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WO2025166244A2 - Compositions et procédés d'acide nucléique flt3l fusionné à l'albumine - Google Patents

Compositions et procédés d'acide nucléique flt3l fusionné à l'albumine

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Publication number
WO2025166244A2
WO2025166244A2 PCT/US2025/014139 US2025014139W WO2025166244A2 WO 2025166244 A2 WO2025166244 A2 WO 2025166244A2 US 2025014139 W US2025014139 W US 2025014139W WO 2025166244 A2 WO2025166244 A2 WO 2025166244A2
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WO
WIPO (PCT)
Prior art keywords
dendritic cells
flt3l
composition
nucleic acid
tumor
Prior art date
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PCT/US2025/014139
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English (en)
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WO2025166244A3 (fr
Inventor
T.C. Wu
Chien-Fu Hung
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Johns Hopkins University
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Johns Hopkins University
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Publication of WO2025166244A3 publication Critical patent/WO2025166244A3/fr
Pending legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the disclosure relates in particular to compositions which induce an anti-tumor immune response by stimulating expansion of circulating dendritic cells (DCs).
  • DCs dendritic cells
  • ICI immune checkpoint inhibitors
  • CAR-T cell chimeric antigen receptor T-cell
  • DCs dendritic cells
  • T-VEC Talimogene laherparepvec
  • composition comprising an expression vector or nucleic acid encoding a fusion polypeptide comprising albumin protein and FMS-like tyrosine kinase 3 ligand (Flt3L) protein.
  • methods of treatment include the delivery of the composition followed by or in conjunction with electroporation to induce the uptake of the expression vector and/or nucleic acid.
  • a composition comprises a therapeutically effective amount of: i) an expression vector encoding a polypeptide comprising an albumin protein linked to an FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof, or ii) a nucleic acid encoding an albumin protein linked to an FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof.
  • the Flt3L is human.
  • the albumin is human.
  • the Flt3L is murine.
  • the albumin is murine.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the composition further comprises at least one chemotherapeutic agent.
  • the composition further comprises at least one immunotherapeutic agent.
  • methods of treating cancer in a subject comprising administering a composition as disclosed herein to a subject, thereby treating the subject, particularly treating cancer in the subject.
  • the administered composition suitably comprises a therapeutically effective amount of: i) an expression vector encoding a polypeptide comprising an albumin protein linked to an FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof, or ii) a nucleic acid encoding an albumin protein linked to an FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof.
  • administered composition may comprise an expression vector or nucleic acid that encodes a polypeptide having up to or at least 70%, 80%, 85%, 90%, 95%, 87%, 98%, 99% or 100% sequence identity to SEQ ID. NO.1.
  • the treatment methods may further comprise comprising applying electroporation to the subject in conjunction with administering the therapeutic composition.
  • an electroporation treatment may be applied to the subject following (e.g. up to 0.25, 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 or 6 minutes or more) after administering the composition to the subject.
  • one or more tumors of the subject is treated by the electroporation, such as after administering the composition to the subject.
  • a method of treating cancer in a subject comprising administering a composition comprising a therapeutically effective amount of an expression vector encoding a polypeptide comprising a sequence having at least 70, 80 or 90% identity to the amino acid sequence of SEQ ID NO: 1 or a nucleic acid encoding a polypeptide comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 to a tumor,
  • the nucleic acid encoding the polypeptide comprises a nucleic acid sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 2.
  • the polypeptide comprises an amino acid sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 1.
  • the polypeptide comprises an amino acid sequence of SEQ ID NO: 1.
  • the method further comprises administering one or more chemotherapeutics.
  • the chemotherapeutics comprise chemotherapeutic agents, radiation, surgery or combinations thereof.
  • the expression vector comprises: a plasmid, viral vector, phage or a bacterial vector.
  • the expression vector is a plasmid.
  • the composition induces an anti-tumor immune response.
  • the composition stimulates expansion of circulating dendritic cells (DCs) as compared to the subject’s baseline circulating dendritic cells.
  • DCs circulating dendritic cells
  • the circulating dendritic cells comprise conventional dendritic cells (cDC1), conventional dendritic cells 2 (cDC2), plasmacytoid dendritic cells (pDC), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or combinations thereof.
  • the circulating dendritic cells comprise conventional dendritic cells (cDC1).
  • the composition is administered intratumorally, subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intravitreallly (i.v.i.), intra-cisterna magna (i.c.m.), or intrasternally. In certain embodiments, the composition is administered intratumorally.
  • a method of treating tumors in a subject comprises administering a composition comprising a therapeutically effective amount of an expression vector encoding a polypeptide comprising an FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof, fused to a protein, or a nucleic acid encoding an FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof, fused to a protein to the tumor; subjecting the tumor
  • the protein comprises albumin or variants thereof.
  • the method further comprises administering one or more chemotherapeutics.
  • the chemotherapeutics comprise chemotherapeutic agents, radiation, surgery or combinations thereof.
  • the expression vector comprises: a plasmid, viral vector, phage or a bacterial vector.
  • the expression vector is a plasmid.
  • the composition induces an anti-tumor immune response.
  • the composition stimulates expansion of circulating dendritic cells (DCs) as compared to the subject’s baseline circulating dendritic cells.
  • the circulating dendritic cells comprise conventional dendritic cells (cDC1), conventional dendritic cells 2 (cDC2), plasmacytoid dendritic cells (pDC), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or combinations thereof.
  • the circulating dendritic cells comprise conventional dendritic cells (cDC1).
  • the composition is administered intratumorally, subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intravitreallly (i.v.i.), intra- cisterna magna (i.c.m.), or intrasternally.
  • the composition is administered intratumorally.
  • an expression vector encodes a fusion polypeptide comprising FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof and albumin.
  • the expression vector is a plasmid.
  • a nucleic acid encodes a fusion polypeptide comprising FMS- like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof, and albumin.
  • a host cell comprises the expression vectors or nucleic acids embodied herein.
  • the albumin protein is mammalian. In certain embodiments, the albumin protein can be murine, porcine, ovine, bovine, human, or combinations thereof.
  • the FMS-like tyrosine kinase receptor 3 ligand (Flt3L) is mammalian.
  • the Flt3L can be murine, porcine, ovine, bovine, human, or combinations thereof.
  • a method of modulating an immune response comprising contact a cell in vitro or administering to a subject, composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, thereby modulating an immune response.
  • the expression vector comprises: a plasmid, viral vector, phage or a bacterial vector. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the composition stimulates expansion of circulating dendritic cells (DCs) as compared to the subject’s baseline circulating dendritic cells.
  • the circulating dendritic cells comprise conventional dendritic cells (cDC1), conventional dendritic cells 2 (cDC2), plasmacytoid dendritic cells (pDC), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or combinations thereof.
  • the circulating dendritic cells comprise conventional dendritic cells (cDC1).
  • the functional portion can comprise, for instance, about 90%, 95%, or more, of the albumin and/or Flt3L polypeptide.
  • the functional portion of the fusion polypeptide composition of the present disclosure can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of either of the wild type albumin and/or FLt3L polypeptides.
  • albumin protein means the full-length expressed polypeptide of the nucleic acid encoding the albumin gene, or a functional portion or fragment, or variant thereof. It will be understood by those of ordinary skill in the art that many different isoforms of both human and murine albumin protein exist and can be used in the compositions disclosed herein. Examples of albumin isoforms include, but are not limited to, human albumin isoforms (NM_000477, AAH41789.1, and AAH35969), for example and mouse albumin ligand isoform (AAH49971), for example.
  • amino acid includes the residues of the natural ⁇ -amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as ⁇ -amino acids, synthetic and unnatural amino acids.
  • ⁇ -amino acids e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val
  • chimeric or “fusion” polypeptide or protein refers to a composition comprising at least one polypeptide or peptide sequence or domain that is chemically bound in a linear fashion with a second polypeptide or peptide domain.
  • One embodiment of this disclosure is an isolated or recombinant nucleic acid molecule encoding a fusion protein comprising at least two domains, wherein the first domain comprises a polypeptide encoding albumin protein, and the second domain comprising a polypeptide encoding FMS-like tyrosine kinase 3 ligand (Flt3L) protein, such as, for example the Human Flt3 ligand (Genbank No. AAA90950.1), or in another embodiment, the Mouse Flt3 ligand (Genbank No. EDL22813.1). Other species of Flt3L peptides are contemplated within the scope of the disclosure.
  • FMS-like tyrosine kinase 3 ligand FMS-like tyrosine kinase 3 ligand
  • the “fusion” can be an association generated by a peptide bond, a chemical linking, a charge interaction (e.g., electrostatic attractions, such as salt bridges, H-bonding, etc.) or the like. If the polypeptides are recombinant, the “fusion protein” can be translated from a common mRNA. Alternatively, the compositions of the domains can be linked by any chemical or electrostatic means. [00040] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term includes an autonomously replicating plasmid or a virus.
  • the term is also construed to include non- plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • Flt3L protein means the full-length expressed polypeptide of the nucleic acid encoding the FMS-like tyrosine kinase 3 ligand protein gene, or a functional portion or fragment, or variant thereof.
  • the term “functional portion or fragment thereof,” with respect to the Flt3L protein, means that the portion or fragment of the Flt3L polypeptide retains its ability to make progenitor cells skewed to alternative dendritic cells subsets, and induce T cell responses and B cell responses in a subject. It will be understood by those of ordinary skill in the art that many different isoforms of both human and murine Flt3L protein exist, and can be used in the compositions disclosed herein.
  • Flt3L isoforms include, but are not limited to, Human Flt3 ligand isoforms (AAA90950.1, NM_001459.3, and NM_001278637.1), for example and mouse Flt3 ligand isoforms (EDL22813.1, S43291, and AAA90952), for example.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • parenteral administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
  • patient or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • polynucleotide is a chain of nucleotides, also known as a “nucleic acid”.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art and include both naturally occurring and synthetic nucleic acids.
  • peptide “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially
  • polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • the peptides provided herein for use in the described and claimed methods and compositions can be cyclic.
  • the term “percent sequence identity” or having “a sequence identity” refers to the degree of identity between any given query sequence and a subject sequence.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.
  • promoter as used herein is defined as a nucleic acid (e.g.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • a “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • a “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
  • the term “transfected” or “transformed” or “transduced” means to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the transfected/transformed/transduced cell includes the primary subject cell and its progeny.
  • “Treatment” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • treating or “treatment” of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human or other mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
  • “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so
  • a variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. [00063] Genes: All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable.
  • mice 13 167460060.1 to GLuc C57BL/6 mice were inoculated with 2xl 0 4 TC-1 cells and utilized for tissue biodistribution experiment once tumors reached approximately 0.5 cm in diameter.
  • alb-Gluc (20 ⁇ g), Gluc (20 ⁇ g) or vehicle control were injected intratumorally, followed by electroporation.
  • Mice blood was collected at indicated timepoints post-injection. On Day 14, mice were euthanized, and tissues were extracted for luminescence analysis.
  • FIG.1B Mice were euthanized and tumor was collected and grinded.
  • FIG.1D Schematic diagram of the albumin-Flt3L fusion protein.
  • FIG.1E SDS-PAGE of alb-Flt3L and Flt3L. All data was presented as the mean ⁇ SEM, (*,p ⁇ 0.05; **,p ⁇ 0.01; ***,p ⁇ 0.001; ****,p ⁇ 0.0001).
  • FIGS.2A-2E are a series of graphs, plots, a schematic and a scan of a photograph demonstrating that alb-Flt3L DNA treatment elicits superior tumor control and better survival compared with that of Flt3L DNA.
  • C57BL/6 mice were inoculated with 2xl0 4 TC-1 cells in thigh.
  • Flt3L DNA (10 ⁇ g) or vehicle control was injected intratumorally and followed by electroporation for a total of three times in 5-day intervals.
  • FIG.2A Schematic of experiment design.
  • FIGS.3A-3L are a series of graphs and plots demonstrating that alb-Flt3L DNA- induced DC expansion leads to tumor control. alb-Flt3L DNA administration displays greater systemic DC population.
  • TC-I-bearing C57BL/6 mice were administered with alb-Flt3L DNA, Flt3L DNA or vehicle control for a total of three cycles in 5-day intervals. Electroporation followed immediately after DNA injection.
  • FIGS.3B, 3C Peripheral blood mononuclear cells
  • FIGS.4A-4F are a series of graphs and plots showing that alb-Flt3L DNA demonstrates robust anti-tumor immunity against TC-1 tumor. Electroporation-medicated alb- Flt3L DNA delivery revealed an increased percentage of IFN- ⁇ -secreting CD8 + T cells. (FIGS.
  • FIGS.5A-5B show the results from histological examination of liver and pancreas from mice treated with Alb-Flat3L DNA or Alb-Flat3L protein. Mice were treated by either Alb-Flat3L DNA administered through intramuscular injection with electroporation or Alb-Flat3L protein administered via intravenous injection three times at 1 week interval. The liver and pancreas from treated mice were harvested 1 week after the last treatment for histological examination.
  • FIG.5A Representative histology of pancreas from mice treated with Alb-Flt3L DNA or protein.
  • FIG.5B Representative histology of liver from mice treated withAlb-Flt3L DNA or protein.
  • Upper panel is the organs from mice treated with Alb-Flt3L DNA.
  • Lower panel is the organs from mice treated with Alb-Flt3L protein.
  • Alb-Flt3L DNA vaccination is well-tolerated in mice while Alb-Flt3L protein vaccination is not well- tolerated in mice.
  • FIGS 6A-6E show the results from histology examination of key organs of mice receiving treatments. Mice were treated with either Alb-Flt3L, Flt3L or without treatment (control) as described in Materials and Methods. One week after the last treatment, vital organs including heart, kidney, lung, pancreas, and liver were harvested and submitted for examination.
  • FIG.6A Representative histology of heart from mice treated with Alb-Flt3L or Flt3L. Mice without treatment is included as control.
  • FIG.6B Representative histology of lung from mice treated with Alb-Flt3L or Flt3L. Mice without treatment is included as control.
  • FIG.6C Representative histology of liver from mice treated with Alb-Flt3L or Flt3L. Mice without treatment is included as control.
  • FIG.6D Representative histology of pancreas from mice treated with Alb-Flt3L or Flt3L. Mice without treatment is included as control.
  • FIG.6E Representative histology of kidney from mice treated with Alb-Flt3L or Flt3L. Mice without treatment is included as control.
  • Upper panel is the organs from mice without treatment as control (CTL).
  • Middle panel is the organs from mice treated with Flt3L.
  • Lower panel is the organs from mice treated with Alb-Flt3L.
  • FMS-like tyrosine kinase receptor 3 (Flt3) is a tyrosine kinase receptor expressed extensively on hematopoietic progenitor cells
  • Flt3 ligand (Flt3L) is a pluripotent growth factor that regulates the maturation and differentiation of DCs in hematopoiesis.
  • Flt3L acts as an essential element in the development of the immune system.
  • One of its critical functions is to stimulate the in vivo expansion of circulating DCs, specifically the addition, serum albumin exhibits the capacity for preferential uptake by tumor cells and accumulates intratumorally.
  • a method of treating cancer in a subject comprises administering a composition comprising a therapeutically effective amount of a vector encoding a polypeptide comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 or a nucleic acid encoding a polypeptide comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 to a tumor; subjecting the tumor to electroporation; thereby treating the subject.
  • functional variants of the inventive polypeptides, and proteins described herein are functional variants of the inventive polypeptides, and proteins described herein.
  • the term “functional variant” as used herein refers to either the albumin and/or Flt3L polypeptide, or fusion protein having substantial or significant sequence identity or similarity to the albumin and/or Flt3L polypeptide, or fusion protein, which functional variant retains the biological activity of the albumin and/or Flt3L polypeptide, or fusion protein of which it is a variant.
  • the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the albumin and/or Flt3L polypeptide, or protein.
  • the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.
  • an amino acid with a polar side chain substituted for another amino acid with a polar side chain e.g., Asp or Glu
  • an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc
  • the nucleic acids of the disclosure can, for example, encode functional variants which also include extensions of the albumin and/or Flt3L polypeptide fusion protein.
  • a functional variant of the albumin and/or Flt3L polypeptide fusion protein can include 1, 2, 3, 4 and 5 additional amino acids from either the N-terminal or C-terminal end of the albumin and/or Flt3L polypeptide fusion protein.
  • the functional variants can comprise the amino acid sequence of the albumin and Flt3L polypeptide fusion protein with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant.
  • the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the albumin and Flt3L polypeptide fusion protein.
  • the albumin and Flt3L polypeptide fusion protein can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the functional variant, e.g., other amino acids, do not materially change the biological activity of the functional variant.
  • the orientation of the two proteins in the fusion protein encoded by the genetic construct can be reversed, i.e., the N- terminal protein can comprise the Flt3L ligand protein and the C-terminal protein can comprise the albumin protein.
  • the nucleic acids encode a mammalian Flt3L protein.
  • the Flt3L protein can be murine, porcine, ovine, bovine, human, or combinations thereof.
  • the present disclosure provides a composition
  • nucleic acid sequence encodes a polypeptide comprising a sequence of at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1.
  • polynucleotides provided herein encoding one or more fusion proteins include codon-optimized sequences.
  • codon-optimized means a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned as compared to a wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence by choosing different codons without altering the amino acid sequence of the encoded protein. Accordingly, codon-optimization generally refers to replacement of codons with synonymous codons to optimize expression of a protein while keeping the amino acid sequence of the translated protein the same.
  • Codon optimization of a sequence can increase protein expression levels (Gustafsson et al., Codon bias and heterologous protein expression.2004, Trends Biotechnol 22: 346-53) of the encoded proteins, for example, and provide other advantages.
  • Variables such as codon usage preference as measured by codon adaptation index (CAI), for example, the presence or frequency of A, G, C, U nucleotides, mRNA secondary structures, cis- regulatory sequences, GC content, and other variables may correlate with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments.2006, BMC Bioinformatics 7:285).
  • Any method of codon optimization can be used to codon optimize polynucleotides and nucleic acid molecules provided herein, and any variable can be altered by codon optimization. Accordingly, any combination of codon optimization methods can be used. Exemplary methods include the high codon adaptation index (CAI) method and others.
  • CAI high codon adaptation index
  • the CAI method chooses a most frequently used synonymous codon for an entire protein coding sequence. As an example, the most frequently used codon for each amino acid can be deduced from 74,218 protein-coding genes from a human genome.
  • Any polynucleotide, nucleic acid sequence, or codon sequence provided herein can be codon optimized.
  • the nucleotide sequence of any region of an RNA or DNA sequence embodied herein may be codon optimized.
  • the primary cDNA template may include reducing the occurrence or frequency of appearance of certain nucleotides in the template strand. For example, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 25% of said nucleotides in the template. In further examples, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 20% of said nucleotides in the template.
  • the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 16% of said nucleotides in the template.
  • the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 15% and may be increased or reduced to a level above or below 12% of said nucleotides in the template.
  • the polynucleotides of the disclosure can comprise one or more chemically modified nucleotides.
  • nucleic acid monomers include non-natural, modified, and chemically modified nucleotides, including any such nucleotides known in the art. Nucleotides can be artificially modified at either the base portion or the sugar portion.
  • RNA polynucleotides comprise nucleotides that are “unmodified” or “natural” nucleotides, which include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). These bases are typically fixed to a ribose or deoxy ribose at the 1' position.
  • A purine
  • G guanine
  • T cytosine
  • U uracil
  • RNA polynucleotides comprising chemically modified nucleotides have also been useful in optimizing protein localization thereby avoiding deleterious bio-responses such as immune responses and/or degradation pathways.
  • modified or chemically modified nucleotides include 5- hydroxycytidines, 5-alkylcytidines, 5-hydroxyalkylcytidines, 5-carboxycytidines, 5- formylcytidines, 5-alkoxycytidines, 5-alkynylcytidines, 5-halocytidines, 2-thiocytidines, N 4 - alkylcytidines, N 4 -aminocytidines, N 4 -acetylcytidines, and N 4 , N 4 -dialkylcytidines.
  • modified or chemically modified nucleotides include 5- hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5- formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-
  • modified or chemically modified nucleotides include 5- hydroxyuridines, 5-alkyluridines, 5-hydroxyalkyluridines, 5-carboxyuridines, 5- carboxyalkylesteruridines, 5-formyluridines, 5-alkoxyuridines, 5-alkynyluridines, 5-halouridines, 2-thiouridines, and 6-alkyluridines.
  • modified or chemically modified nucleotides include 5- hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as “SMeOU”), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine, and 6-methyluridine.
  • modified or chemically-modified nucleotides include 5- methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5- carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 1-methyl-3-(3-amino-3- carboxypropy)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5- methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5- (isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'O-methyluridine, and 3,2'- O-dimethyluridine.
  • modified or chemically-modified nucleotides include N 6 - methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8- oxoadenosine, 8-bromoadenosine, 2-methylthio-N 6 -methyladenosine, N 6 -isopentenyladenosine, 2-methylthio-N 6 -isopentenyladenosine, N 6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N 6 - (cis-hydroxyisopentenyl)adenosine, N 6 -glycinylcarbamoyladenosine, N 6 -threonylcarbamoyl- adenosine, N 6 -methyl-N 6 -threonylcarbamoyl-adenosine, N 6 -
  • modified or chemically modified nucleotides include N 1 - alkylguanosines, N 2 -alkylguanosines, thienoguanosines, 7-deazaguanosines, 8-oxoguanosines, 8- bromoguanosines, O 6 -alkylguanosines, xanthosines, inosines, and N 1 -alkylinosines.
  • modified or chemically modified nucleotides include N 1 - methylguanosine, N 2 -methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8- bromoguanosine, O 6 -methylguanosine, xanthosine, inosine, and N 1 -methylinosine.
  • nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.
  • modified and chemically modified nucleotide monomers include any such nucleotides known in the art, for example, 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'- deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl- nucleotides, and inverted deoxyabasic monomer residues.
  • nucleotides known in the art for example, 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'- deoxy ribonucleotides
  • modified and chemically modified nucleotide monomers include 3'- end stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides, and 3'-inverted thymidine.
  • modified and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'- methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, and 2'- O-methyl nucleotides.
  • the modified monomer is a locked nucleic acid nucleotide (LNA).
  • modified and chemically modified nucleotide monomers include 2',4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-Ethyl (cEt) modified DNAs.
  • modified and chemically modified nucleotide monomers include 2'- amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides, and 2'-O-allyl nucleotides.
  • modified and chemically modified nucleotide monomers include N6- methyladenosine nucleotides.
  • modified and chemically modified nucleotide monomers include nucleotide monomers with modified bases 5-(3-amino)propyluridine, 5-(2- mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.
  • modified and chemically modified nucleotide monomers include 2'- O-aminopropyl substituted nucleotides.
  • modified and chemically modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R, a 2'-OR, a 2'-halogen, a 2'-SR, or a 2'- amino, where R can be H, alkyl, alkenyl, or alkynyl.
  • Example of base modifications described above can be combined with additional modifications of nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers may be found in nature.
  • the nucleic acid is an RNA
  • the RNA molecules can be engineered to comprise one or more modified nucleobases.
  • RNA molecules can be found, for example, in Genes VI, Chapter 9 (“Interpreting the Genetic Code”), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne, eds. (1998, ASM Press, Washington DC).
  • Modified RNA components include the following: 2′-O-methylcytidine; N 4 - methylcytidine; N 4 -2′-O-dimethylcytidine; N 4 -acetylcytidine; 5-methylcytidine; 5,2′-O- dimethylcytidine; 5-hydroxymethylcytidine; 5-formylcytidine; 2′-3-methylcytidine; 2- thiocytidine; lysidine; 2′-O-methyluridine; 2-thiouridine; 2-thio-2′-O-methyluridine; 3,2′-O- dimethyluridine; 3-(3-amino-3-carboxypropyl)uridine; 4-thiouridine; ribosylthymine; 5,2′-O- dimethyluridine; 5-methyl-2-thiouridine; 5-hydroxyuridine; 5-methoxyuridine; uridine 5- oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 5-carboxymethyluridine;
  • Isolated nucleic acid molecules can be produced by standard techniques. For example, PCR techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995.
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to 5′ direction using
  • oligonucleotides 27 167460060.1 phosphoramidite technology
  • oligonucleotides one or more pairs of long oligonucleotides (e.g., >50-100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector, e.g.
  • nucleic acid is a synthetic polynucleotide.
  • the synthetic nucleic acid comprises a modified nucleotide. Modification of the inter-nucleoside linker (i.e., backbone) can be utilized to increase stability or pharmacodynamic properties. For example, inter-nucleoside linker modifications prevent or reduce degradation by cellular nucleases, thus increasing the pharmacokinetics and bioavailability of the nucleic acid.
  • inter-nucleoside linker i.e., backbone
  • inter-nucleoside linker modifications prevent or reduce degradation by cellular nucleases, thus increasing the pharmacokinetics and bioavailability of the nucleic acid.
  • a modified inter-nucleoside linker includes any linker other than other than phosphodiester (PO) liners, that covalently couples two nucleosides together.
  • the modified inter-nucleoside linker increases the nuclease resistance of the nucleic acid compared to a phosphodiester linker.
  • the inter-nucleoside linker includes phosphate groups creating a phosphodiester bond between adjacent nucleosides.
  • the nucleic acid comprises one or more inter- nucleoside linkers modified from the natural phosphodiester.
  • inter-nucleoside linkers of the nucleic acid or contiguous nucleotide sequence thereof are modified.
  • the inter-nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.
  • S Sulphur
  • Modifications to the ribose sugar or nucleobase can also be utilized herein.
  • a modified nucleoside includes the introduction of one or more modifications of the sugar moiety or the nucleobase moiety.
  • the nucleic acids comprise one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA.
  • DNA deoxyribose nucleic acid
  • RNA RNA-derived nucleic acid
  • Numerous nucleosides with modification of the ribose sugar moiety can be utilized, primarily with the aim of improving certain properties of
  • oligonucleotides such as affinity and/or stability.
  • modifications include those where the ribose ring structure is modified. These modifications include replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g. locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA).
  • HNA hexose ring
  • LNA locked nucleic acids
  • Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids or tricyclic nucleic acids.
  • Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
  • Sugar modifications also include modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2′-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2′, 3′, 4′ or 5′ positions.
  • Nucleosides with modified sugar moieties also include 2′ modified nucleosides, such as 2′ substituted nucleosides.
  • a 2′ sugar modified nucleoside is a nucleoside that has a substituent other than H or —OH at the 2′ position (2′ substituted nucleoside) or comprises a 2′ linked biradicle and includes 2′ substituted nucleosides and LNA (2′-4′ biradicle bridged) nucleosides.
  • 2′ substituted modified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O- methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, and 2′-F-ANA nucleoside.
  • the modification in the ribose group comprises a modification at the 2′ position of the ribose group.
  • the modification at the 2′ position of the ribose group is selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′- deoxy, and 2′-O-(2-methoxyethyl).
  • the nucleic acid comprises one or more modified sugars. In some embodiments, the nucleic acid comprises only modified sugars. In certain embodiments, the nucleic acid comprises greater than 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2′-O-methoxyethyl group. In some embodiments, the nucleic acid comprises both inter-nucleoside linker modifications and nucleoside modifications.
  • nucleic acids of the disclosure may be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, 2 nd edition, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 2003.
  • sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid.
  • the nucleic acids also can be chemically synthesized, either as a single nucleic acid (e.g., using automated DNA synthesis in the 3′ to 5′ direction using phosphoramidite technology) or as a series of oligonucleotides.
  • Isolated nucleic acids of the disclosure also can be obtained by mutagenesis of, e.g., a naturally occurring portion RNA, DNA, of an Flt3L and/or albumin encoding DNA.
  • the fusion polypeptides are synthesized from an expression vector encoding the DNA molecule, as described in detail elsewhere herein.
  • NUCLEIC ACIDS AND VECTORS [000123]
  • the composition of the disclosure comprises a nucleic acid encoding one or more elements of the polypeptides described herein.
  • the composition comprises an isolated nucleic acid encoding an alb- Flt3L peptide, or functional fragment or derivative thereof.
  • the composition comprises at least one nucleic acid encoding an alb- Flt3L peptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence homology with SEQ ID NO: 1.
  • the isolated nucleic acid may comprise any type of nucleic acid, including, but not limited to DNA and RNA.
  • the composition comprises
  • an isolated DNA including for example, an isolated cDNA, encoding a peptide of the disclosure, or functional fragment thereof.
  • the composition comprises an isolated RNA encoding a peptide of the disclosure, or a functional fragment thereof.
  • the isolated nucleic acids may be synthesized using any method known in the art. [000126]
  • the present disclosure can comprise use of a vector in which the nucleic acids described herein are inserted. The art is replete with suitable vectors that are useful in the present disclosure.
  • Vectors include, for example, plasmids, viral vectors (such as adenoviruses (“Ad”), adeno-associated viruses (AAV), and vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
  • Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • vectors include those described by Chen et al; BioTechniques, 34: 167-171 (2003). A large variety of such vectors is known in the art and is generally available.
  • expression of natural or synthetic nucleic acids encoding a peptide is typically achieved by operably linking a nucleic acid encoding the peptide or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the vectors of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos.5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
  • the disclosure provides a gene therapy vector.
  • the nucleic acids of the disclosure can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193).
  • a number of viral based systems have been developed for gene transfer into mammalian cells.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • onco-retroviruses such as murine leukemia viruses
  • non-proliferating cells such as hepatocytes.
  • they also have the added advantage of low immunogenicity.
  • 32 167460060.1 includes a vector derived from an adeno-associated virus (AAV).
  • AAV adeno-associated viral
  • AAV vectors have become powerful gene delivery tools for the treatment of various disorders.
  • AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner.
  • Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.
  • nucleic acids encoding the polypeptides described herein.
  • an AAV vector includes to any vector that comprises or derives from components of AAV and is suitable to infect mammalian cells, including human cells, of any of a number of tissue types, such as brain, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas, whether in vitro or in vivo.
  • an AAV vector includes an AAV type viral particle (or virion) comprising a nucleic acid encoding a protein of interest.
  • the AAVs disclosed herein are be derived from various serotypes, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single-stranded or self-complementary).
  • the AAV vector is a human serotype AAV vector.
  • a human serotype AAV is derived from any known serotype, e.g., from AAV1, AAV2, AAV4, AAV6, or AAV9.
  • the serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8.
  • AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.
  • a variety of different AAV capsids have been described and can be used, although AAV which preferentially target the tumors and/or deliver genes with high efficiency are particularly desired.
  • the sequences of the AAV8 are available from a variety of databases. While the examples utilize AAV vectors having the same capsid, the capsid of the gene editing
  • AAV9 see, for example, U.S. Pat. No.7,906,111; US 2011-0236353-A1
  • hu37 see, e.g., U.S. Pat. No.7,906,111; US 2011-0236353-A1
  • the nucleic acid also includes one or more regulatory sequences allowing expression and, in some embodiments, secretion of the protein of interest, such as e.g., a promoter, enhancer, polyadenylation signal, an internal ribosome entry site (“IRES”), a sequence encoding a protein transduction domain (“PTD”), and the like.
  • a promoter, enhancer, polyadenylation signal, an internal ribosome entry site (“IRES”), a sequence encoding a protein transduction domain (“PTD”) and the like.
  • the nucleic acid comprises a promoter region operably linked to the coding sequence to cause or improve expression of the protein of interest in infected cells.
  • a promoter can be ubiquitous, cell- or tissue-specific, strong, weak, regulated, chimeric, etc., for example, to allow efficient and stable production of the protein in the infected tissue.
  • the promoter is homologous to the encoded protein, or heterologous, although generally promoters of use in the disclosed methods are functional in human cells.
  • Examples of regulated promoters include, without limitation, Tet on/off element-containing promoters, rapamycin-inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters.
  • other promoters used include promoters that are tissue specific for tissues such as kidney, spleen, and pancreas.
  • ubiquitous promoters include viral promoters, particularly the CMV promoter, the RSV promoter, the SV40 promoter, etc., and cellular promoters such as the phosphoglycerate kinase (PGK) promoter and the ⁇ -actin promoter.
  • the expression vector is delivered to the tumor, or tissue of interest by, for example, an intratumoral injection, while other times the delivery is via intravenous, transdermal (e.g. skin cancers), intranasal, oral, mucosal, or other delivery methods. Such delivery can be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein can vary greatly depending upon a variety of factors, such as the vector chose, the target cell, organism, or tissue, the general
  • the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus.
  • operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • polyA polyadenylation
  • a great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • promoters typically contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • 35 167460060.1 promoters may also be used. Certain proteins can be expressed using their native promoter. Other elements that can enhance expression can also be included such as an enhancer or a system that results in high levels of expression such as a tat gene and tar element.
  • This cassette can then be inserted into a vector, e.g., a plasmid vector such as, pUC19, pUC118, pBR322, or other known plasmid vectors, that includes, for example, an E. coli origin of replication.
  • a promoter is Elongation Growth Factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatinine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters.
  • Inducible promoters are also contemplated as part of the disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates.
  • Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type.
  • the vector of the present disclosure comprises one or more enhancers to boost transcription of the gene present within the vector.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure.
  • selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic- resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5′ flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • the vectors and nucleic acids embodied herein are introduced into the tumor or tissues via electroporation.
  • Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • a preferred method for the introduction of a polynucleotide into a host cell is electroporation.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl
  • DCP 38 167460060.1 phosphate
  • Choi can be obtained from Calbiochem-Behring
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20° C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates.
  • Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium.
  • Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • the composition comprises a cell genetically modified to express one or more isolated nucleic acids and/or peptides described herein.
  • the cell may be transfected or transformed with one or more vectors comprising an isolated nucleic acid sequence encoding the fusion proteins embodied herein. DELIVERY VEHICLES
  • Delivery vehicles as used herein include any types of molecules for delivery of the compositions embodied herein, both for in vitro and in vivo delivery. Examples, include, without limitation: expression vectors, nanoparticles, colloidal compositions, lipids, liposomes, nanosomes, carbohydrates, organic or inorganic compositions and the like. [000153] Any suitable method can be used to deliver the compositions to the subject. In certain embodiments, the nucleic acids encoding the fusion polypeptides may be delivered to systematic circulation or may be delivered or otherwise localized to a specific tissue type.
  • the compositions of the disclsoure can be formulated as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol modified (PEGylated) low molecular weight LPEI.
  • the compositions can be formulated as a nanoparticle encapsulating the compositions embodied herein.
  • L-PEI has been used to efficiently deliver genes in vivo into a wide range of organs such as lung, brain, pancreas, retina, bladder as well as tumor.
  • liposomes are used to effectuate transfection into a cell or tissue.
  • the pharmacology of a liposomal formulation of nucleic acid is largely determined by the extent to which the nucleic acid is encapsulated inside the liposome bilayer. Encapsulated nucleic acid is protected from nuclease degradation, while those merely associated with the surface of the liposome is not protected.
  • Encapsulated nucleic acid shares the extended circulation lifetime and biodistribution of the intact liposome, while those that are surface associated adopt the pharmacology of naked nucleic acid once they disassociate from the liposome.
  • Nucleic acids may be entrapped within liposomes with conventional passive loading technologies, such as ethanol drop method (as in SALP), reverse-phase evaporation method, and ethanol dilution method (as in SNALP).
  • SALP ethanol drop method
  • SNALP ethanol dilution method
  • hydrophilic and hydrophobic materials such as potential chemotherapy agents
  • hydrophilic and hydrophobic materials such as potential chemotherapy agents. See for example U.S. Pat. No.5,466,468 to Schneider, which discloses parenterally administrable liposome formulation comprising synthetic lipids; U.S. Pat. No.5,580,571, to Hostetler et al. which discloses nucleoside analogues conjugated to phospholipids; U.S. Pat. No. 5,626,869 to Nyqvist, which discloses pharmaceutical compositions wherein the pharmaceutically active compound is heparin or a fragment thereof contained in a defined lipid system comprising at least one amphipathic and polar lipid component and at least one nonpolar lipid component.
  • Liposomes and polymerosomes can contain a plurality of solutions and compounds.
  • the complexes of the invention are coupled to or encapsulated in polymerosomes.
  • polymerosomes are tiny hollow spheres that enclose a solution, made using amphiphilic synthetic block copolymers to form the vesicle membrane.
  • Common polymerosomes contain an aqueous solution in their core and are useful for encapsulating and protecting sensitive molecules, such as drugs, enzymes, other proteins and peptides, and DNA and RNA fragments.
  • the polymersome membrane provides a physical barrier that isolates the encapsulated material from external materials, such as those found in biological systems.
  • Non-viral vectors are modified to effectuate targeted delivery and transfection.
  • PEGylation i.e. modifying the surface with polyethyleneglycol
  • PEGylation is the predominant method used to reduce the opsonization and aggregation of non-viral vectors and minimize the clearance by reticuloendothelial system, leading to a prolonged circulation lifetime after intravenous (i.v.) administration.
  • compositions of the compounds of the disclosure typically comprise a compound of the instant disclosure and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the type of carrier can be selected based upon the intended route of administration.
  • the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, micro emulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethyelene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
  • the compounds can be administered in a time release formulation, for example in a composition which includes a slow release polymer, or in a fat pad described herein.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are generally known to those skilled in the art.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • certain methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the compound may be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the agent.
  • the compound can be administered to a subject in an appropriate carrier or diluent co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluoro-phosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. [000163]
  • the active agent in the composition e.g., alb-Flt3L proteins
  • a therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
  • the active agent is formulated in the composition in a prophylactically effective amount.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects
  • the prophylactically effective amount will be less than the therapeutically effective amount.
  • the amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the instant disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • METHODS OF TREATMENT [000165] The present disclosure provides a method of treating or preventing cancer.
  • the method comprises administering to a subject in need thereof, an effective amount of a composition comprising a nucleic acid, or functional fragment or derivative thereof followed by electroporation at the site of the administration, e.g. a tumor.
  • a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof.
  • the present disclosure provides a method of treating a tumor cell comprising administering to the cell an effective amount of a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, in
  • the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof.
  • a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof.
  • the present disclosure provides fusion protein compositions comprising the polypeptide molecules described herein, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a use of a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, in an amount effective for use in a medicament, and most preferably for use as a medicament for treating a disease or disorder associated with a neoplastic disease in a subject.
  • the neoplastic disease is associated with a solid tumor, a hematological tumor, or wherein the tumor and/or its micro and macrometastases is selected from the group consisting of breast cancer, prostate cancer, pancreatic cancer, colon cancer, hepatoma, glioblastoma, ovarian cancer, leukemia, Hodgkin's lymphoma and multiple myeloma.
  • the term “cancer” includes cancers in tissues that can tolerate high doses of radiation. A high dose of radiation would include doses greater than 2 Gy.
  • the cancers treated by the present disclosure would also include cancers which are resistant to hypoxia, chemotherapy, such as, for example, tamoxifen or taxol resistant cancers, and cancers already resistant to radiation therapy.
  • chemotherapy such as, for example, tamoxifen or taxol resistant cancers
  • cancers already resistant to radiation therapy are known to be capable of treating conditions or diseases discussed above.
  • the compositions of the present disclosure could be used in combination with one or more known therapeutically active agents, to treat a neoplastic or proliferative disease such as a tumor or
  • Non-limiting examples of other therapeutically active agents that can be readily combined in a pharmaceutical composition with the compositions and methods of the present disclosure are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, and an effective amount of at least one PD-1 inhibiting agent or other checkpoint inhibitor.
  • the present disclosure provides methods of treatment of tumors using focused radiation on a subject to initiate an immune response in the subject, followed by administration of a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, and an immunotherapeutic agent, such as a PD-1 antibody, to bypass immune checkpoints and sustain the immune response in the subject.
  • a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, and an immunotherapeutic agent, such as a PD-1 antibody, to bypass immune checkpoints and sustain the immune response in the subject.
  • methods for treating a tumor in a subject in need of treatment thereof comprising administering to the subject a therapeutically effective dose of focused radiation to treat the tumor in combination with a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, at least one immunotherapeutic agent comprising an immune checkpoint inhibitor, and at least one chemotherapeutic agent.
  • the term “immunotherapeutic agent” can include any molecule, peptide, antibody or other agent which can stimulate a host immune system to generate an immune response to a tumor or cancer in the subject.
  • Various immunotherapeutic agents are useful in the compositions and methods described herein.
  • Immune checkpoints means a group of molecules on the cell surface of CD4 and CD8 T cells. These molecules effectively serve as “brakes” to down-modulate or inhibit an anti-tumor immune response. Immune checkpoint molecules include, but are not limited to, Programmed Death 1 (PD-1), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40, and LAG3, which directly inhibit immune cells.
  • PD-1 Programmed Death 1
  • CTL-4 Cytotoxic T-Lymphocyte Antigen 4
  • B7H1, B7H4, OX-40 B7H1, B7H4, OX-40
  • CD137 CD40
  • LAG3 LAG3
  • Immunotherapeutic agents which can act as immune checkpoint inhibitors useful in the methods of the present disclosure, include, but are not limited to, anti-B7-H4; anti-PD1 or anti-B7-H1; anti-CTLA-4 (ipilimumab) and anti-LAG3.
  • Ipilimumab is a fully human, antagonistic monoclonal antibody that binds to CTLA-4.
  • CTLA-4 is a cell-surface protein expressed on certain CD4 and CD8 T cells; when engaged by its ligands (B7-1 and B7-2) on APCs, T-cell activation and effector function are inhibited.
  • an immune checkpoint inhibitor is the inhibitory co- receptor known as programmed death 1 (PD-1 or CD279).
  • PD-1 programmed death 1
  • CD8 T cells that infiltrate the prostate gland in men with cancer, up to 87% express PD-1.
  • Tumor-specific expression of the major ligand of PD-1, B7-H1 is associated with poor prognosis in kidney cancer, as well as in other cancers in humans.
  • MDX-1106 is a genetically engineered, fully human immunoglobulin G4 (IgG4) monoclonal antibody specific for human PD-1 that was recently evaluated in a phase 1, dose-escalation trial.
  • Immunotherapeutic agents can include proteins and/or antibodies to proteins and biomolecules including, for example, B- and T-lymphocyte attenuator protein (BTLA), Tim3, CD160, KIR antagonist antibodies, 4-1BB, OX40, CD27 and CD4.
  • BTLA B- and T-lymphocyte attenuator protein
  • Tim3 Tim3
  • KIR antagonist antibodies 4-1BB
  • 4-1BB 4-1BB
  • OX40 OX40
  • CD27 and CD4 CD27
  • the compositions embodied combine the use of focused radiation for treating cancer. These include stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiation therapy.
  • the focused radiation can have a
  • 47 167460060.1 radiation source selected from the group consisting of a particle beam (proton), cobalt-60 (photon), and a linear accelerator (x-ray).
  • a particle beam proton
  • cobalt-60 photon
  • a linear accelerator x-ray
  • methods for administering a therapeutically effective amount of a composition comprising a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, in combination with the therapeutically effective dose of focused radiation.
  • the immunotherapeutic agents comprise monoclonal antibodies, immune effector cells, vaccines, including dendritic cell vaccines, and cytokines.
  • Monoclonal antibodies used in the inventive compositions and methods can be selected from the group consisting of anti-PD-1 antibody, alemtuzumab, bevacizumab, brentuximab vedotin, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, ipilimumab (anti-CTLA-4), ofatumumab, panitumumab, rituximab, tositumomab, trastuzumab, anti-B7-H4, anti-B7-H1, anti-LAG3, BTLA, anti-Tim3, anti-B7-DC, anti-CD160, MR antagonist antibodies, anti-4-1BB, anti-OX40, anti-CD27, and CD40 agonist antibodies.
  • a pharmaceutical composition comprises a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, and most preferably for use as a medicament for inducing an immune response, or treating cancer, or inhibiting the growth of a tumor, or neoplasm in a subject who receives or will receive focused radiation treatment, when administered to the subject in an effective amount.
  • a pharmaceutical composition comprises a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, and administered in combination with at least one immunotherapeutic agent, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, and most preferably for use as a medicament for inducing an immune response, or treating cancer, or inhibiting the growth of a tumor, or neoplasm in a subject who receives or will receive focused radiation treatment, when administered to the subject in an effective amount.
  • a pharmaceutical composition comprises a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, and is administered in combination a neoadjuvant or treatment regimen for treating cancer.
  • Neoadjuvant therapy includes the administration of a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, with or without the addition of one or more therapeutic or immunotherapeutic agents in combination with focused radiation before, or in conjunction with, traditional chemotherapy/radiation treatment and adjuvant therapy.
  • Neoadjuvant therapy aims to reduce the size or extent of the cancer before using radical treatment intervention, thus making procedures easier and more likely to succeed, and reducing the consequences of a more extensive treatment technique that would be required if the tumor wasn't reduced in size or extent.
  • a patient is administered focused radiation in combination with a first dose of the compositions embodied herein, followed by an antibody days after receiving a result from a biopsy. After a period of time later, for example, a week later, the patient can undergo surgery. Following surgery, for example, two weeks after surgery, the patient is administered focused radiation in combination with a second dose of the nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, followed by an antibody.
  • the patient is administered focused radiation in combination with a third dose of a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, followed by an antibody.
  • a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof followed by an antibody.
  • the treatment regimen presented herein can be adjusted or modified to meet the therapeutic needs of an individual patient. For example, any of the steps disclosed herein can be repeated in series, or individually, to meet such needs.
  • the present inventive methods further comprise administering to the subject additional chemotherapy, immunotherapy and or radiation
  • the method further comprises administering to the subject, adjuvant therapy.
  • methods of treating cancer further comprise administering at least one adjuvant to the subject in combination with a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, with or without the at least one immunotherapeutic agent and/or immune checkpoint inhibitor.
  • Adjuvants include without limitation a cytokine, an interleukin, an interferon, a granulocyte-macrophage colony-stimulating factor (GM-CSF), Bacille Clamette-Guérin (BCG), a keyhole limpet memocyanin (KLH), incomplete Freund's adjuvant (IFA), QS-21, DETOX, and dinitrophenyl.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • BCG Bacille Clamette-Guérin
  • KLH keyhole limpet memocyanin
  • IFA incomplete Freund's adjuvant
  • QS-21 QS-21
  • DETOX dinitrophenyl
  • tumors including solid tumors, lesions, and conditions
  • cancers involving the cervix, ovaries, head and neck, brain cancers involving the spine
  • lung cancers pancreatic cancers
  • prostate cancers prostate cancers
  • liver cancers, kidney cancers breast cancers, melanoma, metastatic orbital tumors, orbital lymphomas, and orbital inflammations.
  • the term “combination” is used in its broadest sense and means that a subject is administered at least two agents, e.g., a dose of radiation and a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, with, or without at least one additional immunotherapeutic agent, e.g., monoclonal antibodies, immune effector cells, vaccines, including dendritic cell vaccines, and cytokines, as described herein or as otherwise known in the art.
  • agents e.g., a dose of radiation and a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof.
  • additional immunotherapeutic agent e.g., monoclonal antibodies, immune effector cells, vaccines, including dendritic cell vaccines, and cytokines, as described herein or as otherwise known in the
  • the phrase “in combination with” refers to the administration of a dose of radiation and at least one immunotherapeutic agent either simultaneously, sequentially, or a combination thereof.
  • a subject administered a combination of a dose of radiation and a nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, with or without at least one additional immunotherapeutic agent can receive a dose of radiation and at least one immunotherapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.
  • nucleic acid encoding albumin protein, or a functional portion or fragment, or variant thereof and an Flt3L protein, or a functional portion or fragment, or variant thereof, with, or without at least one additional immunotherapeutic agent can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another.
  • agents administered sequentially can be administered within 1, 5, 10, 15, 20 or more days of one another.
  • the agents When more than one therapeutic agent is administered in combination with a dose of radiation, and the agents are administered either sequentially or simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either one therapeutic agent and at least one immunotherapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.
  • the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent(s) was administered as a single agent.
  • the effects of multiple agents may, but need not be, additive or synergistic.
  • the agents may be administered multiple times.
  • the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a
  • 51 167460060.1 combination of a dose of radiation and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.
  • a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone.
  • a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.
  • Focused radiation methods suitable for use with the presently disclosed methods include, but are not limited to, stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiation therapy (IMRT).
  • IMRT intensity-modulated radiation therapy
  • stereotactic radiation need not be delivered in a single treatment.
  • the treatment plan can be reliably duplicated day-to-day, thereby allowing multiple fractionated doses of radiation to be delivered.
  • the radiosurgery is referred to as “fractionated stereotactic radiosurgery” or FSR.
  • stereotactic radiosurgery refers to a one-session treatment.
  • the dosage of radiation applied using stereotactic radiosurgery can vary. In some embodiments, the dosage can range from 1 Gy to about 30 Gy, and can encompass intermediate ranges including, for example, from 1 to 5, 10, 15, 20, 25, up to 30 Gy in dose.
  • fractionation allows higher doses to be delivered to tumorous tissue because of an increased tolerance of the surrounding normal tissue to these smaller fractionated doses. Accordingly, while single-dose stereotactic radiation takes advantage of the pattern of radiation given, fractionated stereotactic radiation takes advantage of not only the pattern of radiation, but also of the differing radiosensitivities of normal and surrounding tumorous tissues. Another advantage of fractionated stereotactic radiation is so-called “iterative” treatment, in which the shape and intensity of the treatment plan can be modified during the course of therapy. Fractionated stereotactic radiosurgery can result in a high therapeutic ratio, i.e., a high rate of killing of tumor cells and a low effect on normal tissue. The tumor and the normal tissue respond differently to high single doses of radiation vs. multiple smaller doses of radiation. Single large doses of radiation can kill more normal tissue than several smaller doses
  • the dosage of radiation applied using fractionated stereotactic radiation can vary.
  • the dosage can range from 1 Gy to about 50 Gy, and can encompass intermediate ranges including, for example, from 1 to 5, 10, 15, 20, 25, 30, 40, up to 50 Gy in hypofractionated doses.
  • stereotactic radiosurgery can be characterized by the source of radiation used, including particle beam (proton), cobalt-60 (photon-Gamma Knife®), and linear accelerator (x-ray).
  • a linear accelerator produces high-energy X-ray radiation and is capable of delivering precise and accurate doses of radiation required for radiosurgery.
  • Radiosurgery using a linear accelerator is typically carried out in multi-session, smaller dose treatments so that healthy surrounding tissue is not damaged from too high a dose of radiation.
  • Radiosurgery using linear accelerator technology also is able to target larger brain and body cancers with less damage to healthy tissues.
  • the most common uses of linear accelerator stereotactic radiosurgery are for the treatment of metastatic cancer, some benign tumors and some arterio-venous malformations.
  • Linear accelerator based machines are not dedicated to treatments only within the brain and can be used throughout the body, as well as the head and neck.
  • a “gamma knife” uses multiple, e.g., 192 or 201, highly-focused x-ray beams to make up the “knife” that cuts through diseased tissue.
  • the gamma knife uses precisely targeted beams of radiation that converge on a single point to painlessly “cut” through brain tumors, blood vessel malformations, and other brain abnormalities.
  • a gamma knife makes it possible to reach the deepest recesses of the brain and correct disorders not treatable with conventional surgery.
  • proton beam radiation offers certain theoretical advantages over other modalities of stereotactic radiosurgery (e.g., Gamma Knife® and linear accelerators), because it makes use of the quantum wave properties of protons to reduce doses of radiation to surrounding tissue beyond the target tissue.
  • stereotactic radiosurgery e.g., Gamma Knife® and linear accelerators
  • the proton beam radiation offers advantages for treating unusually shaped brain
  • IMRT intensity-modulated radiation therapy
  • IMRT is an advanced mode of high-precision three-dimensional conformal radiation therapy (3DCRT), which uses computer-controlled linear accelerators to deliver precise radiation doses to a malignant tumor or specific areas within the tumor.
  • 3DCRT the profile of each radiation beam is shaped to fit the profile of the target from a beam's eye view (BEV) using a multileaf collimator (MLC), thereby producing a number of beams.
  • BEV beam's eye view
  • MLC multileaf collimator
  • IMRT allows the radiation dose to conform more precisely to the three-dimensional (3-D) shape of the tumor by modulating the intensity of the radiation beam in multiple small volumes. Accordingly, IMRT allows higher radiation doses to be focused to regions within the tumor while minimizing the dose to surrounding normal critical structures.
  • IMRT improves the ability to conform the treatment volume to concave tumor shapes, for example, when the tumor is wrapped around a vulnerable structure, such as the spinal cord or a major organ or blood vessel.
  • Treatment with IMRT is planned by using 3-D computed tomography (CT) or magnetic resonance (MRI) images of the patient in conjunction with computerized dose calculations to determine the dose intensity pattern that will best conform to the tumor shape.
  • CT computed tomography
  • MRI magnetic resonance
  • combinations of multiple intensity-modulated fields coming from different beam directions produce a custom-tailored radiation dose that maximizes tumor dose while also minimizing the dose to adjacent normal tissues.
  • IMRT typically is used to treat cancers of the prostate, head and neck, and central nervous system. IMRT also has been used to treat breast, thyroid, lung, as well as in gastrointestinal, gynecologic malignancies and certain types of sarcomas.
  • Cancer vaccines are characterized as active immunotherapies because they are meant to trigger a subject's own immune system to respond. Further, cancer vaccines are specific because they should only affect cancer cells. Such vaccines don't just boost the immune system in general; they cause the immune system to attack cancer cells with one or more specific antigens. At this time, only one true cancer vaccine has been approved by the FDA.
  • Sipuleucel-T Provenge®
  • PAP prostatic acid phosphatase
  • cancer vaccines include, but not limited to, tumor cell vaccines, including autologous and allogeneic tumor cell vaccines; antigen vaccines, which boost the immune system by using only one or a few antigens, e.g., proteins or peptides; dendritic cell vaccine, which include special antigen-presenting cells (APCs) that help the immune system recognize cancer cells by breaking down cancer cells into smaller pieces (including antigens), then present these antigens to T cells making it easier for the immune system cells to recognize and attack them; anti-idiotype vaccines, which show promise as a B-cell lymphoma; DNA vaccines, and vector-based vaccines, which use special delivery systems (called vectors) to make them more effective and can include, for example, vector-based antigen vaccines and vector- based DNA vaccines.
  • tumor cell vaccines including autologous and allogeneic tumor cell vaccines
  • antigen vaccines which boost the immune system by using only one or a few antigens, e.g., proteins or peptides
  • the types of cancers for which tumor cell vaccines can be used in conjunction with the inventive fusion proteins include, but are not limited to, melanoma, kidney cancer, ovarian cancer, breast cancer, colorectal cancer, lung cancer, prostate cancer, non-Hodgkin lymphoma, and leukemia.
  • Antigen vaccines are being studied to be used against these cancers, among others: breast cancer, prostate cancer, colorectal cancer, ovarian cancer, melanoma, kidney cancer, pancreatic cancer, and multiple myeloma.
  • the dendritic cell vaccine approach is being studied for use in subjects with these and other cancers: prostate cancer, melanoma, kidney cancer, colorectal cancer, lung cancer, breast cancer, leukemia, and non-Hodgkin lymphoma.
  • Sipuleucel-T Provenge
  • DNA vaccines are now being studied in clinical trials for use against the
  • compositions may be administered individually to a patient, or they may be administered in combination with other drugs, hormones, agents, and the like.
  • the methods of treatment disclosed herein are useful against many mammalian tumors, including, for example, cervical cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer, hepatoma, glioblastoma, ovarian cancer, and head and neck cancers.
  • tumor means a neoplastic growth which may, or may not be malignant.
  • compositions and methods provided herein are not only useful in the treatment of tumors, but in their micrometastses and their macrometastses.
  • micrometastasis is a form of metastasis (the spread of a cancer from its original location to other sites in the body) in which the newly formed tumors are identified only by histologic examination; micrometastases are detectable by neither physical exam nor imaging techniques.
  • macrometastses are usually large secondary tumors.
  • One of Flt3L’s functions is to stimulate the in vivo expansion of circulating DCs, specifically the conventional DCI (cDC1) and plasmacytoid DC (pDC) subsets. 21-26 This expansion is substantial, with expanded cDC1 counts and pDC counts increasing by 130-fold and 6- to 16- fold respectively. 26 In addition, Flt3L administration results in increased numbers of peripheral DC. On the other hand, inhibition of Flt3L signaling leads to the reduction in frequency of DC differentiation and activation.
  • Albumin is a ubiquitous plasma protein with a longer circulatory half-life of approximately 3 weeks, owing to its ability to evade lysosomal degradation through interactions with the neonatal Fe receptor (FcRn) responsible for cellular recycling. 42,42 In addition, serum albumin exhibits the capacity for preferential uptake by tumor cells and accumulates intratumorally. 1, 43 [000219] In this study, the administration of novel alb-Flt3L DNA, a plasmid encoding the alb-Flt3L protein, was explored as an alternative therapeutic strategy to induce an anti-tumor effect.
  • alb-Flt3L DNA mediated by electroporation and without concomitant antigen given, can be delivered and taken up effectively by tumor cells, leading to in vivo production of alb-Flt3L.
  • the treatment effect will be the observation of a controlled tumor response, characterized by enhanced DC mobilization and accumulation in both the lymph nodes and TME, coupled with an increase in the expansion of cDC1 subsets and a heightened level of CD8 + T cell activation.
  • MATERIALS AND METHODS [000221] Mouse experiments: Six- to eight-week-old female C57BL/6 mice were purchased from Charles River Laboratories (Frederick, Maryland, USA).
  • mice were maintained under specific pathogen-free regulation at the Johns Hopkins University School of Medicine Animal Facility (Baltimore, Maryland, USA). All procedures were performed according to preapproved protocols and in accordance with recommendations for the proper use and care of laboratory animals.
  • Cell culture TC-1 cells were maintained in DMEM media supplemented with 1 0% FBS, I% L- glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 2 mM non- essential amino acid, and 2 mM sodium pyruvate.
  • Tumor experiment Subcutaneous tumor models were established through inoculation of 2x10 TC-1 cells in 50 ⁇ l of PBS in the mice thigh.
  • Tumor growth was measured two or three times a week with a digital caliper. Tumor volume was then calculated by the following formula: length x length x width x 0.5. The experiment involving the tumor-bearing mice were terminated when tumor volume exceeds 1,500 mm 3 or 20 mm in length, or significant body weight loss in accordance with the animal protocol.
  • Plasmid DNA constructs and preparation The generation of pcDNA3-alb (albumin), pcDNA3-GLuc (Gaussia Luciferase), pcdna3-albGluc, and pcDNA3-Flt3L have been described previously. 39, 40 Next, for the generation of pcDNA3-albFlt3L, mouse Flt3 ligand was
  • TC-1 tumors were surgically removed from the mice and placed in RPMI-1640 medium. The tumor was grounded into smaller pieces and incubated with tissue digestion buffer containing collagenase I (0.05mg/ml), collagenase IV (0.05mg/ml) and DNase I (l00U/ml). Tumor specimen was then dissociated using a gentleMACSTM Tissue Dissociator according to manufacturing protocol.
  • Gaussia Luciferase activity in vivo Gaussia luciferase (20 ⁇ g) or alb-GLuc (20 ⁇ g) were diluted in 30ul PBS and injected intratumorally in TC- 1-bearing mice, which was followed by electroporation. Blood from mice was collected at indicated time after injection.
  • 59 167460060.1 membrane was hybridized with anti-Flt3L antibody (from R&Dsystems) or anti-b-actin (from ThermoFisher scientific).
  • Cell preparation for flow cytometry analysis Mice blood samples were collected from the submandibular vein using a lancet and mixed with 10% EDTA solution. RBC lysis was performed twice using RBC lysis buffer (Cell Signaling Technology, Danvers, Massachusetts). Zombie Aqua live/dead (BioLegend, San Diego, CA) was used for live cell detection according to manufacturing instructions. Flow cytometry data was collected using a 13-color Beckman Coulter CytoFLEX S with CellQuest software.
  • FlowJo 10.4 software (FlowJo LLC) was used for data analysis.
  • Intracellular cytokine staining and flow cytometry analysis To detect HPV16 E7-specific CD8 + T cell responses by IFN- ⁇ intracellular staining, splenocytes from mice were incubated with HPV16 E7 (aa 49 to 57) peptide (1 mg/ml), in the presence of GolgiPlug (l ml/ml, 1:1,000 dilution of the antibody; BD Pharmingen, San Diego, CA) at 37°C overnight.
  • the stimulated splenocytes were then washed with PBS containing 0.5% BSA and stained further with PE-conjugated anti-mouse CD8a (1 ml/sample, 1:800 dilution of the antibody) at 4°C for 30 min. After washing, the cells were fixed and permeabilized using the Cytofix/Cytoperm kit according to the manufacturer's instructions (eBioscience, San Diego, CA) at 37°C for 30 min. Furthermore, intracellular IFN- ⁇ was stained with FITC-conjugated anti- mouse IFN- ⁇ at 4°C for 30 min. After washing, the cells were pipetted and resuspended in PBS plus 0.5% BSA.
  • TC-1-bearing mice were treated with murine albumin-fused Gaussia luciferase (alb-GLuc) or GLuc intratumorally, followed by electroporation. Mice blood
  • mice were euthanized, and tissues were extracted for luminescence analysis. Tumor samples were dissociated and measured with fixed protein weight (l00 ⁇ g). Alb-GLuc demonstrated significantly higher concentration in tumors compared to tumors administered with GLuc and vector control (FIG.1B). Similar luminescence detection results were also found in tumor-drainage lymph nodes (FIG.1C).
  • alb-Flt3L DNA was generated, a plasmid encoding the alb-Flt3L protein (FIG.1D).
  • HEK293 cells were transfected with alb- Flt3L DNA.
  • Western blot verified the formation of the alb-Flt3L protein (FIG.1E).
  • alb-Flt3L DNA treatment demonstrates superior tumor control and survival: Subsequently, it was sought to determine whether alb-Flt3L DNA delivery can generate tumor control and an anti-tumor response in TC-1-bearing mice.
  • alb-Flt3L DNA was vaccinated intratumorally followed by electroporation for a total of three times in 5-day intervals (FIG.2A). It was found that the alb-Flt3L DNA group demonstrated superior anti-tumor effect, in terms of tumor volume and tumor weight (FIGS.2B, 2C, 2D).
  • Survival analysis revealed that mice administered with alb-Flt3L DNA had a superior overall survival compared to other groups (FIG.2E).
  • alb-Flt3L DNA treatment provides a promising therapeutic anti-tumor effect in vivo.
  • alb-Flt3L DNA treatment elicited robust expansion of type I conventional dendritic cells in vivo: Tumor antigen presentation via antigen processing cells is a critical step for the initiation of an anti-tumor cytotoxic immune response.
  • Flt3L regulates signaling for DC differentiation it was further investigated the biological effects of alb-Flt3L DNA on DCs. Mice blood was collected from tumor-bearing mice treated with the protocol described and immune cells were analyzed with flow cytometry.
  • 61 167460060.1 gain greater insight into the immune context within the TME, TC-1 tumors and their corresponding tumor drainage lymph nodes were harvested at the end of the experiment. As expected, higher proportions of DCs were presented in tumor-infiltrated lymphocytes after alb- Flt3L DNA treatment. (FIGS.3E, 3F). An increased level of cDC1 differentiation was also noted accordingly (FIGS.3G, 3H). Similarly, elevated numbers of DC (FIGS.3I, 3J) and cDC1 (FIGS. 3K, 3L) were found in tumor-drainage lymph nodes.
  • alb-Flt3L activates DCs, predominantly cDC1 populations, to elicit the anti-tumor immunity observed.
  • alb-Flt3L treatment generates potent CD8 + cytotoxic T cells against tumor: Cytotoxic T cell reaction is a major component upon DC activation against tumor. Therefore, TC-1-bearing mice were used to confirm the immune reaction responsible for alb- Flt3L delivery. After treatment, mice were euthanized and their splenocytes were isolated.
  • Nucleated hematopoietic cells significantly increased in alb-Flt3L DNA-treated mice, which is related to enhanced hematopoietic production (FIGS.4A, 4B). Furthermore, the level of IFN- ⁇ -secreting CD8 + splenocyte was significantly greater after alb-Flt3L injection among all groups, compared with naked DNA (FIGS.4C, 4D). As expected, IFN- ⁇ -secreting CD8 + lymphocytes also increased in locoregional lymphoid tissue (FIGS.4E, 4F).
  • FIG.6A Histological examination of key vital organs, including heart (FIG.6A), lung (FIG.6B), liver (FIG.6C), pancreas (FIG.6D), and kidney (FIG.6E) revealed no abnormal findings, with results within normal limits for mice in the control group, the Flt3L treatment group, or the Alb-Flt3L treatment group. Taken together, these findings suggest that treatment with Flt3L or Alb-Flt3L is safe and well tolerated in mice. [000238] DISCUSSION [000239] This study illustrated preliminary results in the electroporation-mediated delivery of alb- Flt3L DNA without giving additional tumor-specific antigens. This success is underscored by the demonstration of an enhanced tumor control and increased overall survival
  • 62 167460060.1 achieved through the administration of alb-Flt3L DNA when compared to naked Flt3L DNA. Further analysis of the immunological responses revealed that alb-Flt3L DNA delivery expands more DCs -specifically cDC1 populations- consistently in the tumor, lymph nodes, and bloodstream. The ability to expand cDC1 population is crucial in antitumor immunity, as this DC subset cross-presents to CD8 + T cells, thereby activating a tumor-specific cytotoxic response. 6-8, 44 Consistent with the established impact of expanding the cDC1 population, the alb-Flt3L DNA- induced antitumor response exhibited a significantly higher percentage of activated and tumor- specific CD8 + T cells.
  • TME T cell immunoglobulin mucin receptor 3
  • HMGB1 high mobility group protein B1
  • cDC1s are associated with superior antigen cross- presentation and are crucial for cellular immunity against tumors. 22, 45 Evidence revealed that the augmentation and recruitment of cDC1s is beneficial for tumor control, and can even overcome resistance to anti-PD1 therapy. 9, 46 Therefore, this alb-Flt3L-regulated cDC1 effect is an attractive response that can innovate the current lines of immunotherapy.
  • cytokines such as IL-2 and IL-12
  • plasmid delivery presents a streamlined alternative that circumvents the time and cost-intensive processes of transfecting optimized plasmids into bacterial hosts, as well as the subsequent isolation and purification of proteins.
  • These logistical simplifications of plasmid delivery, coupled with their potent antitumor responses, underscore their important biological value and warrant further exploration of this approach as a combination therapy with mainstream immunotherapeutic strategies to bolster the current fight against solid tumors.
  • Transfection efficiency is a major determinant of successful DNA delivery. In terms of transfection efficacy, intramuscular (IM) injection followed by electroporation is regarded as a superior approach compared with conventional IM injection alone.
  • intratumoral electroporation has been regarded as a favorable approach to generate sufficiently high concentration of gene expression with less off-target toxicity, one that reaches therapeutic levels. 59 The work herein revealed that intratumoral electroporation is a feasible strategy for alb- Flt3L DNA delivery.
  • one viable approach is to deliver alb-Flt3L self-replicating messenger RNA enclosed within lipid nanoparticles or cationic nanoemulsions.
  • This strategy as evidenced by the development of SARS-CoV2 vaccines and cancer therapeutic vaccines, offers rapid and low-cost manufacturing, along with safe administration and the potential to elicit high potency in humans.
  • electroporation-mediated plasmid delivery is a therapeutic technique known for its high transfection efficiency and demonstrated effectiveness in both preclinical and clinical studies.
  • mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood 95: 3489-3497. 21. C. Waskow, K. Liu, G. Darrasse-Jeze, P. Guermonprez, F. Ginhoux, M. Merad, T.
  • DNGR-1 limits Flt3L-mediated antitumor immunity by restraining tumor- infiltrating type I conventional dendritic cells.
  • J lmmunother Cancer 9. 25. E. Maraskovsky, E. Daro, E. Roux, M. Teepe, C.R. Maliszewski, J. Hoek, D. Caron, M. E. Lebsack, and H. J. McKenna (2000). In vivo generation of human dendritic cell subsets by Flt3 ligand. Blood 96: 878-884. 26. G. Breton, J. Lee, Y. J. Zhou, J. J. Schreiber, T. Keler, S. Puhr, N. Anandasabapathy, S.

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Abstract

Des vecteurs ou des compositions d'acide nucléique codant pour des polypeptides d'albumine fusionnés à Flt3L, induisent des cellules dendritiques circulantes et génèrent une réponse antitumorale. Dans des aspects préférés, les compositions sont administrées par électroporation et possèdent une bioactivité plus persistante dans des organes ciblés.
PCT/US2025/014139 2024-01-31 2025-01-31 Compositions et procédés d'acide nucléique flt3l fusionné à l'albumine Pending WO2025166244A2 (fr)

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