS5A as the antigen of the immune response domain was made by digesting the plasmid pFastBacHTa-gp64 TBD-HBV Core protamine with Xba I and Hind III and the TBD-HBV Core protamine tail fragment was isolated. The plasmid pFastBacHTa-gp64 HCV NS5A-TBD was digested with Xba I and Hind III and the TBD-HBV Core protamine tail fragment was cloned in.
Chimeric antigens encompassing the HBV Core protamine-tail domain located at various positions in the molecule were designed as follows (see
Step 2. Production of Recombinant Baculovirus for the Expression of Chimeric Antigen
The expression of chimeric antigen NS5A-TBD-HBV Core protamine domain protein was performed using a baculovirus expression system in Sf9 insect cells. To generate recombinant baculoviruses encoding the chimeric antigen for expression, the Bac-To-Bac system (Invitrogen, Carlsbad, Calif., USA) was used. This system uses site-specific transposition with the bacterial transposon Tn7 to generate recombinant baculovirus in E. coli strain DH10Bac. The pFastBacHTa-64 HCV NS5A-TBD-HBV Core protamine plasmid has mini-Tn7 elements flanking the cloning site. The plasmid was used to transform E. coli strain DH10Bac, which has a baculovirus shuttle plasmid (bacmid) containing the attachment site of Tn7 within a LacZα gene. Transposition disrupted the LacZα gene so that only recombinant bacmids produced white colonies on plates containing X-gal/IPTG, and are easily selected for. The advantage of using transposition in E. coli is that single colonies contain only recombinant bacmids. The recombinant bacmid was isolated using standard plasmid isolation protocols and was used for the transfection of Sf9 insect cells to generate baculoviruses that express the chimeric antigen. The efficiency of the transfection was verified by checking for production of baculovirus DNA by PCR to screen for the inserted gene of interest. The expression of the heterologous protein in the transfected Sf9 cells was verified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots using the 6× His tag monoclonal antibody or anti mouse IgG1 (Fc specific) antibody as the probe. Once the production of recombinant baculovirus and the expression of chimeric protein were confirmed, recombinant virus was amplified to produce a concentrated stock of baculovirus.
Step 3. Production of Chimeric Antigen in Insect Cells
High titre recombinant baculovirus stocks were used to infect insect cells (eg. Sf9, High Five™). The infection is optimized, with respect to the MOI of the baculovirus, the period of the infection and the viability of the host cells. It is important to keep the viability of the insect cells at high levels to prevent degradation of the recombinant protein. The expressed proteins are purified by protocols developed for 6× His tagged proteins using affinity chromatography methods.
Some of the Chimigen® Molecules used in testing include:
- Chimigen® HBV Core Vaccine—Chimigen® Molecule structure with HBV Core protein present in the immune response domain 10 as the antigen 11 and as a NAID. Chimigen® HBV S1/S2 Core Vaccine—Chimigen® Molecule structure with HBV core protein and HBV S1/S2 protein in immune response domain. The structure of this Vaccine is shown in
FIG. 4 a, and the sequence is shown inFIG. 4 b. - Chimigen® HBV Core Protamine Tail—Chimigen® Molecule structure with HBV Core protein present in the immune response domain 10 as the antigen 11 and a NAID, and with HBV Core protamine domain 32 also located at the C-terminus of the Chimigen® Molecule as a NAID.
- Chimigen® HBV NS5A Protamine Vaccine—Chimigen® Molecule structure with HCV NS5A protein present in the immune response domain 10 as the antigen 11, and with HBV Core protamine domain 32 located at the C-terminus of the Chimigen® Molecule as a NAID.
- Chimigen® HBV Protamine tail HCV NS5A vaccine—Chimigen® Molecule structure with both the protamine domain 32 of HBV Core and HCV NS5A in the immune response domain 10.
Chimigen® HBV Core Vaccine and Chimigen® HBV S1/S2 Core Vaccine were visualized using Tapping Mode Atomic Force Microscopy (TM-AFM). The images generated are shown in
SureSilencing shRNA plasmid was mixed with Chimigen HBV S1/S2 Core Vaccine under denaturing conditions. After removal of the denaturing conditions, encapsulation was evaluated by DNase treatment and PCR amplification of GFP DNA. The shRNA vector plasmid and results are shown in
Binding of Chimigen® HBV S1/S2 Core Vaccine to immature DCs was investigated. Vaccine at 1-50 mg/ml was added for 1 hr at 4° C. to two day cultured PBMC-derived immature DCs. Bound vaccine was detected using anti-mouse IgG1-biotin and SA-PECy5 by flow cytometry. As shown in
Binding of Chimigen® HBV S1/S2 Core Vaccine to the liver cell line HepG2 was investigated. Vaccine at 1-50 μg/ml was added for 1 hr at 4° C. to HepG2 cells, and bound vaccine detected using anti-mouse IgG1-biotin and SA-PECy5 by flow cytometry. As shown in
Binding of Chimigen® HBV S1/S2 Core Vaccine with encapsulated shRNA plasmid to immature DCs was investigated. Vaccine at 1-50 μg/ml with and without encapsulated shRNA plasmid was added for 1 hr at 4° C. to two day cultured PBMC-derived immature DCs. Vaccine binding was detected using anti-mouse IgG1-biotin and SA-PECy5 by flow cytometry. As shown in
Chimigen® HBV S1/S2 Core Vaccine was combined with SOCS1 shRNA plasmids (Thermo Fisher Scientific) or with a control plasmid.
Chimigen® HBV S1/S2 Core Vaccine was combined with SOCS1 siRNA (commercially available). For non-targeting control siRNA, a pool of four double stranded RNAs was provided:
For SOCS siRNA, a pool of four double stranded RNAs. The sense sequences were as follows:
Binding of Chimigen® HBV S1/S2 Core Vaccine with encapsulated siRNA to immature DCs was investigated. Chimigen® HBV S1/S2 Core Vaccine (1-50 μg/ml) with and without encapsulated siRNA (GAPDH) was added for 1 hr at 4° C. to two day cultured PBMC-derived immature DCs. Vaccine binding was detected using anti-mouse IgG1-biotin and SA-PECy5 by flow cytometry. Vaccine was either encapsulated with GAPDH siRNA or was incubated with GAPDH for 60 min at room temperature (6:1 mole ratio of siRNA:vaccine). As shown in
Chimigen® HCV NS5A-Protamine Tail Vaccine was combined with CD86 siRNA. The vaccine was incubated with CD86siRNA for 1 hour at room temperature at a 6:1 molar ratio.
Example 7 Antigen Presentation AssayPBMC-derived monocytes were differentiated to immature dendritic cells, which were then loaded with vaccine; shRNA plasmid; or vaccine with shRNA plasmid (SOCS1 or non-targeting). T cells were isolated from autologous PBMC's and cultured with antigen-loaded dendritic cells. T cells were restimulated after 11 days of culture.
T cells were investigated for their functional response to DCs loaded with Chimigen® shRNA plasmid encapsulated Vaccine. PBMC-derived monocytes were differentiated to immature DCs which were then loaded with vaccine, shRNA plasmid, or Chimigen® HBV S1/S2-Core Vaccine encapsulated with shRNA plasmid (SOCS1 or non-targeting). T cells were isolated from autologous PBMCs and cultured with antigen-loaded DCs. Following 11 days of culture, T cells were re-stimulated with antigen-loaded DCs. For these experiments the DCs were not matured and exogenous IL-2 was not added to cell cultures.
IFN-γ secretion was measured by ELISA after one and two stimulations. Results are shown in
Following one and two stimulations, the production of IFN-γ in the T cell cultures was assessed by ELISA. As shown in
IFN-γ and TNF-α expression in CD8+ and CD4+ T cells was measures by intracellular cytokine labelling after two stimulations. Results are shown in
Six hours following a second stimulation, the production of IFN-γ and TNF-α in CD8+ and CD4+ T cells was assessed by intracellular cytokine labelling with detection by flow cytometry. As shown in
An evaluation of the relative number of T cells in cultures treated with vaccine versus encapsulated vaccine is shown in
Immature dendritic cells were loaded with Chimigen® HBV S1/S2-Core Vaccine, CD86 siRNA, non-targeting siRNA, Chimigen® HBV S1/S2-Core Vaccine and CD86 siRNA, or Chimigen® HBV S1/S2-Core Vaccine and non-targeting siRNA. The DCs were then matured with LPS and assessed for CD86 expression by flow cytometry. Results are shown in
Chimigen® HBV Core Protamine Tail Vaccine was prepared by incorporation of the protamine-like domain of HBV core protein within the target binding domain. Specifically, the protamine-like domain was incorporated at the C-terminus of the target binding domain and the HBV NS5A antigen was incorporated within the immune response domain.
Example 11 Antigen Presentation AssayT cells were investigated for their functional response to DCs loaded with Chimigen® NS5A Protamine Tail Vaccine with and without SOCS1 or non-targeting siRNA. PBMC-derived monocytes were differentiated to immature DCs which were then loaded with vaccine, siRNA, or Chimigen® HCV NS5A Protamin Tail Vaccine encapsulated with siRNA (SOCS1 or non-targeting). T cells were isolated from autologous PBMCs and cultured with antigen-loaded DCs. Following 11 days of culture, T cells were re-stimulated with antigen-loaded DCs. For these experiments the DCs were not matured and exogenous IL-2 was not added to cell cultures.
Following one and two stimulations, the production of IFN-γ in the T cell cultures was assessed by ELISA. As shown in
Six hours following a second stimulation, the production of IFN-γ and TNF-α in CD8+ and CD4+ T cells was assessed by intracellular cytokine labelling with detection by flow cytometry. As shown in
An approximation of the relative number of T cells in cultures treated with vaccine versus vaccine and siRNA is shown in
Immature dendritic cells were loaded with Chimigen® HCV NS5A Protamine Tail Vaccine, CD86 siRNA, non-targeting siRNA, Chimigen® NS5A Protamine Tail Vaccine and CD86 siRNA, or Chimigen® NS5A Protamine Tail Vaccine and non-targeting siRNA. The DCs were then matured with LPS and assessed for CD86 expression by flow cytometry. Results are shown in
Binding of Chimigen® HBV S1/S2 Core Vaccine with biotin-labelled CD86 siRNA to immature DCs was investigated. Vaccine, vaccine and biotin-labelled CD86 siRNA, or CD86 siRNA was added for 1 hr at 4° C. to two day cultured PBMC-derived immature DCs. Binding was detected using SA-PECy5 by flow cytometry. As shown in
Internalization of Chimigen® HBV S1/S2 Core Vaccine with biotin-labelled CD86 siRNA to immature DCs was investigated. Vaccine, vaccine and biotin-labelled CD86 siRNA, or CD86 siRNA was added for 1 hr at 4° C. and then 2 hr at 37° C. to PBMC-derived immature DCs (2 day cultured). Binding and internalization was detected by FACS after addition of SA-PECy5 with and without prior fixation and permeablization. As shown in
Encapsulated or naked siRNA was digested with Benzonase for 10 minutes at RT. The digested siRNA was separated on a SDS-PAGE gel containing 1 mM EDTA and was stained with 0.2% methylene blue. As shown in
The above-described embodiments of the present invention are intended to be examples only. Alterations, modifications and variations may be effected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto.
Claims
1.-38. (canceled)
39. A fusion protein comprising:
- (a) a target binding domain for binding to a receptor on the surface of the target cell, the target binding domain comprising an antibody fragment comprising the hinge region, at least a portion of a CH1 region and an Fc fragment comprising a CH2 and a CH3 domain;
- (b) an immune response domain operatively attached to the target binding domain;
- (c) a first nucleic acid interaction domain corresponding to a HBV core protein or a fragment thereof operatively attached to the immune response domain; and
- (d) a second nucleic acid interaction domain operatively attached at the C-terminus of the target binding domain corresponding to the HBV core protein or a fragment thereof having at least the protamine domain of the HBV core protein.
40. The fusion protein of claim 39, wherein the target binding domain is a xenotypic antibody fragment.
41. The fusion protein of claim 39, wherein the immune response domain comprises an antigenic amino acid sequence.
42. The fusion protein of claim 39, wherein the immune response domain provides targeting to a secondary target cell.
43. The fusion protein of claim 39, wherein the first nucleic acid binding domain comprises a fragment of the assembly domain of the HBV core protein and wherein the second nucleic acid binding domain comprises a fragment of the assembly domain and the protamine domain of the HBV core protein.
44. The fusion protein of claim 39, wherein the first nucleic acid binding domain comprises at least amino acids 1 to 78 of the HBV core protein (SEQ ID NO:2) and wherein the second nucleic acid binding domain comprises at least amino acids 81 to 183 of the HBV core protein (SEQ ID NO:2).
Type: Application
Filed: Dec 19, 2013
Publication Date: Jul 3, 2014
Applicant: Akshaya Bio Inc. (Edmonton)
Inventors: Rajan George (Edmonton), Antoine Noujaim (Edmonton)
Application Number: 14/134,867
International Classification: C07K 16/28 (20060101); C07K 14/005 (20060101);