Patent Grant
9688991
Throughout this application various publications, patents, patent application publications and books are referred to. Full citations for the publications may be found at the end of the specification. The disclosures of the publications, patents, patent application publications and books are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Poor immunogenicity of conventional protein vaccines, in particular an inability to elicit robust T cell-mediated immunity, has limited their use as vaccines targeting diverse diseases including viral infections and cancers. One approach, which has recently been utilized to activate T cell responses, is targeting of antigen to dendritic cells (DCs), a cell type that is pivotal for eliciting T cell activation. Indeed, DC-targeted approaches have recently attracted significant research interest and are rapidly becoming important therapeutic approaches (1-4). DCs possess the capability of processing self and foreign antigens resulting in presentation of antigen to its cognate T cell receptor. Targeting antigen uptake to DCs via specific DC-enriched receptors has been shown to enhance antigen presentation on major histocompatibility complex (MHC) class I and II molecules by as much as 1000-fold and 50-fold, respectively (5). Depending on the antigenic stimulus, DCs can induce tolerance or activate the immune system (6), making them important targets in the development of novel treatments to autoimmune diseases, viral infections and cancer. Targeting antigens to DCs most often involves coupling the antigen of interest to a delivery agent specific for a readily endocytosed cell surface receptor on the DCs.
Typically, targeted antigen delivery has made use of antibodies as the targeting agent. However, large size and immunogenicity have presented problems in this approach.
The present invention addresses the need to providing improved targeting methods by providing aptamers for preferential vaccine targeting.
A composition is provided comprising an oligonucleotide aptamer conjugated to an antigen, wherein the aptamer is directed against a cell-surface target of an antigen-presenting cell.
A method is provided of presenting an antigen to a dendritic cell, comprising contacting the dendritic cell with a composition comprising an oligonucleotide aptamer conjugated to the antigen, wherein the aptamer is directed against a cell-surface target of a dendritic cell.
A method is also provided of eliciting an immune response in a subject comprising administering to the subject a composition comprising an oligonucleotide aptamer conjugated to an antigen, wherein the aptamer is directed against a cell-surface target of an antigen-presenting cell, in an amount effective to elicit an immune response.
Additional objects of the invention will be apparent from the description which follows.
6. Mutant forms of min.2 bind DEC205 with lower affinity and are less effective at cross-presentation when compared with min.2. (a) Schematic representation showing min.2 mutation sites. The conserved heptamer is underlined. Mutations are highlighted in red (b) Uptake of fluorescently labeled mutant aptamers by CHO-DEC205 following a 1 hour incubation with 20 nM conjugate as measured by flow cytometry. (c-e) Activation of adoptively transferred OT-I cells following injection of 20 μg multimerized aptamer:OVA or control constructs was determined by their ability to proliferate, as measured by CFSE dilution (c) and intracellular cytokine staining (d, e). Multimerized aptamers conjugated to OVA were injected into B6.SJL-ptprc mice that had received 106 CFSE-labeled OT-I cells. Proliferation and cytokine production of OT-I cells was determined 3 days later by flow cytometry. The percentage of TCRβ+CD45.2+ cells present in the total splenic population (OT-I cells) is reported on each plot. Production of IFNγ (d) and IL2 (e) by OT-I cells. The percentage of OT-I cells that have undergone 3 or more divisions and are producing IFNγ (d) and IL2 (e) is indicated on each dot plot. Data shown are representative of three experiments.
A composition is provided comprising an oligonucleotide aptamer conjugated to an antigen, wherein the aptamer is directed against a cell-surface target of an antigen-presenting cell.
In an embodiment, the aptamer comprises an oligoribonucleotide.
In an embodiment, the aptamer is PEGylated. In an embodiment, the PEG is 200-400 daltons, 400-800 daltons, 800-1000 daltons, 1000-2000 daltons, 2000-5000 daltons, or 5000-10,000 daltons. In an embodiment, the PEG is in the form of a (succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester. In an embodiment, the PEG is in the form of a (succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol]ester), a (succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester), a (succinimidyl-[(N-maleimidopropionamido)-hexaethyleneglycol]ester), a (succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol]ester), or a succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol]ester).
In an embodiment, the aptamer is multimerized. In an embodiment, the multimerized aptamer comprises 2, 3 or 4 aptamers.
In an embodiment, the antigen-presenting cell is a dendritic cell. In an embodiment, the cell is a CD8alpha+ dendritic cell.
In an embodiment, the aptamer comprises the sequence set forth in SEQ ID NO:1 (GGGAGGUGUGUUAGCACACGAUUCAUAAUCAGCUACCCUCCC). In an embodiment, the aptamer comprises the sequence set forth in SEQ ID NO:1 in the description hereinbelow.
In an embodiment, the aptamer further comprises a 3′ inverted dT.
In an embodiment, the antigen is conjugated to the 5′ end of the aptamer.
In an embodiment, the antigen is conjugated to the aptamer by a thioether or by a disulfide bond.
In an embodiment, the cell-surface target is a DEC-205 receptor molecule.
In an embodiment, the cell-surface target is a human macrophage mannose receptor.
In an embodiment the aptamer-antigen conjugate further comprises one or more additional oligonucleotide aptamers conjugated to the antigen, wherein the aptamers are directed against the cell-surface target of an antigen-presenting cell.
In an embodiment, the antigen is a vaccine molecule.
In an embodiment, the antigen is a tumor antigen, an autoantigen, or a component of a pathogen.
In an embodiment, the composition further comprises an immunological adjuvant.
In an embodiment, the cell surface target is a DEC-205 receptor having the sequence set forth in GenBank: AAC17636.1.
A method is provided of presenting an antigen to a dendritic cell, comprising contacting the dendritic cell with a composition comprising an oligonucleotide aptamer conjugated to the antigen, wherein the aptamer is directed against a cell-surface target of a dendritic cell.
A method is also provided of eliciting an immune response in a subject comprising administering to the subject a composition comprising an oligonucleotide aptamer conjugated to an antigen, wherein the aptamer is directed against a cell-surface target of an antigen-presenting cell, in an amount effective to elicit an immune response.
In an embodiment of the methods or compositions, the antigen-presenting cell is a dendritic cell. In an embodiment, the cell is a CD8alpha+ dendritic cell.
In an embodiment of the methods or compositions, the aptamer comprises an oligoribonucleotide. In an embodiment, the aptamer is multimerized. In an embodiment, the multimerized aptamer comprises 2, 3 or 4 aptamers. In an embodiment, the aptamer is PEGylated. In an embodiment, the PEG is 200-400 daltons, 400-800 daltons, 800-1000 daltons, 1000-2000 daltons, 2000-5000 daltons, or 5000-10,000 daltons. In an embodiment, the PEG is in the form of a (succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester. In an embodiment, the PEG is in the form of a (succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol]ester), a (succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester), a (succinimidyl-[(N-maleimidopropionamido)-hexaethyleneglycol]ester), a (succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol]ester), or a succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol]ester).
In an embodiment of the methods or compositions, the antigen is a protein, polypeptide, peptide or carbohydrate. In an embodiment, the antigen is a vaccine molecule. In an embodiment, the antigen is a tumor antigen, an autoantigen, or a component of a pathogen. In an embodiment, the cell-surface target is a receptor molecule. In an embodiment, the cell-surface target is a DEC-205 receptor molecule. In an embodiment, the cell-surface target is a human macrophage mannose receptor. In an embodiment, the composition further comprises an immunological adjuvant. In an embodiment, the cell surface target is a DEC-205 having the sequence set forth in GenBank: AAC17636.1.
In an embodiment of the methods or compositions, the antigen is a peptide of less than 50 amino acids. In an embodiment the antigen is a peptide of less than 45 amino acids. In an embodiment the antigen is a peptide of less than 40 amino acids. In an embodiment the antigen is a peptide of less than 35 amino acids. In an embodiment the antigen is a peptide of less than 30 amino acids. In an embodiment the antigen is a peptide of less than 25 amino acids. In an embodiment the antigen is a peptide of less than 20 amino acids. In an embodiment the antigen is a peptide of less than 15 amino acids. In an embodiment the antigen is a peptide of less than 10 amino acids. In an embodiment the antigen is a peptide of at least 5 amino acids.
In an embodiment of the methods or compositions, the cell surface target is a DEC-205 comprising the following amino acid sequence, or comprising an amino acid sequence having 95% or greater identity to the following amino acid sequence:
In an embodiment of the methods or compositions, the aptamer is less than 60, less than 50, less than 40 or less than 30 nucleotides in length.
Aptamers, unless otherwise specified, are RNA or DNA molecules, or comprise both ribonucleotide residues and deoxyribonucleotide residues, and are generally generated from large combinatorial libraries (1014-1015) of nucleic acids. In an embodiment, the aptamer comprises RNA. This may be done by a process of in vitro selection or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) which targets a specific protein or molecular target. They are generated though a process that relies on binding. Aptamers are thus nucleic acids (oligonucleotides, often oligoribonucleotides—a ribonucleic acid aptamer) which bind a specific protein or molecular target, typically with nanomolar or subnanomolar affinity. Generally, aptamers discriminate against molecules closely related to the target molecule.
The aptamers of the invention may comprise nucleosides. A “nucleoside” as used herein is a glycosylamine consisting of a base bound to a ribose or deoxyribose sugar via a beta-glycosidic linkage. Examples include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (—CH2—OH), producing nucleotides, which are the molecular building blocks of DNA and RNA. Nucleosides can be produced by de novo synthesis pathways, particularly in the liver, but they are more abundantly supplied via ingestion and digestion of nucleic acids in the diet, whereby nucleotidases break down nucleotides (such as the thymine nucleotide) into nucleosides (such as thymidine) and phosphate.
The aptamers of the invention may comprise nucleoside analogs. A “nucleoside analog” is a nucleoside structurally similar to the naturally occurring residues in RNA and DNA, used in medicine and in molecular biology, and which can be incorporated, e.g. chemically, into an oligonucleotide or nucleic acid by formation of a phosphodiester bond or equivalent with one or two residues of the residue chain depending on whether the nucleoside analog is in a terminal or intra-chain position, respectively. Nucleic acids are chains of nucleotides, which are composed of three parts: a phosphate backbone, a pucker-shaped pentose sugar, either ribose or deoxyribose, and one of five nucleobases. A nucleoside analogue differs from a nucleoside by having any one or more of its hydroxyl, base or sugar groups altered, as long as the alteration does not prevent the nucleoside analogue from being incorporated into an oligonucleotide such as an aptamer, internalizing nucleic acid or tumor-homing nucleic acid. In an embodiment of the invention the nucleoside analogue(s) are one or more of the following: a deoxyadenosine analog, a deoxycytidine analog, a deoxyguanosine analog, a (deoxy-)thymidine analog, and/or a deoxyuridine analog. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking proprieties. The ribonucleic acid aptamers of the invention may thus comprise nucleoside analogs.
Nucleoside analogs as envisaged in the current invention include, but are not limited to, cytosine arabinoside, fludarabine, cladribine, acyclovir, 2′,3′-dideoxyinosine; 9-β-D-ribofuranosyladenine; β-arabinofuranosylcytosine; arabinosylcytosine; 4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one; 2′,3′-dideoxy-3′-thiacytidine; 2′-3′-dideoxycytidine; {(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]cyclopent-2-en-1-yl}methanol; 2-Amino-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylidenecyclopentyl]-6,9-dihydro-3H-purin-6-one; 2′-3′-didehydro-2′-3′-dideoxythymidine; 1-(2-deoxy-β-L-erythro-pentofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione; 1-[(2R,4S,5S)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione; 1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-iodo-1,2,3,4-tetrahydropyrimidine-2,4-dione; 1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-(trifluoromethyl) pyrimidine-2,4-dione; 5-Fluoro-2′-deoxycytidine; 5-Fluorodeoxycytidine; Floxuridine (5-Fluoro-1-[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-pyrimidine-2,4-dione); 4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)pyrimidin-2(1H)-one; 2′,2′-difluoro-2′-deoxycytidine; (8R)-3-(2-deoxy-β-D-erythro-pentofuranosyl)-3,4,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol.
The ribonucleic acid aptamers of the invention may comprise, on their constituent nucleotides, 2′ modifications. Preferred modifications are 2′ F on pyrimidines and 2′ H or 2′ OMe on purines.
In an embodiment, of the methods, compositions and aptamers described herein, the aptamer comprises SEQ ID NO:1 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional nucleotide residues at the 3′ end thereof, and, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional nucleotide residues at the 5′ end thereof.
The present invention provides aptamers that are ribonucleic acid or deoxyribonucleic acid or mixed ribonucleic acid and deoxyribonucleic acid or which are analogs thereof, especially via modification of 2′ groups on purines and/or pyrimidines. Aptamers of the invention may be single stranded. In some embodiments, the aptamers of the invention comprises at least one chemical modification (other than the included nucleoside analog(s), if included). In some embodiments, the chemical modification is selected from the group consisting of: a chemical substitution at a sugar position; a chemical substitution at a phosphate position; and a chemical substitution at a base position, of the nucleic acid. In other embodiments, the chemical modification is selected from the group consisting of: incorporation of a modified nucleotide, 3′ capping, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, and incorporation of phosphorothioate into the phosphate back bone. In a preferred embodiment, the non-immunogenic, high molecular weight compound is polyalkylene glycol, more preferably polyethylene glycol. In an embodiment the chemical modification is an inverted thymidine cap. In an embodiment the chemical modification is once or more phosphorothioate backbone modification(s). In a preferred embodiment, the aptamer is modified at a 2′ group of one or more purines thereof and/or at a 2′ of one or more pyrimidines thereof.
Aptamers of the present invention can be administered by any appropriate route or means, including systemically, topically, parentally or enterally. In non-limiting examples, administration is by subcutaneous injection (aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker et al., J. Chromatography B. 732: 203-212, (1999)), intravenously, intranasally (lower and/or upper epithelia), or by direct injection into the desired body system, tissue or organ. Any vaccine dosing schedule may be employed, including those used in the art for the administration of the aptamer-antigen conjugates of the present invention.
The present invention additionally provides a pharmaceutical composition comprising a therapeutically effective amount of the aptamer-antigen composition and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier is preferably compatible with the aptamer-antigen conjugate and not significantly deleterious to the subject. Examples of acceptable pharmaceutical carriers include liposomes (which may encapsulate the aptamer-antigen conjugate, or which may be attached the aptamer-antigen conjugate) saline, carboxymethylcellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate, sucrose, starch, talc, and water, among others. Formulations of the pharmaceutical composition may conveniently be presented in unit dosage and may be prepared by any method known in the pharmaceutical art. For example, the aptamer, or aptamer conjugate or aptamer-liposome composition may be brought into association with a carrier or diluent, as a suspension or solution. Optionally, one or more accessory ingredients, such as buffers, flavoring agents, surface-active ingredients, and the like, may also be added. The choice of carriers will depend on the method of administration. The pharmaceutical composition can be formulated for administration by any method known in the art, including but not limited to, intravenously and orally. In one embodiment, there may be a number of active pharmaceutical ingredients in the formulation or composition aside from the aptamer-antigen conjugate. In this embodiment, the other active pharmaceutical ingredients in the formulation or composition must be compatible with the aptamer.
The aptamers of the invention can be chemically synthesized if desired, or transcribed from appropriate encoding nucleic acids and modified during or after sequence synthesis if desired. The aptamers of the present invention, with or without included one or more nucleoside analogs, can be stored in a variety of forms, including as lyophilized powders.
The term “antigen” means all, or parts, of a protein, polypeptide, peptide or carbohydrate, and/or vaccine molecule capable of causing an immune response in a vertebrate, preferably a mammal. In an embodiment, the antigen is a protein, polypeptide or peptide. In a further embodiment, the protein, polypeptide or peptide may be glycosylated. In an embodiment, the antigen is a vaccine molecule. A “vaccine molecule” as used herein is a chemical molecule capable of eliciting an immune response in an animal, preferably a mammal, when administered thereto as a vaccine. In non-limiting examples, the vaccine molecule is an intact but inactivated (non-infective) or attenuated form of a biological pathogen, a purified or isolated component of a biological pathogen that is immunogenic (e.g., an outer coat protein of a virus), a toxoids (e.g. a modified tetanospasmin toxin of tetanus which is non-toxic itself).
In an embodiment of the methods, T cell stimulation is effected. In an embodiment of the methods, CD8+ T cell stimulation is effected.
In an embodiment, the immune response comprises a Th1 response. In an embodiment, the immune response comprises an adaptive immunity response. In an embodiment, the immune response comprises interferon gamma production. In an embodiment, the immune response comprises interleukin-2 production. In an embodiment, the immune response comprises T-cell proliferation. In an embodiment, the composition is administered in an amount sufficient to induce cytokine release by dendritic cells. In an embodiment, the method evokes an immune response comprising presentation of the antigen as a component of an MHC-I or MHC-II conjugate. n an embodiment, the immune response is an adaptive immunity response.
Antigen-presenting cells are well-known in the art, and are generally considered as cells of the immune system that displays foreign antigen complexes with major histocompatibility complex (MHC) on their surfaces. Examples include dendritic cells, macrophages and B-cells. In a preferred embodiment of the invention, the antigen-presenting cell is of the same species as the subject.
As used herein “and/or”, for example as in option A and/or option B, means the following embodiments: (i) option A, (ii) option B, and (iii) the option A plus B, and any subset of such options, including only one option.
The subject may be any subject. Preferably, the subject is a mammal More preferably, the subject is a human.
All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Where a numerical range is provided herein, it is understood that all numerical subsets of that range, and all the individual integers contained therein, are provided as part of the invention. Thus, for example, an oligonucleotide which is less than 30 nucleotides in length includes the subset of oligonucleotides which are 18 to 22 nucleotides in length, the subset of oligonucleotides which are 20 to 25 nucleotides in length etc. as well as an oligonucleotide which is 10 nucleotides in length, an oligonucleotide which is 11 nucleotides in length, an oligonucleotide which is 12 nucleotides in length, etc. up to and including an oligonucleotide which is 29 nucleotides in length. This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
Introduction
Effective therapeutic vaccines often require activation of T cell-mediated immunity. However, poor immunogenicity of conventional protein-based vaccines has necessitated the development of alternative approaches. Robust T cell activation can be achieved by using antibodies or antibody fragments to direct antigens of interest to professional antigen presenting cells. Here, aptamers have been selected that specifically bind DEC205, a C-type lectin expressed predominantly on the surface of CD8α+ dendritic cells (DCs) that has been shown to be particularly efficient at facilitating antigen cross-presentation and subsequent CD8+ T cell activation. A minimized DEC205 aptamer was conjugated to the model antigen ovalbumin (OVA), and the DEC205 aptamer-OVA conjugate was delivered in vitro to murine CD11c+ splenocytes. Antigen cross-presentation was verified by proliferation and cytokine production by primary murine CD8+ T cells expressing a T cell receptor (TCR) specific for the MHC I-restricted OVA257-264 peptide SIINFEKL (SEQ ID NO:12). Compared with a nonspecific modified RNA of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and interferon gamma (IFNγ) secretion.
Nucleic acid aptamers, however, due to their unique chemical properties and low immunogenicity, may provide an effective alternative to antibody-based antigen delivery (7,8). Aptamers are short ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) sequences generated in vitro by an iterative selection process (systematic evolution of ligands by exponential enrichment, SELEX) to bind with high affinity and specificity to a given target. Aptamers have demonstrated a wide range of flexibility, finding applications in flow cytometry staining, activating signaling pathways through cell surface receptor ligation, drug or siRNA delivery, blocking protein-protein interactions, and inhibiting enzyme function (7-9). Several aptamer-based therapeutics are currently undergoing clinical trials, with one already approved for use (10).
Results
With the goal of utilizing aptamers for targeted antigen delivery, herein are disclosed aptamers that recognize the murine receptor DEC205 (mDEC205) and are adapted for use as a targeted antigen delivery agent. DEC205 is an excellent target receptor for this proof of principle study. This C-type lectin is expressed mainly by CD8α+DCs, with lower expression by other cells in the hematopoietic lineage (>1 log less)(11). More importantly, studies targeting this receptor with anti-DEC205 antibodies fused to a variety of different antigens have demonstrated that targeting this receptor leads to efficient cross-presentation of these molecular cargoes including cancer (12,13) and HIV epitopes (14,15). Indeed, a cancer vaccine utilizing anti-human DEC205 antibody is already being tested in the clinic (16).
A sequential selection strategy of recombinant mDEC205 protein followed by cells expressing mDEC205 was used to identify nuclease stabilized RNA aptamers specific for the mDEC205 receptor. Minimized aptamers conjugated to the model chicken antigen ovalbumin (OVA) specifically bound mDEC205-expressing cell lines and primary cells. In the presence of the adjuvants lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly(I:C)), incubation of anti-DEC205 aptamer-OVA conjugate with primary DCs in vitro resulted in cross presentation of antigen, as determined by T cell proliferation assays and cytokine secretion.
Identification of Anti-mDEC205 Aptamers: Pool enrichment—In order to identify aptamers that specifically recognized mDEC205 and that were readily internalized by cells that naturally express this receptor, a three-stage selection procedure was employed (
In order to further enrich the population for aptamers that bound to the receptor in the context of the cell surface, for Round 4 a cell-based selection utilizing CHO/mDEC205 cells was switched to. Prior to the selection, a negative selection step was performed on CHO cells to deplete the population of any nonspecific cell binders. Following positive selection on CHO/mDEC205 cells, bound RNA was recovered amplified and assayed by flow cytometry.
The Round 4 RNA showed no improvement over Round 3 (
Clone analysis: Flow cytometric analysis (
In order to verify the specificity of the selected aptamers for mDEC205, the expression of mDEC205 was knocked down on CHO/mDEC205 cells using siRNA and assessed the level of surface staining 24 hrs later using either an mDEC205-specific antibody, NLDC145, or the anti-mDEC205 aptamer, Clone 1. Cells treated with the anti-DEC205 siRNA displayed a ˜50% knockdown in protein expression as determined by flow cytometry, with the data generated using the aptamer, Clone 1 paralleling those of the antibody (
In addition, the ability of Clone 1 to bind A20.Kb cells, a B cell lymphoma line that natively expresses mDEC205, was investigated as well as A20.Kb/mDEC205 cells, a variant of A20.Kb cells engineered to express higher levels of mDEC205. As expected, when treated with the aptamer, A20.Kb cells showed significant staining as determined by flow cytometry with even greater levels observed for the A20.Kb/mDEC205 cells (
Minimization: Sequence analysis of the Round 5 clones revealed a seven base sequence (5′-UUCAUAA-3′) (SEQ ID NO:3) that recurred in several clones (
The minimized aptamer, min.2 (
Enhancing Cross Presentation with Anti-mDEC205 Aptamers: Aptamer-OVA conjugation—Antibodies that target DEC205 have been shown to greatly enhance the cross-presentation of antigens (5,6,19). To this end the well characterized ovalbumin (OVA) system was utilized as a model to investigate the ability of min.2 to deliver cargo for cross-presentation.
Using the 5′-thiol-modified min.2, aptamer-OVA conjugates were generated using the heterobifunctional crosslinker N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS). In short, OVA protein was activated by reaction with a 10-fold molar excess of sulfo-GMBS. The activated OVA was subsequently desalted and then reacted with the 5′-thiolated min.2 in a ratio of 5:1 OVA:RNA (
As a control to ensure the specificity of min.2-mediated antigen delivery, an RNA of similar length but unrelated sequence (‘cntrl.36’) was chemically synthesized and conjugated to OVA in a manner similar to that of min.2. Conjugation efficiency and purification of cntrl.36 was very similar to that of min.2, indicating that likely the only significant difference between the two conjugates is the RNA sequence itself (
To ensure that the aptamer-protein conjugates still functioned, fluorescently labeled conjugates were generated and assessed binding to CHO/mDEC205 cells. As shown in
Aptamer-Mediated OVA Delivery: The ability of anti-mDEC205 aptamer to enhance delivery and cross presentation was assessed in vitro using murine CD11c+ splenocytes, a subset of which are CD8α+DEC205+. In short, DCs were incubated with either the aptamer-OVA conjugates (min.2:OVA or cntrl.36:OVA) or antibody-OVA fusions (NLDC145:OVA or GL117:OVA, a rat IgG2a anti-Escherichia coli β-galactosidase antibody which served and an isotype control).
CD69 is expressed within hours of T cell stimulation (20), making it one of the earliest cell surface markers to be upregulated and a useful indicator of T cell activation. Cytometric analysis of lymph node cells isolated from RAG1−/− OT-I+/+ (‘OT-I cells’) exposed to DC's treated with as little as 10 nM min.2:OVA for ˜20 hrs demonstrated the potential for this aptamer to induce T cell activation, although the observed effect was less than that seen with the mDEC205-targeted antibody-OVA chimera, NLDC145:OVA (
A much more dramatic effect was observed when the effect on OT-I cell proliferation was examined (
Activation of naïve T cells results in acquisition of effector functions including expression of activation markers, cell proliferation and cytokine production. Intracellular cytokine flow cytometry (ICFC) was used to measure interleukin 2 (IL-2) and interferon gamma (IFNγ) present in OT-I T cells following incubation with DCs treated with aptamer conjugates or antibody chimeras. Significant intracellular IL-2 and IFNγ was observed for OT-I cells in response to OVA delivery by aptamer or antibody, and was similar to the response seen with SIINFEKL peptide (
Interestingly, analysis of secreted cytokine levels by ELISA presented a slightly different picture. Targeted delivery by both NLDC145:OVA chimera and min.2:OVA conjugate induced significant secretion of IFNγ by OT-I cells (
Several aptamers that have been used for the targeted delivery of molecular cargoes have not triggered innate immune responses in vitro (21) or in vivo (22,23), and while the FDA-approved aptamer pegaptanib has shown good a safety profile (24), innate immune stimulation may be an aptamer-specific property (25) that requires testing on a case-by-case basis. However, innate immune stimulation may be triggered by specific aptamers, dependent on properties such as RNA sequence (25). Therefore, testing is required on a case-by-case basis. To ensure that the aptamers, which specifically target a DC receptor and are endocytosed, did not provoke any adverse effect on the cells, DCs treated for 24 hrs were monitored with the aptamer-OVA conjugates for upregulation of the activation markers CD80, CD86, CD40 and MHC II (I-A). Importantly, treatment with the aptamer or control conjugates did not produce any changes in expression level beyond that observed by treatment with DBPS alone. (CD11c+ splenocytes were isolated from C57BL/6 mice implanted with Flt3L-expressing B16 melanoma. After isolation, splenocytes were incubated overnight with 10 nM RNA:OVA conjugates in the absence or presence of LPS and poly(I:C). DPBS, OVA and NLDC145:OVA+LPS and poly(I:C) served as reference treatments. Neither cntrl.36:OVA nor min.2:OVA induced DC activation (as measured by CD80 and CD86 expression) above background (DPBS, OVA) levels). Furthermore, neither cntrl.36:OVA+LPS and poly(I:C) nor min.2:OVA+LPS and poly (I:C) induced DC activation above that seen with NLDC145:OVA+LPS and poly(I:C). As expected, only samples treated with polyinosinic:polycytidylic acid (poly(I:C)) and lipopolysaccharide (LPS) showed activation and upregulation of these receptors. Similarly, when cytokine secretion into the media by these same cells was monitored, INFγ, IL-6, IL-10, IL-12, macrophage chemoattractant protein 1 (MCP-1) and tumor necrosis factor alpha (TNFα) all remained at background levels.
Min.2:OVA is functional for cross-presentation in vivo: The ability of min.2:OVA to enhance cross-presentation in vivo was assessed using adoptively transferred OT-I cells. In short, lymph node-derived OT-I cells were labeled with CFSE and injected i.v. into congenic mice (B6.SJL-ptprc). Twenty-four hours later, the mice were injected, i.v., with min.2:OVA or controls. Three days later, spleens were harvested and T cell proliferation and cytokine production was determined by flow cytometry. Consistent with our in vivo targeting results (
To further confirm that the response observed with min.2:OVA was due to min.2 interacting with DEC205, two mutants of min.2 were generated which largely maintained the sequence composition of the parent aptamer but disrupted the predicted folded structure or the order of nucleotide in the conserved heptamer (
To determine how these mutations would affect T cell activation, the mutated aptamers were conjugated to OVA at a 3:1 ratio and were injected into mice that had received OT-I cells. T cell activation was measured 3 days after aptamer:OVA injection (as in
Discussion
Strategies to specifically target DCs and enhance or control the presentation of antigens by these cells are already finding their way into the clinical trials (16,26). Of particular interest are approaches in which antigens are specifically directed to bind DC cell surface receptors using targeting agents. Receptors such as DEC205, the mannose receptor, CD207, DC-SIGN, Clec9a and DCIR2 and others have been shown to be good molecular targets for directing cargoes to intracellular pathways, which can lead to enhanced antigen presentation (19,26-29). Interestingly, different receptors differ in their ability to stimulate cellular or humoral immunity. This is likely due to factors including cellular expression patterns of the various receptors and to their relative ability to deliver antigenic cargoes to MHC I and II following internalization. For example, in mice, delivery of antigen via the DCIR2 receptor promotes antigen presentation on MHC II, whereas targeting the DEC205 receptor favors MHC I presentation (19). Moreover, co-stimulatory signals can be used to affect downstream T cell responses. For example, co-delivery of adjuvants such as poly(I:C) or anti-CD40 antibodies with antigens conjugated to DEC205-specific antibody leads to DC maturation and the production of sustained CD8+ T cell responses (5,27). However in the absence of such additional signals, the CD8+ proliferation is followed by deletion, leading to tolerance (6). Thus these targeted approaches offer the potential to more finally tune the immunological outcome.
Aptamers that that are specific for surface receptors have previously been used for targeted delivery of molecular cargoes, including small molecule drugs (30), toxins and other proteins (31,32), as well as nanoparticles (33). And while aptamers have been developed to target some immunologically relevant receptors and modulate immune responses (9,34,35), aptamers have yet to be utilized for the targeted delivery of antigens.
To test the potential to utilize aptamers in this capacity, nuclease-stabilized aptamers were generated that targeted mDEC205. Using a minimized variant readily amenable to chemical synthesis, aptamer:OVA conjugates were generated and their ability to specially target CD11C+ DEC205+ DCs and enhance cross-presentation to CD8α+ OT-I T cells in vitro assessed. The anti-mDEC205 aptamer proved efficient at specifically enhancing T cell activation at concentrations as low as 1 nM (
It is interesting to compare the aptamer-targeted results with those of the anti-DEC205 antibody, NLDC145. In the in vitro assays the antibody performed at least 10-fold better than the aptamer showing almost complete activation of the T cell population at 100pM. This fact is perhaps not too surprising considering that the observed binding constant of the aptamer is ˜20 nM, and thus appreciable aptamer binding and subsequent uptake would not be expected at concentrations much lower than observed. While the binding affinity of NLDC145 has not been reported, comparable antibodies that target human DEC205 have reported dissociation constants of 100pM-1 nM, 10-100-fold lower than the aptamer (15). A portion of the increased affinity observed with antibodies is a result of their bivalent nature. In the case of the experiments, aptamer-OVA conjugates were generated at a 1:5 aptamer to OVA ratio such that conjugates likely bore only a single aptamer. To the extent that OVA possesses 20 surface lysines, it would be expected that the addition of multiple aptamers could result in enhanced receptor binding and subsequent functional delivery.
It is also interesting to note the differences observed in the production of IFNγ or IL-2 by OT-I cells ˜48 hr following exposure of DCs to min.2:OVA conjugate or NLDC145:OVA chimera. IFNγ was detected both intracellularly and in culture supernatants. In contrast, although intracellular IL-2 was observed for both min.2:OVA and NLDC145:OVA, no IL-2 was detected in the culture supernatants for DCs incubated with aptamer-mediated antigen delivery. This apparent discrepancy likely reflects a difference in the magnitude or kinetics of secretion, as IL-2 was detected, albeit at reduced levels, 24 hrs following OT-I incubation with DCs cultured with min.2:OVA. IL-2 is utilized as a T cell growth factor and the lower levels of IL-2 detected in the culture supernatants, when compared with ICFC, coincide with T cell expression of IL-2 receptor (36). While it is known that IL-2 may be dispensable for the initial T cell proliferative response (37), this may have implications for the in vivo testing of aptamer-mediated antigen delivery.
In summary, the work demonstrates a new approach for targeting antigens to specific DC receptors using nucleic acid aptamers. Our anti-mDEC205 aptamers perform well in vitro and demonstrate the ability to target mDEC205+ cells and enhance antigen delivery. Although the aptamer-mediated antigen delivery had a lower efficacy than that observed with the anti-mDEC205 antibody NLDC145, this difference likely rests in differences in the binding affinity of these two targeting agents. To the extent that aptamers have been reported with binding constants in the pM range, it seems likely that future improvements can and will be achieved.
Materials and Methods
Protein expression and purification: Chinese hamster ovary (CHO) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; GIBCO Invitrogen no. 11995) supplemented with 5-10% fetal bovine serum or 5% Ultra-Low IgG FBS supplemented with antibiotic-antimycotic, and nonessential amino acids (all from GIBCO Invitrogen).
CHO cells, stably expressing an open reading frame for the full-length mouse DEC205 (CHO/mDEC205) were generated as previously described (15). Similarly, the extracellular domain (residues 1-1667) of mDEC205 was fused in-frame with the hIgG1 FC domain. The mDEC205/hIgG1FC construct was inserted into the pCMV expression vector (Clontech), transfected into CHO cells to generate stably expressing CHO/mDEC205/hIgG1FC cells. Culture supernatant from these cells was used to purify the mDEC205/hIgG1FC fusion protein by affinity to Protein A Sepharose (GE Healthcare).
Aptamer Selection: The sequence of the N50 library used for selection against mDEC205 was: 5′ GGGAGGTGAATGGTTCTACGAT-N50-TTACATGCGAGATGACCACGTAATTGAATTAAATGCCCGCCATGACCAG-3′. (SEQ ID NO:4) The single stranded DNA library was synthesized such that N regions contained an equal probability of containing A, T, G, or C, as previously described (38). Following deprotection, the library was gel purified by denaturing (7M urea) gel electrophoresis on an 8% polyacrylamide gel. The single stranded DNA library was amplified by PCR to generate a double stranded DNA bearing a T7 promoter and transcribed in vitro using the Y639F mutant of T7 RNA polymerase (39,40) and 2′-fluoro (2′-F) pyrimidines. Following transcription, the RNA was purified on a denaturing (7 M urea) 8% polyacrylamide gel.
For Round 1, ˜3 copies of an RNA library encompassing ˜1×1014 sequences (˜20 μg) were utilized. The library, was diluted in 20 μL HBSS (GIBCO) and then thermally equilibrated by incubation at 70° C. for five minutes, followed by room temperature for fifteen minutes prior to addition to the immobilized protein target (mDEC205/hIgG1FC).
Immobilized protein was prepared by incubating 25 pmol mDEC205/hIgG1FC with 25 μL of washed Dynabeads Protein G resin in 200 uL of washing/blocking buffer (WB, 0.1 M NaPO4 pH 8.2, 0.01% Tween 20). This mixture was incubated 30 minutes at room temperature with rotation and then washed three times with 200 μL DPBS without Ca2+ and Mg2+ (GIBCO). After the third wash, buffer was removed and replaced with the RNA library. The resin was then incubated for an additional 30 minutes at room temperature with rotation, followed by three washes with 200 μL HBSS. After the third wash, protein and RNA were eluted by a 5 minute incubation with 20 μL 200 mM glycine pH 2.5. The eluant was combined with 400 μL 0.3 M NaOAc, containing 4 μg glycogen and the RNA recovered by ethanol precipitation.
The recovered RNA was reverse transcribed to single-stranded DNA (ssDNA) using Moloney Murine Lukemia Virus reverse transcriptase (M-MLV RT, Invitrogen), the ssDNA was amplified by polymerase chain reaction (PCR) using Taq DNA polymerase, and PCR product was transcribed into 2′-F-Y-RNA by a modified T7 RNA polymerase (Y639F39 and P266L41 mutations). For subsequent rounds, 1 μg of the previous round's selection product was prepared as above for Round 1.
Prior to Rounds 2 and 3 a negative selection step was used to deplete resin binders of aptamers that bound the FC region from the population. The negative selection step was performed by incubating the library with 20 μL Dynabeads Protein G resin prepared as above but substituting 50 pmol human IgG1 FC region (hIgG1FC) for mDEC205/hIgG1FC fusion protein. Protein G resin loaded with hIgG1FC was then incubated with the prepared RNA for 30 minutes at room temperature with rotation, after which the resin was spun down and the supernatant was removed and added to 20 μL Dynabeads Protein G resin loaded with 25 pmol mDEC205/hIgG1FC as above. RNA was recovered, reverse-transcribed, amplified and transcribed as described above.
Round 4 was performed using CHO cells that were stably transfected to express mDEC205. Prior to the positive selection, a negative selection was performed on the parental CHO cells, which no not express this receptor. In short, 5 μg of RNA was combined with a two-fold molar excess of reverse primer used for amplification (T5OR). The mixture was diluted to a final volume of 50 μL in HBSS, denatured 5 minutes at 70° C., and allowed to anneal for 15 minutes at room temperature. Re-annealed RNA was added to ˜1×105 CHO cells in a 24 well plate containing 450 μL and incubated for 30 minutes at 37° C. Following incubation the media (containing non-bound RNA) was removed from the CHO cells and transferred to a well containing ˜1×105 CHO/mDEC205 cells. The RNA was incubated with CHO/mDEC205 for 1 hour at 37° C., after which cells were washed three times with 1 mL HBSS, and cells were lysed for 5 minutes at room temperature with 500 μL TRIzol. Recovered RNA was reverse-transcribed, amplified and transcribed as described above.
For Round 5 an ‘internalization selection’ was performed in which bone marrow derived dendritic cells (BMDC's) were targeted. BMDC's were prepared from mouse bone marrow by treatment for 5 days with GM-CSF as previously described (17). RNA (5 μg) was combined with a two-fold molar excess of reverse primer used for amplification (T50R). The mixture was diluted to a final volume of 50 μL in HBSS, denatured 5 minutes at 70° C., and allowed to anneal for 15 minutes at room temperature. The RNA was subsequently added to one well of a 24 well plate containing ˜5×104 BMDC's in 450 μL Advanced RPMI 1640 containing 10% FBS and 5% J5 medium (supernatant from B16 melanoma cells transfected to express GM-CSF, courtesy of the Palliser lab) supplemented with single-stranded DNA and transfer RNA (1 mg/mL each).
After a 1 hour incubation at 37° C., the media was removed, and cells were washed three times with 1 mL HBSS containing 0.1% NaN3 followed by a single wash with 1 mL cold 200 mM glycine, 150 mM NaCl, pH 4. The cells were subsequently washed an additional three times with 1 mL HBSS containing 0.1% NaN3 followed by 1 mL DPBS without Ca2+ and Mg2+ and trypsinized by the addition of 500 μL 0.05% trypsin, 0.53 mM ethylenediaminetetraacetic acid (EDTA) for 15 minutes at 37° C. Following an additional 1mL wash with 1 mL HBSS containing 0.1% NaN3, the cells were pelleted and resuspended in 100 μL with HBSS containing 5 μL RiboShredder ribonuclease cocktail (EpiCentre). The reaction was incubated for 15 minutes at room temperature, after which the cells were washed three times with 1 mL HBSS+0.1% NaN3 and lysed for five minutes at room temperature with 500 μL TRIzol. RNA was recovered, reverse-transcribed, amplified and transcribed as described above.
PCR product from Round 5 was cloned into the pCR2.1-TOPO TA vector by TOPO TA cloning (Invitrogen).
Chemical synthesis of RNA aptamers: Minimized aptamers and controls were synthesized in-lab on an Expedite 8909 DNA synthesizer (Applied Biosystems, Carlsbad, Calif.) using 2′-fluoro-deoxycytidine and 2′-fluoro-deoxyuridine phosphoramidites (Metkinin, Kuusisto, Finland). Unless noted otherwise, all reagents were purchased from Glen Research (Sterling, Va.). The aptamer was synthesized bearing a 5′ thiol modification using a thiol-modifier C6 S—S phosphoramidite and a 3′ inverted dT residue for added serum stability. The sequences of the minimized aptamer, min.2, and a non-binding aptamer, c36, were: 5S GGGAGGUGUGUUAGCACACGAUUCAUAAUCAGCUACCCUCCCt (SEQ ID NO:1 with inverted dT) and SSGGCGUAGUGAUUAUGAAUCGUGUGCUAAUACACGCCt (SEQ ID NO:5), respectively, where ‘t’ is a 3′inverted dT and ‘5S’ is the 5′ thiol. All aptamers were synthesized with the final 4,4′-dimethoxytrityl (DMT) protecting on left on. Following deprotection, aptamers were purified by reversed phase HLPC on a 10×50 mm Xbridge C18 column (Waters, Milford, Mass.) using a linear gradient of acetonitrile in 0.1M triethylammonium acetate (TEAA) at pH 7.0.
Aptamer Binding by Flow Cytometry: Aptamer binding was assessed by flow cytometery. Rounds from each selection or isolated clones were first hybridized to a biotinylated labeled oligonucleotide that was complementary to the 3′ end of the library (TSOR). In a typical assay, 10 pmol of transcribed RNA pool or aptamer was added to 11 pmol biotinylated TSOR (B-TSOR) in 10 μL HBSS or DPBS. The RNA was thermally equilibrated by heating to 70° C. for 3 minutes and then allowed to cool on the bench for 15 minutes at room temperature. The RNA was subsequently added to cells of interest resuspended at 1×105 cells in 100 μL flow cytometry buffer (HBSS containing 1% BSA and 0.1% NaN3) supplemented with 1 mg/mL ssDNA and/or tRNA. The RNA and cells were incubated for 15 minutes on ice, pelleted by centrifugation and washed once with 1 mL flow cytometry buffer. Cells were resuspended in 100 μL flow cytometry buffer containing 0.5 μL PhycoLink streptavidin-R-phycoerythrin (SA-PE) or PhycoLink streptavidin-allophycocyanin (SA-APC) and incubated an additional 15 minutes on ice, washed with 1 mL flow cytometry buffer, resuspended with 500 μL flow cytometry buffer, and analyzed by flow cytometry, with exclusion of dead cells by 7-aminoactinomycin D (7-AAD) or 4′,6-diamidino-2-phenylindole (DAPI) staining.
Flow Cytometry: Fluorophore-conjugated RNA: Thiolated aptamers (5S.min2 or 5S.cntrl36) were used to generate the Alexa Fluor 488 (AF488) aptamers used for flow cytometry. Labeling was performed using AF488-05-malemide (Invitrogen) as follows: thiolated aptamer was reduced using 10 mM tris(2-carboxyethyl)phosphine (TCEP) in 100 μL, of 0.1M TEAA. Samples were heated at 70° C. for 3 minutes followed by incubation at room temperature for 1 hour. The reduced aptamers were desalted using a Biospin 6 column (BioRad, Hercules, Calif.) into PBS supplemented with 50 mM phosphate pH 7.5. To this, AF488-05-maleimide in DMSO was added to a 10-fold molar excess over RNA. Following an overnight reaction at 4° C., the aptamer was desalted an additional time using a Biospin 6 column. In a typical assay, 10 pmol of dye-conjugated RNA was diluted into 10 μL DPBS or HBSS, denatured for 3 minutes at 70° C. and allowed to cool for 15 minutes at room temperature. Cell staining was performed as described above but without the addition of SA-PE or SA-APC.
mDEC205 Knockdown Measurement in CHO/mDEC205: CHO/mDEC205 were transfected in wells of a 12-well plate using Lipofectamine™ 2000 (Invitrogen) and one of two siRNA's directed against mDEC205 (si-mDEC205.1 sense CUAACAACAUGUUGUGGAAGUGGGT (SEQ ID NO:6) si-mDEC205.1 antisense ACCCACUUCCACAACAUGUUGUUAGUU (SEQ ID NO:7) and si-mDEC205.2 sense GGACUUUGUGCAUUUGUUAAAGGAC, (SEQ ID NO:8) si-mDEC205.1 antisense GUCCUUUAACAAAUGCACAAAGUCCUU; (SEQ ID NO:9) where underlined residues are DNA), siRNA against the unrelated enhanced green fluorescent protein (EGFP, sense AAGCUGACCCUGAAGUUCAUCUGCACC, (SEQ ID NO:10) antisense GGUGCAGAUGAACUUCAGGGUCAGCUU) (SEQ ID NO:11), or a mock (no siRNA) transfection, according to the manufacturer's protocol. Two days after transfection, cells were washed with DPBS without Ca2+ and Mg2+, lifted from the plate with 5 mM EDTA in DPBS without Ca2+ and Mg2+, washed with flow cytometry buffer, and resuspended in 100 μL. Cells were then stained with Clone 1 hybridized to B-T5OR or with biotinylated NLDC145 antibody clone (anti-mDEC205), and processed as described above.
mDEC205 Cell Surface Binding Assay: A20.Kb cells or A20.Kb.mDEC205 cells which are engineered to express higher amounts of mDEC205 were washed with RPMI, resuspended at 1×105 cells in 100 μL RPMI supplemented with 1 mg/mL ssDNA, and stored on ice while Alexa Fluor 488-conjugated min.2 and Alexa Fluor 488-conjugated cntrl.36 were prepared as described previously. After addition of refolded RNA to cells, the RNA-cell mixture was incubated for 15 minutes at 37° C., washed with and resuspended in flow cytometry buffer, and analyzed by flow cytometry. Median Alexa Fluor 488 fluorescence of live (DAPI−) cells was plotted versus min.2 or cntrl.36 concentration using GraphPad Prism, and dissociation constant (KD) was calculated by fitting a one site binding model to the data.
A20.Kb cells and A20.Kb.mDEC205 cells were generated from A20 cells by viral transduction. The cell lines were generated by superinfecting retroviruses made from pMX-Kb and/or pMX-mDEC205 as described previously (42).
Synthesis of Aptamer Ovalbumin Conjugate: Thiolated aptamers (5S.min2 or 5S.cntrl36) were used to generate the aptamer OVA conjugates. Pierce Imject Ovalbumin (OVA) was activated with a ten-fold molar excess of N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS, Pierce) in 1 mM EDTA in DPBS without Ca2+ and Mg2+ according to the manufacturer's protocol, and excess sulfo-GMBS was removed with a Micro Bio-Spin 6 column.
Thiolated aptamers were reduced using 10 mM TCEP and subsequently desalted using a Bio-Spin 6 column into DPBS without Ca2+ and Mg2+. The reduced aptamers were incubated with a 5-fold molar excess of sulfo-GMBS-activated OVA. The reaction mixture was incubated 30 minutes at room temperature or overnight at 4° C. and then quenched by incubation with excess L-cysteine or BODIPY FL L-cystine (Invitrogen) that had previously been reduced with TCEP. When the latter was used, the conjugate could be used for flow cytometry staining.
The reaction mixture was desalted to 20 mM Tris, pH 7 with a Micro Bio-Spin 6 column and then purified on a Mini Q™ 4.6/50 PE column (GE Healthcare) equilibrated with the same buffer. After washing out the flowthrough, the NaCl concentration was increased linearly to 0.5 M with a steep gradient and held at that concentration until all unconjugated OVA was eluted. Salt concentration was then increased from 0.5 M to 2.5 M over 20 column volumes (CV), at a flow rate of 1 CV per minute. Elution was tracked by monitoring absorbance at 260 nm. Elution fractions were analyzed by electrophoresis on a 7 M urea, 8% acrylamide gel, which was stained with GelStar nucleic acid stain (Lonza) and then scanned on a Storm 840 imager (GE Healthcare). Fractions were pooled, concentrated using Amicon Ultra 10K centrifugal filtration units (Millipore), and buffer exchanged to DPBS before storage at −20° C.
In vitro Proliferation Assays: A single-cell suspension of splenocytes from C57BL/6 mice aged 6 weeks to 6 months was prepared using 400 U/mL collagenase D (Roche), and CD11c+ were isolated using biotinylated antibody clone N418 and MACS streptavidin microbeads, or MACS CD11c+ microbeads alone, according to the manufacturer's protocol (Miltenyi). CD11c+ splenocytes were plated in a 96-well U-bottom plate at 4×105 in 100 μL Advanced RPMI 1640 (Invitrogen) containing 10% FBS, Pen Strep (Invitrogen), GlutaMAX-I (Invitrogen) and 10 mM HEPES (MPBio) (complete culture medium), 1 μg/mL lipopolysaccharides from Escherichia coli O111:B4 (LPS, Sigma), and 10 μg/mL polyinosinic-polycytidylic acid (poly(I:C), Sigma). The cells were incubated for 4 hours at 37° C., after which the media was replaced with media of the same formulation containing additional 1 mg/mL ssDNA and 10 nM of either Imject OVA, rat IgG2a isotype antibody-OVA chimera (Iso:OVA, courtesy of the Palliser lab), NLDC145-OVA chimera (DEC:OVA, courtesy of the Palliser lab), cntrl.36:OVA, or min.2:OVA (several wells received an equivalent volume of DPBS as a negative control). The cells were incubated for an additional 16-20 hours at 37° C.
The next day, a single-cell suspension of lymph node cells was prepared from the axillary, brachial, inguinal, popliteal and mesenteric lymph nodes of 6 week-old to 6 month-old C57BL/6 RAG−/− OT-I+/+ (OT-I) mice. The OT-I cells were resuspended in DPBS containing 5% FBS, carboxyfluorescein succinimidyl ester (CFSE) was added to 5 μM, and the cells were incubated for 5 minutes at 37° C. protected from light. The reaction was quenched by adding ten volumes of complete culture medium and incubating for five minutes at room temperature. Cells were washed three times in complete culture medium and resuspended in complete culture medium at 1×106 cells/mL. After their 16-20 hour incubation, the CD11c+ splenocytes were washed carefully three times with DPBS, 1×105 CFSE-labeled OT-I cells were added, and the cells were incubated an additional 2-3 days at 37° C. At the end of this incubation, cells were resuspended, washed with flow cytometry buffer, stained with DAPI and antibodies against CD8α and TCRβ, and analyzed by flow cytometry.
In vitro Cytokine Production: CD11c+ splenocytes were prepared as described above and treated in triplicate with 10 nM of either of the following: Imject OVA, SIINFEKL (OVA257-264) peptide (SEQ ID NO:12), rIgG2a:OVA, NLDC145:OVA, cntrl.36:OVA, or min.2:OVA. OT-I cells were prepared as described above, except for CFSE staining. After 16-20 hour incubation with antigen at 37° C., the CD11c+ splenocytes were washed carefully three times with DPBS, and 1×105 OT-I cells were added. Following incubation at 37° C. for two additional days, media was removed and saved, it was replaced with the same media containing 10 μg/mL brefeldin A (BFA, Invitrogen), and cells were incubated for 5 hours at 37° C. Cells were resuspended, fixed with 4% paraformaldehyde (Sigma-Aldrich) in pH 7 PBS for 10 minutes at 37° C., washed, permeabilized with 0.1% saponin (Sigma) in flow cytometry buffer, stained with antibodies against CD8α, TCRβ, interleukin-2 (IL-2) and interferon γ (IFNγ), and analyzed by flow cytometry. The saved media was analyzed for secreted IL-2 and IFNγ by enzyme-linked immunosorbent assay (ELISA).
22. Dassie, J. P. et al. Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors. Nat Biotechnol 27, 839-849 (2009).
This application is a U.S. national stage entry under 35 U.S.C. §371 of PCT International Patent Application No. PCT/US2013/049212, filed Jul. 3, 2013, which claims benefit of U.S. Provisional Application No. 61/671,239, filed Jul. 13, 2012, the contents of each of which are incorporated herein by reference into the subject application.
This invention was made with government support under grant numbers AI093539 and AI099567 and awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2013/049212 | 7/3/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2014/011465 | 1/16/2014 | WO | A |
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| 20040031072 | La Rosa et al. | Feb 2004 | A1 |
| 20130123478 | Levy et al. | May 2013 | A1 |
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| 2014011465 | Jan 2014 | WO |
| 2015066001 | May 2015 | WO |
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| PCT International Search Report and Written Opinion, dated Dec. 20, 2013 in connection with PCT International Application No. PCT/US2013/49212 11 pages. |
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| 20150191730 A1 | Jul 2015 | US |
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| 61671239 | Jul 2012 | US |
