WO2025166244A2 - Albumin-fused flt3l nucleic acid compositions and methods - Google Patents

Abstract

Vectors or nucleic acid compositions encoding polypeptides of albumin fused to Flt3L, induce circulating dendritic cells and generate an anti-tumor response. In preferred aspects, the compositions are delivered via electroporation and possess more persistent bioactivity in targeted organs.

Description

PCT APPLICATION ATTORNEY DOCKET NO.348358.17502 ALBUMIN-FUSED FLT3L NUCLEIC ACID COMPOSITIONS AND METHODS The present application claims the benefit of U.S. provisional application no.63/627,732 filed January 31, 2024, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0001] This invention was made with government support under grants HL 151530 and HL132055 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD [0002] The present disclosure relates in general to methods of cancer treatment. The disclosure relates in particular to compositions which induce an anti-tumor immune response by stimulating expansion of circulating dendritic cells (DCs). BACKGROUND [0003] Over the past decade, immunotherapy has changed the landscape of cancer management, characterized by the notable breakthroughs achieved through the utilization of immune checkpoint inhibitors (ICI) and chimeric antigen receptor T-cell (CAR-T cell) therapy. The clinical success of immunotherapy has unveiled a deeper understanding of the complexity and diverse immune components within the tumor microenvironment (TME). These current achievements have paved the way to utilize different immune cells to act on the TME for antitumor immunity.^{1, 2} Tumor-specific CD8⁺ T cell infiltration into the TME is a major component of tumor-specific immune reaction and serves as a prognosis for tumor regression in cancer patients.^3-5 Since a robust T cell response depends on adequate antigen identification, numerous strategies have been developed to target dendritic cells (DCs), thereby expanding the existing array of cancer treatment options. [0004] DCs are a heterogeneous group of antigen-presenting cells to mediate the intersection of the innate and adaptive immune responses. Upon the recognition of tumor antigens, DCs engage in cross-priming T cells, activating them for subsequent infiltration into 167460060.1 tumor lesions.^6-8 There are bodies of evidence describing the association between increased levels of DCs in the TME to better prognosis in cancer patients undergoing ICI treatments.^{9, 10} A deeper understanding of the TME revealed that DC functions were impaired by the tumor-infiltrative immunosuppressive cells and various counteractive cytokines produced by tumor cells.^11-13 Recently, several therapeutic strategies have been developed to target DCs to overcome these treatment limitations. For instance, STAT3 inhibitor has demonstrated efficacy to reverse the dysregulated DCs in the TME14 while mTOR inhibitor could enhance DC activation.¹⁵ There are also cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) which directly stimulates DC differentiation, activation, and migration.¹⁶ In clinical trials, Talimogene laherparepvec (T-VEC), an oncolytic virus encoding GM-CSF expression, has demonstrated durable clinical responses and has been approved for treatment in patients with advanced melanoma.¹⁷ Moreover, nanoparticle delivery of free antigens, in combination with adjuvants, can stimulate DCs and activate the tumor-specific T cells.¹⁸ Adoptive transfer of blood and monocyte- derived DCs loaded with a recombinant fusion tumor antigen has demonstrated improved tumor control and better patient survival, providing another feasible strategy for DC-based therapeutic cancer vaccination.¹⁹ Collectively, these novel DC-derived therapies offer both theoretically sound and clinically practical approaches in the modern era of immunotherapy. SUMMARY [0005] In one aspect, we now provide a composition comprising an expression vector or nucleic acid encoding a fusion polypeptide comprising albumin protein and FMS-like tyrosine kinase 3 ligand (Flt3L) protein. [0006] In certain preferred aspects, 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. [0007] In certain aspects, 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.

2 167460060.1 [0008] In certain embodiments, the Flt3L is human. [0009] In certain embodiments, the albumin is human. [00010] In certain embodiments, the Flt3L is murine. In certain embodiments, the albumin is murine. [00011] In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises at least one chemotherapeutic agent. In certain embodiments, the composition further comprises at least one immunotherapeutic agent. [00012] In a further aspect, methods of treating cancer in a subject is provided , 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. In certain preferred aspects, 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. [00013] In preferred aspect, the treatment methods may further comprise comprising applying electroporation to the subject in conjunction with administering the therapeutic composition. For example, 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. In preferred aspects, one or more tumors of the subject is treated by the electroporation, such as after administering the composition to the subject. [00014] In certain aspects, a method of treating cancer in a subject is provided, 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,

3 167460060.1 thereby treating the subject. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 1. [00015] In certain embodiments, the method further comprises administering one or more chemotherapeutics. [00016] In certain embodiments, the chemotherapeutics comprise chemotherapeutic agents, radiation, surgery or combinations thereof. [00017] In certain embodiments, 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 induces an anti-tumor immune response. [00018] In certain embodiments, the composition stimulates expansion of circulating dendritic cells (DCs) as compared to the subject’s baseline circulating dendritic cells. In certain embodiments, 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. In certain embodiments, the circulating dendritic cells comprise conventional dendritic cells (cDC1). In certain embodiments, 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. [00019] In another aspect, 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

4 167460060.1 to electroporation; thereby treating the subject. In certain embodiments, the protein comprises albumin or variants thereof. [00020] In certain embodiments, the method further comprises administering one or more chemotherapeutics. In certain embodiments, the chemotherapeutics comprise chemotherapeutic agents, radiation, surgery or combinations thereof. [00021] In certain embodiments, the expression vector comprises: a plasmid, viral vector, phage or a bacterial vector. In certain embodiments, the expression vector is a plasmid. [00022] In certain embodiments, the composition induces an anti-tumor immune response. [00023] In certain embodiments, the composition stimulates expansion of circulating dendritic cells (DCs) as compared to the subject’s baseline circulating dendritic cells. In certain embodiments, 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. In certain embodiments, the circulating dendritic cells comprise conventional dendritic cells (cDC1). [00024] In certain embodiments, 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. [00025] In another aspect, an expression vector encodes a fusion polypeptide comprising FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof and albumin. In certain embodiments, the expression vector is a plasmid. [00026] In another aspect, a nucleic acid encodes a fusion polypeptide comprising FMS- like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof, and albumin. [00027] In another aspect, a host cell comprises the expression vectors or nucleic acids embodied herein. [00028] In another aspect, the albumin protein is mammalian. In certain embodiments, the albumin protein can be murine, porcine, ovine, bovine, human, or combinations thereof.

5 167460060.1 [00029] In another aspect, the FMS-like tyrosine kinase receptor 3 ligand (Flt3L) is mammalian. In certain embodiments, the Flt3L can be murine, porcine, ovine, bovine, human, or combinations thereof. [00030] In another aspect, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, In certain embodiments, the circulating dendritic cells comprise conventional dendritic cells (cDC1). [00031] In reference to the expression vector encoding the fusion polypeptide composition of the present disclosure, 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. [00032] Definitions [00033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. [00034] It is also to be understood that the terminology used herein is for the purpose of

6 167460060.1 describing particular embodiments only and is not intended to be limiting. [00035] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, recitation of “a cell”, for example, includes a plurality of the cells of the same type. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” [00036] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/−20%, +/−10%, +/−5%, +/−1%, or +/−0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. [00037] As used herein, the term “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. [00038] The term, “amino acid” includes the residues of the natural α-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 β-amino acids, synthetic and unnatural amino acids. Many types of amino acid residues are useful in the adipokine polypeptides and the disclosure is not limited to natural, genetically-encoded amino acids. Examples of amino acids that can be utilized in the peptides described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the reference cited therein.

7 167460060.1 Another source of a wide array of amino acid residues is provided by the website of RSP Amino Acids LLC. [00039] The term “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. 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. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements—or, as appropriate, equivalents thereof— and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc. [00041] “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. Thus, 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. Both 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

8 167460060.1 template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. [00042] The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter. [00043] As used herein, “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. Examples of vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, 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. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. [00044] As used herein, the term “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. Examples of 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.

9 167460060.1 [00045] The term “functional portion or fragment thereof,” with respect to the albumin protein, means that the portion or fragment of the albumin polypeptide retains its ability to bind to the neonatal Fc receptor and traffic through the lymphatic system. [00046] Unless otherwise specified, a “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. The phrase 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). [00047] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. [00048] “Parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques. [00049] The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates. [00050] The term “polynucleotide” is a chain of nucleotides, also known as a “nucleic acid”. As used herein 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. [00051] The terms “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

10 167460060.1 homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The 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. [00052] 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. [00053] The terms “pharmaceutically acceptable” (or “pharmacologically acceptable”) refer 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. The term “pharmaceutically acceptable carrier,” as used herein, 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. [00054] The term “promoter” as used herein is defined as a nucleic acid (e.g. DNA) sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. [00055] As used herein, the term “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. [00056] 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. [00057] 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.

11 167460060.1 [00058] 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. [00059] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs. [00060] 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. [00061] “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. Accordingly, “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. The benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician. [00062] “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

12 167460060.1 that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. 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. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, are intended to encompass homologous and/or orthologous genes and gene products from other species. [00064] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. [00065] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS [00066] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. [00067] FIGS.1A-1E are a series of graphs and a blot demonstrating that albumin-fused Gaussia luciferase (alb-GLuc) exhibits extended biodistribution under local injection compared

13 167460060.1 to GLuc. C57BL/6 mice were inoculated with 2xl 0⁴ TC-1 cells and utilized for tissue biodistribution experiment once tumors reached approximately 0.5 cm in diameter. alb-Gluc (20µg), Gluc (20µg) 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.1A) At different timepoints post-injection, serum luciferase activity was determined in all groups (n=4-5). Luminescence was measured as described. (FIG.1B) Mice were euthanized and tumor was collected and grinded. Tumor protein (100ug) was taken from each sample. Kinetics curve was shown in relative light unit (n=4-5). (FIG.1C) Tumor- drainage lymph node was collected. Kinetics curve was shown in relative light unit (n=4-5). (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). [00068] 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 2xl0⁴ TC-1 cells in thigh. alb-Flt3L DNA (10µg), Flt3L DNA (10µg) 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. (FIG.2B) Mice were euthanized and tumor sample was isolated. Illustration of tumor appearance following the described treatment protocol (n=5). (FIG.2C) Tumor weight for the indicated treatment groups (n=5). (FIG.2D) Tumor growth curve following the described treatment protocol (n=5). (FIG.2E) Survival curve was presented in percentages (n=5). All data was presented as the mean± SEM, (*,p < 0.05; **,p< 0.01; ***, p< 0.001; ****,p< 0.0001). [00069] 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. (FIG.3A) Peripheral blood mononuclear cells (PBMCs) were collected on Day 12. Representative gating of DC population on flow cytometry. Total DC counts as shown among the indicated treatment groups (n=4-5). (FIGS.3B, 3C)

14 167460060.1 Representative flow gating and quantification of blood cDC1 for the indicated treatment groups (n=4-5). (D) Quantification of blood cDC2 for the indicated treatment groups (n=4-5). (FIGS. 3E, 3F) Mice were euthanized on Day 18 and tumor-infiltrated immune cells were isolated. Representative flow gating and quantification of tumor- infiltrated DC (n=4-5) and (FIGS.3G, 3H) cDC1 population for the indicated treatment groups (n=4- 5). (FIGS.3I, 3J) Mice were euthanized on Day 18 and tumor-drainage lymph nodes were isolated. Representative flow gating and quantification of tumor-infiltrated DC (n=4-5) and (FIGS.3K, 3L) cDC1 for the indicated treatment groups (n=4-5). All data was presented as the mean± SEM, (*,p < 0.05; **,p< 0.01; ***,p< 0.001; ****,p< 0.0001). [00070] 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-γ-secreting CD8⁺ T cells. (FIGS. 4A, 4B) TC-1-bearing C57BL/6 mice following the described treatment protocol were euthanized while splenocytes were isolated. Numbers of CD45⁺ splenocyte for the indicated treatment groups (n=4-5). (FIGS.4C, 4D) Mice PBMC was collected on Day 18 following described treatment protocol. Representative flow gating and quantification of IFN-γ-secreting CD8⁺ T cell for the indicated treatment groups (n=4-5). (FIGS.4E, 4F) Representative flow gating and quantification of IFN-r-secreting cos+ splenocyte for the indicated treatment groups (n=4-5). All data was presented as the mean± SEM, (*, p < 0.05; **,p< 0.01; ***,p< 0.001; ****,p< 0.0001). [00071] 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. (200X). Alb-Flt3L DNA vaccination is well-tolerated in mice while Alb-Flt3L protein vaccination is not well- tolerated in mice.

15 167460060.1 [00072] 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. (200X) DETAILED DESCRIPTION [00073] The FMS-like tyrosine kinase receptor 3 (Flt3) is a tyrosine kinase receptor expressed extensively on hematopoietic progenitor cells, and Flt3 ligand (Flt3L) is a pluripotent growth factor that regulates the maturation and differentiation of DCs in hematopoiesis. Notably, Flt3L acts as an essential element in the development of the immune system.^{20, 21} 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.^{1, 43} The disclosure herein is based on the finding that a plasmid encoding a fusion polypeptide comprising Flt3L displays therapeutic effects against cancer. [00074] Accordingly, in certain embodiments, 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.

16 167460060.1 [00075] Also included in the scope of the disclosure 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. In reference to the original albumin and/or Flt3L polypeptide, or protein, 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. [00076] Amino acid sequence of Alb-Flt3L, SEQ ID NO: 1 Human albumin-linker(italics)-Human Flt3 ligand (underlined) MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIA FAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCT VATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTA FHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAA CLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKC CTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQ TALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGLEFTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQ DEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQP PPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPVETVF

17 167460060.1 HRVSQDGLDLLTS [00077] Nucleic acid sequence of Human albumin (black)-linker(italics)-Human Flt3 ligand (underlined) SEQ ID NO: 2: atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcacacaagagtgaggttgctca tcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaa ttagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaa ttatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaac acaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttga aaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgt tgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactca agtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgca gaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggacctt gccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccg aagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaag gatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaacc actctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaattt aatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtg tcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgc agaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaat ccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgc agatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaa gagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggaggg taaaaaacttgttgctgcaagtcaagctgccttaggcttagaattcacccaggactgctccttccaacacagccccatctcctccgacttcgct gtcaaaatccgtgagctgtctgactacctgcttcaagattacccagtcaccgtggcctccaacctgcaggacgaggagctctgcgggggcc tctggcggctggtcctggcacagcgctggatggagcggctcaagactgtcgctgggtccaagatgcaaggcttgctggagcgcgtgaac acggagatacactttgtcaccaaatgtgcctttcagcccccccccagctgtcttcgcttcgtccagaccaacatctcccgcctcctgcaggag acctccgagcagctggtggcgctgaagccctggatcactcgccagaacttctcccggtgcctggagctgcagtgtcagcccgtagagacg gtgtttcaccgtgtcagccaggatggtctcgatctcctgacctcg [00078] Mouse albumin (black)-linker(italics)-Mouse Flt3 ligand (underlined) amino acid sequence, SEQ ID NO: 3: MKWVTFLLLLFVSGSAFSRGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCS YDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTK QEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAP ELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERA FKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCEN QATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDV FLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEP KNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPE DQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEF KAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKA ADKDTCFSTEGPNLVTRCKDALAEFGTPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTV

18 167460060.1 AVNLQDEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPEC LRFVQTNISHLLKDTCTQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIALEATE LPEPRPRQ [00079] Mouse albumin (black)-linker(italics)-Mouse Flt3 ligand (underlined) nucleic acid sequence, SEQ ID NO: 4: atgaagtgggtaacctttctcctcctcctcttcgtctccggctctgctttttccaggggtgtgtttcgccgagaagcacacaagagtgagatcgc ccatcggtataatgatttgggagaacaacatttcaaaggcctagtcctgattgccttttcccagtatctccagaaatgctcatacgatgagcatg ccaaattagtgcaggaagtaacagactttgcaaagacgtgtgttgccgatgagtctgccgccaactgtgacaaatcccttcacactctttttgg agataagttgtgtgccattccaaacctccgtgaaaactatggtgaactggctgactgctgtacaaaacaagagcccgaaagaaacgaatgttt cctgcaacacaaagatgacaaccccagcctgccaccatttgaaaggccagaggctgaggccatgtgcacctcctttaaggaaaacccaac cacctttatgggacactatttgcatgaagttgccagaagacatccttatttctatgccccagaacttctttactatgctgagcagtacaatgagatt ctgacccagtgttgtgcagaggctgacaaggaaagctgcctgaccccgaagcttgatggtgtgaaggagaaagcattggtctcatctgtcc gtcagagaatgaagtgctccagtatgcagaagtttggagagagagcttttaaagcatgggcagtagctcgtctgagccagacattccccaat gctgactttgcagaaatcaccaaattggcaacagacctgaccaaagtcaacaaggagtgctgccatggtgacctgctggaatgcgcagatg acagggcggaacttgccaagtacatgtgtgaaaaccaggcgactatctccagcaaactgcagacttgctgcgataaaccactgttgaagaa agcccactgtcttagtgaggtggagcatgacaccatgcctgctgatctgcctgccattgctgctgattttgttgaggaccaggaagtgtgcaa gaactatgctgaggccaaggatgtcttcctgggcacgttcttgtatgaatattcaagaagacaccctgattactctgtatccctgttgctgagac ttgctaagaaatatgaagccactctggaaaagtgctgcgctgaagccaatcctcccgcatgctacggcacagtgcttgctgaatttcagcctc ttgtagaagagcctaagaacttggtcaaaaccaactgtgatctttacgagaagcttggagaatatggattccaaaatgccattctagttcgctac acccagaaagcacctcaggtgtcaaccccaactctcgtggaggctgcaagaaacctaggaagagtgggcaccaagtgttgtacacttcct gaagatcagagactgccttgtgtggaagactatctgtctgcaatcctgaaccgtgtgtgtctgctgcatgagaagaccccagtgagtgagcat gttaccaagtgctgtagtggatccctggtggaaaggcggccatgcttctctgctctgacagttgatgaaacatatgtccccaaagagtttaaag ctgagaccttcaccttccactctgatatctgcacacttccagagaaggagaagcagattaagaaacaaacggctcttgctgagctggtgaag cacaagcccaaggctacagcggagcaactgaagactgtcatggatgactttgcacagttcctggatacatgttgcaaggctgctgacaagg acacctgcttctcgactgagggtccaaaccttgtcactagatgcaaagacgccttagccgaattcgggacacctgactgttacttcagccaca gtcccatctcctccaacttcaaagtgaagtttagagagttgactgaccacctgcttaaagattacccagtcactgtggccgtcaatcttcagga cgagaagcactgcaaggccttgtggagcctcttcctagcccagcgctggatagagcaactgaagactgtggcagggtctaagatgcaaac gcttctggaggacgtcaacaccgagatacattttgtcacctcatgtaccttccagcccctaccagaatgtctgcgattcgtccagaccaacatc tcccacctcctgaaggacacctgcacacagctgcttgctctgaagccctgtatcgggaaggcctgccagaatttctctcggtgcctggaggt gcagtgccagccggactcctccaccctgctgcccccaaggagtcccatagccctagaagccacggagctcccagagcctcggcccagg cag [00080] An example of an Flt3L variant is the Homo sapiens FMS-related receptor tyrosine kinase 3 ligand (FLT3LG), transcript variant 3, mRNA (Accession: NM_001459 XM_005258679, Version: NM_001459.4) SEQ ID NO: 5: 1 cctttcactt tcggtctctg gctgtcaccc ggcttggccc cttccacacc caactggggc 61 aagcctgacc cggcgacagg aggcatgagg ggcccccggc cgaaatgaca gtgctggcgc 121 cagcctggag cccaacaacc tatctcctcc tgctgctgct gctgagctcg ggactcagtg 181 ggacccagga ctgctccttc caacacagcc ccatctcctc cgacttcgct gtcaaaatcc 241 gtgagctgtc tgactacctg cttcaagatt acccagtcac cgtggcctcc aacctgcagg 301 acgaggagct ctgcgggggc ctctggcggc tggtcctggc acagcgctgg atggagcggc

19 167460060.1 361 tcaagactgt cgctgggtcc aagatgcaag gcttgctgga gcgcgtgaac acggagatac 421 actttgtcac caaatgtgcc tttcagcccc cccccagctg tcttcgcttc gtccagacca 481 acatctcccg cctcctgcag gagacctccg agcagctggt ggcgctgaag ccctggatca 541 ctcgccagaa cttctcccgg tgcctggagc tgcagtgtca gcccgactcc tcaaccctgc 601 cacccccatg gagtccccgg cccctggagg ccacagcccc gacagccccg cagccccctc 661 tgctcctcct actgctgctg cccgtgggcc tcctgctgct ggccgctgcc tggtgcctgc 721 actggcagag gacgcggcgg aggacacccc gccctgggga gcaggtgccc cccgtcccca 781 gtccccagga cctgctgctt gtggagcact gacctggcca aggcctcatc ctggggagga 841 tactgaggca cacagagggg agtcaccagc cagaggatgc atagcctgga cacagaggaa 901 gttggctaga ggccggtccc ttccttgggc ccctctcatt ccctccccag aatggaggca 961 acgccagaat ccagcaccgg ccccatttac ccaactctgt acaaagccct tgtccccatg 1021 aaattgtata taaatcatcc ttttctacca [00081] Another example is the Homo sapiens FMS-related receptor tyrosine kinase 3 ligand (FLT3LG), transcript variant 1, mRNA (NCBI Reference Sequence: NM_001204502.2, Version: NM_001204502.2) SEQ ID NO: 6: 1 cctttcactt tcggtctctg gctgtcaccc ggcttggccc cttccacacc caactggggc 61 aagcctgacc cggcgacagg aggcatgagg ggcccccggc cgaaatgaca gtgctggcgc 121 cagcctggag cccaacaacc tatctcctcc tgctgctgct gctgagctcg ggactcagtg 181 ggacccagga ctgctccttc caacacagcc ccatctcctc cgacttcgct gtcaaaatcc 241 gtgagctgtc tgactacctg cttcaagatt acccagtcac cgtggcctcc aacctgcagg 301 acgaggagct ctgcgggggc ctctggcggc tggtcctggc acagcgctgg atggagcggc 361 tcaagactgt cgctgggtcc aagatgcaag gcttgctgga gcgcgtgaac acggagatac 421 actttgtcac caaatgtgcc tttcagcccc cccccagctg tcttcgcttc gtccagacca 481 acatctcccg cctcctgcag gagacctccg agcagctggt ggcgctgaag ccctggatca 541 ctcgccagaa cttctcccgg tgcctggagc tgcagtgtca gcccgactcc tcaaccctgc 601 cacccccatg gagtccccgg cccctggagg ccacagcccc gacagccccg cagccccctc 661 tgctcctcct actgctgctg cccgtgggcc tcctgctgct ggccgctgcc tggtgcctgc 721 actggcagag gacgcggcgg aggacacccc gccctgggga gcaggtgccc cccgtcccca 781 gtccccagga cctgctgctt gtggagcact gacctggcca aggcctcatc ctgcggagcc 841 ttaaacaacg cagtgagaca gacatctatc atcccatttt acaggggagg atactgaggc 901 acacagaggg gagtcaccag ccagaggatg catagcctgg acacagagga agttggctag 961 aggccggtcc cttccttggg cccctctcat tccctcccca gaatggaggc aacgccagaa 1021 tccagcaccg gccccattta cccaactctg tacaaagccc ttgtccccat gaaattgtat 1081 ataaatcatc cttttctacc a [00082] The nucleic acids of the disclosure can, for example, encode the amino acid sequence of the albumin and/or Flt3L polypeptide fusion protein with at least one or more conservative amino acid substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same chemical or physical

20 167460060.1 properties. For instance, 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. [00083] 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. For example, 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. [00084] Alternatively or additionally, 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. Preferably, 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. [00085] It will be understood by those of ordinary skill in the art that 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. [00086] In some embodiments, the nucleic acids encode a mammalian Flt3L protein. In certain embodiments, the Flt3L protein can be murine, porcine, ovine, bovine, human, or combinations thereof. [00087] In accordance with an embodiment, the present disclosure provides a composition

21 167460060.1 comprising a nucleic acid encoding a human albumin protein, or a functional portion or fragment, or variant thereof, and a human Flt3L protein, or a functional portion or fragment, or variant thereof, such as, for example, SEQ ID NO: 1. [00088] In certain embodiments, the 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. [00089] In some embodiments, the polynucleotides provided herein encoding one or more fusion proteins include codon-optimized sequences. As used herein, the term “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). [00090] 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. 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.

22 167460060.1 [00091] In some embodiments, the nucleotide sequence of any region of an RNA or DNA sequence embodied herein may be codon optimized. In certain embodiments, 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. In some examples, 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. [00092] In certain embodiments, the polynucleotides of the disclosure can comprise one or more chemically modified nucleotides. Examples of 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. In nature, most 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. The use of RNA polynucleotides comprising chemically modified nucleotides have been shown to improve RNA expression, expression rates, half-life and/or expressed protein concentrations. 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. [00093] Examples of modified or chemically modified nucleotides include 5- hydroxycytidines, 5-alkylcytidines, 5-hydroxyalkylcytidines, 5-carboxycytidines, 5- formylcytidines, 5-alkoxycytidines, 5-alkynylcytidines, 5-halocytidines, 2-thiocytidines, N⁴- alkylcytidines, N⁴-aminocytidines, N⁴-acetylcytidines, and N⁴, N⁴-dialkylcytidines. [00094] Examples of 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-

23 167460060.1 thiocytidine; N⁴-methylcytidine, N⁴-aminocytidine, N⁴-acetylcytidine, and N⁴, N⁴- dimethylcytidine. [00095] Examples of 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. [00096] Examples of 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. [00097] Examples of 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. [00098] Examples of modified or chemically-modified nucleotides include N⁶- methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8- oxoadenosine, 8-bromoadenosine, 2-methylthio-N⁶-methyladenosine, N⁶-isopentenyladenosine, 2-methylthio-N⁶-isopentenyladenosine, N⁶-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N⁶- (cis-hydroxyisopentenyl)adenosine, N⁶-glycinylcarbamoyladenosine, N⁶-threonylcarbamoyl- adenosine, N⁶-methyl-N⁶-threonylcarbamoyl-adenosine, 2-methylthio-N⁶-threonylcarbamoyl- adenosine, N⁶,N⁶-dimethyladenosine, N⁶-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N⁶- hydroxynorvalylcarbamoyl-adenosine, N⁶-acetyl-adenosine, 7-methyl-adenine, 2-methylthio- adenine, 2-methoxy-adenine, alpha-thio-adenosine, 2'-0-methyl-adenosine, N⁶,2'-O-dimethyl- adenosine, N⁶,N⁶,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine,

24 167460060.1 2-amino-N⁶-methyl-purine, 1-thio-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara- adenosine, and N⁶-(19-amino-pentaoxanonadecyl)-adenosine. [00099] Examples of modified or chemically modified nucleotides include N¹- alkylguanosines, N²-alkylguanosines, thienoguanosines, 7-deazaguanosines, 8-oxoguanosines, 8- bromoguanosines, O⁶-alkylguanosines, xanthosines, inosines, and N¹-alkylinosines. [000100] Examples of modified or chemically modified nucleotides include N¹- methylguanosine, N²-methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8- bromoguanosine, O⁶-methylguanosine, xanthosine, inosine, and N¹-methylinosine. [000101] Examples of nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art. [000102] Examples of 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. [000103] Examples of modified and chemically modified nucleotide monomers include 3'- end stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides, and 3'-inverted thymidine. [000104] Examples of 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. In an exemplary embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA). [000105] Examples of modified and chemically modified nucleotide monomers include 2',4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-Ethyl (cEt) modified DNAs. [000106] Examples of modified and chemically modified nucleotide monomers include 2'- amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides, and 2'-O-allyl nucleotides.

25 167460060.1 [000107] Examples of modified and chemically modified nucleotide monomers include N6- methyladenosine nucleotides. [000108] Examples of 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. [000109] Examples of modified and chemically modified nucleotide monomers include 2'- O-aminopropyl substituted nucleotides. [000110] Examples of 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. [000111] 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. [000112] In certain embodiments, when the nucleic acid is an RNA, the RNA molecules can be engineered to comprise one or more modified nucleobases. For example, known modifications of 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; N⁴- methylcytidine; N⁴-2′-O-dimethylcytidine; N⁴-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; 5- methoxycarbonylmethyluridine; 5-methoxycarbonylmethyl-2′-O-methyluridine; 5- methoxycarbonylmethyl-2′-thiouridine; 5-carbamoylmethyluridine; 5-carbamoylmethyl-2′-O- methyluridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl) uridinemethyl

26 167460060.1 ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methylaminomethyl-2- thiouridine; 5-methylaminomethyl-2-selenouridine; 5-carboxymethylaminomethyluridine; 5- carboxymethylaminomethyl-2′-O-methyl-uridine; 5-carboxymethylaminomethyl-2-thiouridine; dihydrouridine; dihydroribosylthymine; 2′-methyladenosine; 2-methyladenosine; N⁶Nmethyladenosine; N⁶,N⁶-dimethyladenosine; N⁶,2′-O-trimethyladenosine; 2 methylthio- N⁶Nisopentenyladenosine; N⁶-(cis-hydroxyisopentenyl)-adenosine; 2-methylthio-N⁶-(cis- hydroxyisopentenyl)-adenosine; N⁶-glycinylcarbamoyl)adenosine; N⁶ threonylcarbamoyl adenosine; N⁶-methyl-N⁶-threonylcarbamoyl adenosine; 2-methylthio-N⁶-methyl-N⁶- threonylcarbamoyl adenosine; N⁶-hydroxynorvalylcarbamoyl adenosine; 2-methylthio-N⁶- hydroxnorvalylcarbamoyl adenosine; 2′-O-ribosyladenosine (phosphate); inosine; 2′O-methyl inosine; 1-methyl inosine; 1; 2′-O-dimethyl inosine; 2′-O-methyl guanosine; 1-methyl guanosine; N²-methyl guanosine; N²,N²-dimethyl guanosine; N²,2′-O-dimethyl guanosine; N²,N²,2′-O- trimethyl guanosine; 2′-O-ribosyl guanosine (phosphate); 7-methyl guanosine; N²; 7-dimethyl guanosine; N²; N²; 7-trimethyl guanosine; wyosine; methylwyosine; under-modified hydroxywybutosine; wybutosine; hydroxywybutosine; peroxywybutosine; queuosine; epoxyqueuosine; galactosyl-queuosine; mannosyl-queuosine; 7-cyano-7-deazaguanosine; archaeosine [also called 7-formamido-7-deazaguanosine]; and 7-aminomethyl-7-deazaguanosine. [000113] 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. Generally, 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. [000114] 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

27 167460060.1 phosphoramidite technology) or as a series of oligonucleotides. For example, 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. a plasmid. Isolated nucleic acids of the disclosure also can be obtained by mutagenesis of, e.g., a naturally occurring portion of Flt3L and/or albumin DNA. [000115] In some embodiments, the nucleic acid is a synthetic polynucleotide. In some embodiments, 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. Generally, a modified inter-nucleoside linker includes any linker other than other than phosphodiester (PO) liners, that covalently couples two nucleosides together. In some embodiments, the modified inter-nucleoside linker increases the nuclease resistance of the nucleic acid compared to a phosphodiester linker. For naturally occurring oligonucleotides, the inter-nucleoside linker includes phosphate groups creating a phosphodiester bond between adjacent nucleosides. In some embodiments, the nucleic acid comprises one or more inter- nucleoside linkers modified from the natural phosphodiester. In some embodiments all of the inter-nucleoside linkers of the nucleic acid or contiguous nucleotide sequence thereof, are modified. For example, in some embodiments the inter-nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage. [000116] Modifications to the ribose sugar or nucleobase can also be utilized herein. Generally, a modified nucleoside includes the introduction of one or more modifications of the sugar moiety or the nucleobase moiety. In some embodiments, the nucleic acids, as described, 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. Numerous nucleosides with modification of the ribose sugar moiety can be utilized, primarily with the aim of improving certain properties of

28 167460060.1 oligonucleotides, such as affinity and/or stability. Such 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). 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. [000117] 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. Indeed, much focus has been spent on developing 2′ substituted nucleosides, and numerous 2′ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity. 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. Examples of 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. By way of further example, in some embodiments, the modification in the ribose group comprises a modification at the 2′ position of the ribose group. In some embodiments, 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). [000118] In some embodiments, 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.

29 167460060.1 [000119] The nucleic acids of the disclosure, including DNA, RNA or nucleic acids encoding the fusion polypeptide, 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. Generally, 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. [000120] 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. [000121] In certain embodiments, the fusion polypeptides are synthesized from an expression vector encoding the DNA molecule, as described in detail elsewhere herein. [000122] NUCLEIC ACIDS AND VECTORS [000123] In some embodiments, the composition of the disclosure comprises a nucleic acid encoding one or more elements of the polypeptides described herein. For example, in some embodiments, the composition comprises an isolated nucleic acid encoding an alb- Flt3L peptide, or functional fragment or derivative thereof. [000124] In some embodiments, 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. [000125] The isolated nucleic acid may comprise any type of nucleic acid, including, but not limited to DNA and RNA. For example, in some embodiments, the composition comprises

30 167460060.1 an isolated DNA, including for example, an isolated cDNA, encoding a peptide of the disclosure, or functional fragment thereof. In some embodiments, 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. Other 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. [000127] In brief summary, the 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.

31 167460060.1 [000128] 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. In another embodiment, the disclosure provides a gene therapy vector. [000129] The nucleic acids of the disclosure can be cloned into a number of types of vectors. For example, 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. [000130] Further, 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. In general, 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). [000131] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, 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. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. For example, 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. In some embodiments, the composition

32 167460060.1 includes a vector derived from an adeno-associated virus (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. [000132] Further provided are nucleic acids encoding the polypeptides described herein. Also contemplated are adeno-associated virus (AAV) vectors comprising nucleic acids encoding the polypeptides described herein. In certain instances, 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. In certain instances, an AAV vector includes an AAV type viral particle (or virion) comprising a nucleic acid encoding a protein of interest. In some embodiments, as further described herein, 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). In some embodiments, the AAV vector is a human serotype AAV vector. In such embodiments, a human serotype AAV is derived from any known serotype, e.g., from AAV1, AAV2, AAV4, AAV6, or AAV9. In some embodiments, the serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8. [000133] 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. [000134] 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

33 167460060.1 vector and the AAV targeting vector are the same AAV capsid. Another suitable AAV is, e.g., rh10 (WO 2003/042397). Still other AAV sources include, e.g., AAV9 (see, for example, U.S. Pat. No.7,906,111; US 2011-0236353-A1), and/or hu37 (see, e.g., U.S. Pat. No.7,906,111; US 2011-0236353-A1), AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, (U.S. Pat. Nos.7,790,449; 7,282,199, WO 2003/042397; WO 2005/033321, WO 2006/110689; U.S. Pat. Nos.7,790,449; 7,282,199; 7,588,772). Still other AAV can be selected, optionally taking into consideration tissue preferences of the selected AAV capsid. [000135] In some embodiments, 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. Thus, in some embodiments, 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. Such 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. In certain embodiments, 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. In certain embodiments. other promoters used include promoters that are tissue specific for tissues such as kidney, spleen, and pancreas. Examples of 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 β-actin promoter. [000136] In some embodiments, 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. One skilled in the art understands that 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

34 167460060.1 condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc. [000137] In certain embodiments, 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. As used herein, “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. 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. [000138] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to 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. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. [000139] The selection of appropriate promoters can readily be accomplished. In certain aspects, one would use a high expression promoter. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. The Rous sarcoma virus (RSV) and MMT

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. [000140] Another example of a promoter is Elongation Growth Factor-1α (EF-1α). However, other 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. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. [000141] 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. In some embodiments, the vector of the present disclosure comprises one or more enhancers to boost transcription of the gene present within the vector. [000142] In order to assess the expression of the nucleic acid and/or peptide, 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. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both

36 167460060.1 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. [000143] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, 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. In general, 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. [000144] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, 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. In certain embodiments, the vectors and nucleic acids embodied herein, are introduced into the tumor or tissues via electroporation. [000145] 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.

37 167460060.1 [000146] 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. [000147] 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). [000148] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, 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. For example, 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. [000149] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl

38 167460060.1 phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). 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). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes. [000150] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant nucleic acid sequence in the host cell, a variety of assays may be performed. Such 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. [000151] In certain embodiments, the composition comprises a cell genetically modified to express one or more isolated nucleic acids and/or peptides described herein. For example, 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

39 167460060.1 [000152] 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 nucleic acids encoding the fusion polypeptides may be modified or programmed to be active under only certain conditions such as by using a tissue-specific promoter so that the encoded nuclease is preferentially or only transcribed in certain tissue types. [000154] In some embodiments, 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. In some embodiments, 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. [000155] In certain embodiments, 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). [000156] Liposomal delivery systems provide stable formulation, provide improved pharmacokinetics, and a degree of ‘passive’ or ‘physiological’ targeting to tissues. Encapsulation

40 167460060.1 of hydrophilic and hydrophobic materials, such as potential chemotherapy agents, are known. 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. [000157] Liposomes and polymerosomes can contain a plurality of solutions and compounds. In certain embodiments, the complexes of the invention are coupled to or encapsulated in polymerosomes. As a class of artificial vesicles, 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. Polymerosomes can be generated from double emulsions by known techniques, see Lorenceau et al., 2005, Generation of Polymerosomes from Double-Emulsions, Langmuir 21(20):9183-6, incorporated by reference. [000158] In certain embodiments, non-viral vectors are modified to effectuate targeted delivery and transfection. PEGylation (i.e. modifying the surface with polyethyleneglycol) 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. PEGylated nanoparticles are therefore often referred as “stealth” nanoparticles. PHARMACEUTICAL COMPOSITIONS [000159] Certain aspects of the instant disclosure pertain to pharmaceutical compositions of the compounds of the disclosure. The pharmaceutical compositions of the disclosure typically comprise a compound of the instant disclosure and a pharmaceutically acceptable carrier. As

41 167460060.1 used herein “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. In various embodiments, 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. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the instant disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions. [000160] Therapeutic 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. In many cases, 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. Moreover, 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. 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.

42 167460060.1 [000161] 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. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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. [000162] Depending on the route of administration, 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. For example, 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) preferably is formulated in the composition in a therapeutically effective amount. 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. In another embodiment, 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

43 167460060.1 prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. [000164] 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. In some embodiments, 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. [000166] In accordance with an embodiment, 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. [000167] In accordance with an embodiment, 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

44 167460060.1 combination either simultaneously or serially with at least one other chemotherapeutic or radiation treatment. [000168] In accordance with an embodiment, 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. [000169] In accordance with some embodiments, the present disclosure provides fusion protein compositions comprising the polypeptide molecules described herein, and a pharmaceutically acceptable carrier. [000170] In certain embodiments, 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. In certain embodiments, 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. [000171] In certain embodiments, 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. [000172] In yet another embodiment, 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. [000173] In certain embodiments, the compositions and methods of the present disclosure can be used in combination with one or more additional therapeutically active agents which are known to be capable of treating conditions or diseases discussed above. For example, 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

45 167460060.1 cancer. 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. [000174] In certain embodiments, 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. [000175] In accordance with one or more embodiments, 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. [000176] In certain embodiments, methods for treating a tumor in a subject in need of treatment thereof, the method 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. [000177] As used herein, 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.

46 167460060.1 [000178] It will be understood by those of ordinary skill in the art, that the term “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. 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. [000179] Ipilimumab (anti-CTLA-4) 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. When an antibody to CTLA-4 is administered, the CTLA-4 receptor can no longer bind to these ligands, and T-cell responses are unrestrained. Ipilimumab has been evaluated in a number of clinical trials in melanoma, renal cell cancer, and more recently in prostate cancer. [000180] In certain embodiments, an immune checkpoint inhibitor is the inhibitory co- receptor known as programmed death 1 (PD-1 or CD279). Among the 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. Conversely, in multiple systems blocking PD-1: B7-H1 interactions causes tumors to regress. 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. [000181] 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. [000182] In certain embodiments, 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). [000183] In certain embodiments, 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. [000184] In certain embodiments, the immunotherapeutic agents comprise monoclonal antibodies, immune effector cells, vaccines, including dendritic cell vaccines, and cytokines. [000185] 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. [000186] In certain embodiments, 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. [000187] In certain embodiments, 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.

48 167460060.1 [000188] In certain embodiments, 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. It will be understood by those of ordinary skill in the art, that the term “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. [000189] For example, in one non-limiting strategy, 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. After another period of time, for example two weeks later, 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. One of ordinary skill in the art would recognize upon review of the presently disclosed subject matter that 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. [000190] In accordance with another embodiment, the present inventive methods further comprise administering to the subject additional chemotherapy, immunotherapy and or radiation

49 167460060.1 treatment. In other embodiments, the method further comprises administering to the subject, adjuvant therapy. [000191] In certain embodiments, 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. [000192] It will be understood that the methods can be used to treat many tumors, both benign and malignant. In one or more embodiments, the disclosure provides methods and compositions for treating cancers, including, for example, cancers which exist as solid tumors in a subject. One of ordinary skill in the art, upon review of the presently disclosed subject matter, would understand that other tumors, including solid tumors, lesions, and conditions can be treated by the presently disclosed methods including, but not limited to, cancers involving the cervix, ovaries, head and neck, brain; cancers involving the spine; lung cancers; pancreatic cancers; prostate cancers; liver cancers, kidney cancers; breast cancers, melanoma, metastatic orbital tumors, orbital lymphomas, and orbital inflammations. [000193] 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. [000194] In certain embodiments, the timing of administration 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

50 167460060.1 one additional immunotherapeutic agent, can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, 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. Therefore, 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. [000195] It will be understood by those of ordinary skill, that when administered sequentially, 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 be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. 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. [000196] When administered in combination, 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. [000197] As used herein, 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. [000198] Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, 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. [000199] 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). Because of noninvasive fixation devices, 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. When used to treat a tumor over time, the radiosurgery is referred to as “fractionated stereotactic radiosurgery” or FSR. In contrast, stereotactic radiosurgery refers to a one-session treatment. [000200] In certain embodiments, 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. The main advantage of fractionation is that it 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

52 167460060.1 of radiation can. Accordingly, multiple smaller doses of radiation can kill more tumor cells while sparing normal tissue. [000201] In certain embodimenst, the dosage of radiation applied using fractionated stereotactic radiation can vary. In some embodiments, 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. [000202] It will be understood by those of ordinary skill in the art that 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. [000203] As used with the inventive methods and compositions provided herein, 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. [000204] In accordance with the inventive methods and compositions, use of 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. In practice, the proton beam radiation offers advantages for treating unusually shaped brain

53 167460060.1 tumors and arteriovenous malformations. The homogeneous doses of radiation delivered by a proton beam source also make fractionated therapy possible. Proton beam radiosurgery also has the ability to treat tumors outside of the cranial cavity. These properties make proton beam radiosurgery efficacious for post-resection therapy for many chordomas and certain chondrosarchomas of the spine and skull base, as well as a mode of therapy for many other types of tumors. [000205] In accordance with another embodiment of the inventive methods and compositions, intensity-modulated radiation therapy (IMRT) can be used. 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. In 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. More particularly, 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. [000206] 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. Typically, 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. Because the ratio of normal tissue dose to tumor dose is reduced to a minimum with the IMRT approach, higher and more effective radiation doses can safely be delivered to tumors with fewer side effects compared with conventional radiotherapy techniques. 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.

54 167460060.1 [000207] 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®) is used to treat advanced prostate cancer. In this vaccine, white blood cells (cells of the immune system) are removed from the patient's blood and exposed to a protein from prostate cancer cells called prostatic acid phosphatase (PAP). These exposed cells are then given back to the patient by infusion into a vein (IV). Once in the body, the cells make other immune system cells attack the patient's prostate cancer. [000208] Other types of 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. [000209] 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), which is approved to treat advanced prostate cancer, is an example of a dendritic cell vaccine. DNA vaccines are now being studied in clinical trials for use against the

55 167460060.1 following cancers, among others: melanoma, leukemia, prostate cancer, and head and neck cancers. [000210] The precise effective amount of the nucleic acid compositions for a human subject will depend upon the severity of the subject's disease state, general health, age, weight, gender, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance or response to therapy. A routine experimentation can determine this amount and is within the judgment of the medical professional. Compositions may be administered individually to a patient, or they may be administered in combination with other drugs, hormones, agents, and the like. [000211] It is also contemplated that in an embodiment of the present disclosure, 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. [000212] It will be understood by those of ordinary skill in the art that the term “tumor” as used herein means a neoplastic growth which may, or may not be malignant. Additionally, the compositions and methods provided herein are not only useful in the treatment of tumors, but in their micrometastses and their macrometastses. Typically, 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. In contrast, macrometastses are usually large secondary tumors. [000213] In accordance with an embodiment, the present disclosure provides compositions and methods for the prevention and/or treatment of tumors, and their micrometastses and their macrometastses. [000214] While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth

56 167460060.1 and scope of the present disclosure should not be limited by any of the above-described embodiments. [000215] The following examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods. EXAMPLES [000216] Example 1 Electroporation-Mediated Novel Albumin-fused Flt3L DNA Delivery Promotes cDC1-Associated Anticancer Immunity. [000217] 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.^29-30 Both lymphoid CD8⁺ DC and tissue CD103⁺ Dc are derived from Flt3L signaling, and they are efficient at cross-presenting antigens to facilitate CD8⁺ T cell activation subsequently.^31.32 Altogether, Flt3L is crucial for DC-initiated antigen identification and the regulation of adaptive immune activation. [000218] Flt3L has been previously incorporated as a vaccine adjuvant, in combination with selected antigens, to potentiate the immunological effect through the augmentation of DC recruitment.^33-35 However, the in vivo administration of Flt3L itself is limited by its short serum half-life.³⁶ The majority of Flt3L treatments require daily administration, a process that demands a substantial investment in time and resources during the phases of recombinant protein expression and purification. Moreover, Flt3L has impaired mobility to secondary lymphoid tissues and the TME.^37-38 To address these major drawbacks, the invetors developed a novel fusion protein albumin-Flt3L (alb-Flt3L), which is the conjugation of albumin to Flt3L.^{39, 40}

57 167460060.1 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. The hypothesis herein is that 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. [000220] MATERIALS AND METHODS [000221] Mouse experiments: Six- to eight-week-old female C57BL/6 mice were purchased from Charles River Laboratories (Frederick, Maryland, USA). All 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. [000222] 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. [000223] Tumor experiment: Subcutaneous tumor models were established through inoculation of 2x10 TC-1 cells in 50µl 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 mm³ or 20 mm in length, or significant body weight loss in accordance with the animal protocol. [000224] 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

58 167460060.1 amplified by PCR using cDNA template of pcDNA3-Flt3L³⁹ and a set of primers, 5'- TTTGAATTCGGGACACCTGACTGTTACTTC-3' (SEQ ID NO: 7) and 5'- AAACTTAAGCTACTGCCTGGGCCGAGGCTCTGG -3' (SEQ ID NO: 8). The amplified product was then cloned into the EcoRI/Afl II sites of pcDNA3-alb. [000225] Intratumoral DNA delivery via electroporation: alb-Flt3L, Flt3L, or empty vector DNA plasmids (l0µg per mice) were diluted in 30µl PBS and injected intratumorally. Electroporation was immediately performed at the injection site using the BTX830 (BTX Harvard Apparatus, Holliston, MA). A needle electrode was used to deliver the following electroporation parameter: 106V at 20ms/pulse for 8 pulses. [000226] Isolation of tumor-infiltrating immune cells: 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 gentleMACS™ Tissue Dissociator according to manufacturing protocol. The digested tumor tissues are filtered through a 70-µM nylon cell strainer. Mononuclear cells are further separated from the resultant tumor immune cells with Ficoll-Paque plus (GE Healthcare Life Sciences, Marlborough, MA). Tumor protein content level was further checked with luminescence assay. [000227] Measurement of Gaussia Luciferase activity in vivo: Gaussia luciferase (GLuc) (20µg) or alb-GLuc (20µg) 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. Blood serum was isolated and mixed with coelenterazine-H (Regis), and GLuc activity was measured subsequently using the GloMax Luminometer (Promega, Madison, WI, USA). The total luminescence was normalized to tissue weight. [000228] Western blot analysis: Western blotting was performed for Flt3L in HEK293 cells transfected with pcDNA3- Flt3L or pcDNA3-albFlt3L. To identify the protein expression level of Flt3L, HEK293 cells were transfected with 10 µg of each plasmid. Forty-eight hours after transfection, total cell lysates were collected for Western blot analysis. Equivalent amounts of total cell lysates were separated on a precast Tris-HCI protein gel (Life Technology, Rockville, MD, USA) and then transferred onto a nitrocellulose membrane (Bio-Rad). After blocking, the

59 167460060.1 membrane was hybridized with anti-Flt3L antibody (from R&Dsystems) or anti-b-actin (from ThermoFisher scientific). [000229] 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. [000230] Intracellular cytokine staining and flow cytometry analysis: To detect HPV16 E7-specific CD8⁺ T cell responses by IFN-γ 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-γ was stained with FITC-conjugated anti- mouse IFN-γ at 4°C for 30 min. After washing, the cells were pipetted and resuspended in PBS plus 0.5% BSA. [000231] Statistical analysis: All data are expressed as mean± standard error of the mean (S.E.M). The statistical significance is determined by one-way ANOVA with Tukey-Kramer multiple comparison or Student's t-test using the GraphPad Prism 9 software (GraphPad, CA, USA). All p-values: <.0.05 were considered significant (*,p < 0.05; **,p< 0.01; ***,p< 0.001; ****,p< 0.0001). [000232] RESULTS [000233] Albumin-fusion exhibits favorable biodistribution in tumor and lymph nodes: Generally, Flt3L administration requires a daily schedule due to its short half-life. To overcome this major drawback, albumin conjugation was used to achieve better bioactivity. First, biodistribution assay was performed. TC-1-bearing mice were treated with murine albumin-fused Gaussia luciferase (alb-GLuc) or GLuc intratumorally, followed by electroporation. Mice blood

60 167460060.1 was collected over the first two weeks after treatment. Serum analysis revealed that serum luciferase activity of the alb-GLuc group was at least 10-fold higher than GLuc and vector control group at different indicated timepoints (FIG.1A). On Day 14, mice were euthanized, and tissues were extracted for luminescence analysis. Tumor samples were dissociated and measured with fixed protein weight (l00µg). 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). Furthermore, alb-Flt3L DNA was generated, a plasmid encoding the alb-Flt3L protein (FIG.1D). To confirm the presence of alb-Flt3L protein in vivo, HEK293 cells were transfected with alb- Flt3L DNA. Western blot verified the formation of the alb-Flt3L protein (FIG.1E). Taken together, the data provides evidence that intratumoral albumin-fused DNA injection is a feasible and favorable method to provide sustained bioactivity and widespread distribution in the blood, tumor, and lymph nodes. [000234] 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. On Day 5, 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). Therefore, these results provide evidence that alb-Flt3L DNA treatment provides a promising therapeutic anti-tumor effect in vivo. [000235] 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. Given that 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. Under alb-Flt3L DNA treatment, more dendritic cell expansion was noted compared to naked Flt3L DNA and vector control (FIG.3A). Subpopulation analysis revealed that the alb-Flt3L DNA group presented significantly higher cDC1 population (FIGS.3B, 3C), but not cDC2 population (FIG.3D). To

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. Based on these tumor-bearing experiments, the results herein provide evidence that the administration of alb-Flt3L activates DCs, predominantly cDC1 populations, to elicit the anti-tumor immunity observed. [000236] 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-γ-secreting CD8⁺ splenocyte was significantly greater after alb-Flt3L injection among all groups, compared with naked DNA (FIGS.4C, 4D). As expected, IFN-γ-secreting CD8⁺ lymphocytes also increased in locoregional lymphoid tissue (FIGS.4E, 4F). These findings indicate that nucleated hematopoietic cells, especially IFN-y-secreting CD8⁺ T cells, have been significantly promoted in response to alb-Flt3L treatment, contributing to its therapeutic effect against tumors. [000237] Flt3L and Alb-Flt3L treatments are well tolerated: To ensure that Flt3L or Alb-Flt3L DNA treatment is well tolerated in mice, the health of the treated mice was closely monitored. A necropsy was conducted, and histology examinations were performed following the final treatment. 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. [000240] One major barrier for immunotherapy is the underlying suppressive network in the TME, which perturbs DC function and leads to ineffective T cell activation.¹¹ This process, for instance, is regulated by the T cell immunoglobulin mucin receptor 3 (TIM3) expression, which segregates high mobility group protein B1 (HMGB1) from subsequent DC activation. The Flt3/Flt3L axis is a potential targeting pathway to improve current lines of immunotherapy by enhancing DC-related tumor antigen expression and presentation. The results herein not only illustrated a robust anti-tumor effect via alb-Flt3L delivery, but also demonstrated that cDC1s play a pivotal role in alb-Flt3L-mediated anti-tumor immunity. [000241] Among the different types of DCs, 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. [000242] Currently, there is a wealth of research dedicated to immunogene therapy involving cytokines, such as IL-2 and IL-12, all with the objective of enhancing antitumor responses.^47-49 This study stands alone in its investigation of another family of cytokines, namely, the hematopoietic growth factors such as Flt3L. The work here, introduces the electroporation- mediated delivery of alb-Flt3L DNA, which has several benefits compared to direct protein delivery. Firstly, this treatment strategy can mitigate the previous requirement of daily Flt3L protein administration to achieve the antitumor response.^{3, 50} The electroporation-mediated delivery of alb-Flt3L DNA provides cells with the amino acid sequence for recurrent translation of the alb-Flt3L protein. Consequently, the treatment intervals can be extended, and the total number of treatment cycles can be reduced while an effective antitumor response is still

63 167460060.1 expressed. Secondly, 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. [000243] 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.^{51, 52} During electrotransfer, plasmid enters target cells through the transient destabilization of the cell membrane generated by short-lasting, high-intensity electric field.⁵³ Simultaneously, the minimally damaged cells will release pro-inflammatory signals to recruit DCs, creating more opportunities for antigen presentations.⁵⁴ When appropriate field strengths are applied, which vary depending on tissue and plasmid types, this method can produce between 10 and 1000- fold increases in gene expression with minimal tissue damage.^55, Altogether, 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. [000244] The best administration route for cancer vaccine is still under debate. There are two major challenges of systemic administration. First, the systemic circulation of cytokines typically results in limited intratumoral accumulation. Second, systemic cytokine treatment can elevate the risk of toxicity and associated adverse effects due to systemic immune activation.⁵⁹ Instead, intratumoral delivery of vaccines, shown to have high therapeutic index and low toxicity, is an alternative option for cancer treatment.⁶⁰ Based on the results herein, Flt3L- induced immune cells, both cDC1 and IFN-γ-secreting T cells, were present in abundance in the tumor drainage lymph nodes and splenocytes, supporting intratumoral delivery as a preferred method for novel therapeutic vaccine development in the future. [000245] A significant limitation of electroporation-mediated delivery of alb-Flt3L DNA is the requirement for peritumoral sites to be readily accessible without the need for invasive procedures. This logistical challenge highlights the importance of innovating plasmid delivery as

64 167460060.1 potential next steps to expand the applicability of this FLT3L-based approach. For future research, 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.^{61 62} [000246] In summary, electroporation-mediated plasmid delivery is a therapeutic technique known for its high transfection efficiency and demonstrated effectiveness in both preclinical and clinical studies. This study revealed that electroporation-mediated delivery of alb-Flt3L DNA can drive a potent anti-tumor response characterized by greater cDC1 expansion and tumor- specific CD8⁺ T cell activation. This approach explored the intersection of gene therapy and cytokine delivery as an additional modality of immunotherapy for cancer treatment. [000247] References 1. R. A. Morgan, M. E. Dudley, J. R. Wunderlich, M. S. Hughes, J. C. Yang, R. M. Sherry, R. E. Royal, S. L. Topalian, U.S. Kammula, N. P. Restifo, et al. (2006). Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314: 126-129. 2. A. D. Waldman, J.M. Fritz, and M. J. Lenardo (2020). A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol 20: 651-668. 3. A. I. Daud, K. Loo, M. L. Pauli, R. Sanchez-Rodriguez, P. M. Sandoval, K. Taravati, K. Tsai, A. Nosrati, L. Nardo, M. D. Alvarado, et al. (2016). Tumor immune profiling predicts response to anti-PD-I therapy in human melanoma. J Clin Invest 126: 3447- 3452. 4. W. H. Fridman, L. Zitvogel, C. Sautes-Fridman, and G. Kroemer (2017). The immune contexture in cancer prognosis and treatment. Nat Rev Clin Oncol 14: 717-734. 5. R. Sun, E. J. Limkin, M. Vakalopoulou, L. Dercle, S. Champiat, S. R. Han, L. Verlingue, D. Brandao, A. Lancia, S. Ammari, et al. (2018). A radiomics approach to assess tumour- infiltrating CDS cells and response to anti-PD-I or anti-PD-L1 immunotherapy: an imaging biomarker, retrospective multicohort study. Lancet Oncol 19: 1180-1191. 6. Y. Wang, Y. Xiang, V. W. Xin, X. W. Wang, X. C. Peng, X. Q. Liu, D. Wang, N. Li, J. T. Cheng, Y. N. Lyv, et al. (2020). Dendritic cell biology and its role in tumor immunotherapy. J Hematol Oncol 13: 107. 7. T. L. Murphy, and K. M. Murphy (2022). Dendritic cells in cancer immunology. Cell Mol Immunol 19: 3-13. 8. K. Palucka, and J. Banchereau (2012). Cancer immunotherapy via dendritic cells. Nat Rev Cancer 12: 265-277. 9. J.P. Bottcher, E. Bonavita, P. Chakravarty, H. Blees, M. Cabeza-Cabrerizo, S. Sammicheli, N. C. Rogers, E. Sahai, S. Zelenay, and C. Reise Sousa (2018). NK Cells Stimulate

65 167460060.1 Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control. Cell 172: 1022-1037 el 014. 10. K. C. Barry, J. Hsu, M. L. Broz, F. J. Cueto, M. Binnewies, A. J. Combes, A. E. Nelson, K. Loo, R. Kumar, M. D. Rosenblum, et al. (2018). A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments. Nat Med 24: 1178-1191. 11. S. Chiba, M. Baghdadi, H. Akiba, H. Yoshiyama, I. Kinoshita, H. Dosaka-Akita, Y. Fujioka, Y. Ohba, J. V. Gorman, J. D. Colgan, et al. (2012). Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarm in HMGB1. Nat Immunol 13: 832-842. 12. B. Ruffell, D. Chang-Strachan, V. Chan, A. Rosenbusch, C. M. Ho, N. Pryer, D. Daniel, E. S. Hwang, H. S. Rugo, and L. M. Coussens (2014). Macrophage IL-10 blocks CD8+ T cell- dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells. Cancer Cell 26: 623-637. 13. F. Veglia, V. A. Tyurin, D. Mohammadyani, M. Blasi, E. K. Duperret, L. Donthireddy, A. Hashimoto, A. Kapralov, A. Amoscato, R. Angelini, et al. (2017). Lipid bodies containing oxidatively truncated lipids block antigen cross-presentation by dendritic cells in cancer. Nat Commun 8: 2122. 14. Y. Nefedova, P. Cheng, D. Gilkes, M. Blaskovich, A. A. Beg, S. M. Sebti, and D. I. Gabrilovich (2005). Activation of dendritic cells via inhibition of Jak2/STAT3 signaling. J Immunol 175: 4338-4346. 15. Y. Wang, X. Y. Wang, J. R. Subjeck, P.A. Shrikant, and H. L. Kim (2011). Temsirolimus, an mTOR inhibitor, enhances anti-tumour effects of heat shock protein cancer vaccines. Br J Cancer 104: 643-652. 16. M. Merad, P. Sathe, J. Helft, J. Miller, and A. Mortha (2013). The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol 31: 563-604. 17. R.H. Andtbacka, H. L. Kaufman, F. Collichio, T. Amatruda, N. Senzer, J. Chesney, K. A. Delman, L. E. Spitler, I. Puzanov, S.S. Agarwala, et al. (2015). Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma. J Clin Oncol 33: 2780- 2788. 18. M.A. Aznar, L. Planelles, M. Perez-Olivares, C. Molina, S. Garasa, I. Etxeberria, G. Perez, I. Rodriguez, E. Bolanos, P. Lopez-Casas, et al. (2019). Immunotherapeutic effects of intratumoral nanoplexed poly I:C. J Immunother Cancer 7: 116. 19. M.A. Cheever, and C. S. Rigano (2011). PROVENGE (Sipuleucel-T) in prostate cancer: the first FDA-approved therapeutic cancer vaccine. Clin Cancer Res 17: 3520- 3526. 20. H.J. McKenna, K. L. Stocking, R. E. Miller, K. Brasel, T. De Smedt, E. Maraskovsky, C.R. Maliszewski, D. H. Lynch, J. Smith, B. Pulendran, et al. (2000). 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.

66 167460060.1 Shengelia, K. Yao, and M. Nussenzweig (2008). The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues. Nat Immunol 9: 676-683. 22. S. K. Wculek, J. Amores-Iniesta, R. Conde-Garrosa, S. C. Khouili, I. Melero, and D. Sancho (2019). Effective cancer immunotherapy by natural mouse conventional type- 1 dendritic cells bearing dead tumor antigen. J lmmunother Cancer 7: 100. 23. M. L. Broz, M. Binnewies, B. Boldajipour, A. E. Nelson, J. L. Pollack, D. J. Erle, A. Barczak, M. D. Rosenblum, A. Daud, D. L. Barber, et al. (2014). Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell 26: 638-652. 24. F. J. Cueto, C. Del Fresno, P. Brandi, A. J. Combes, E. Hernandez-Garcia, A. R. Sanchez-Paulete, M. Enamorado, C. P. Bromley, M. J. Gomez, R. Conde-Garrosa, et al. (2021). 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. Schlesinger, M. Caskey, K. Liu, et al. (2015). Circulating precursors of human CDle+ and CD141+ dendritic cells. J Exp Med 212: 401-413. 27. E. Maraskovsky, K. Brasel, M. Teepe, E. R. Roux, S. D. Lyman, K. Shortman, and H. J. McKenna (1996). Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J Exp Med 184: 1953- 1962. 28. K. Brasel, H.J. McKenna, P. J. Morrissey, K. Charrier, A. E. Morris, C. C. Lee, D. E. Williams, and S. D. Lyman (1996). Hematologic effects of flt3 ligand in vivo in mice. Blood 88: 2004-2012. 29. K. A. Whartenby, P.A. Calabresi, E. Mccadden, B. Nguyen, D. Kardian, T. Wang, C. Mosse, D. M. Pardoll, and D. Small (2005). Inhibition ofFLT3 signaling targets DCs to ameliorate autoimmune disease. Proc Natl Acad Sci USA 102: 16741-16746. 30. R. Tussiwand, N. Onai, L. Mazzucchelli, and M. G. Manz (2005). Inhibition of natural type I IFN-producing and dendritic cell development by a small molecule receptor tyrosine kinase inhibitor with Flt3 affinity. J lmmunol 175: 3674-3680. 31. N. Anandasabapathy, R. Feder, S. Mollah, S. W. Tse, M. P. Longhi, S. Mehandru, I. Matos, C. Cheong, D. Ruane, L. Brane, et al. (2014). Classical Flt3L-dependent dendritic cells control immunity to protein vaccine. J Exp Med 211: 1875-1891. 32. B. U. Schraml, and C. Reise Sousa (2015). Defining dendritic cells. Curr Opin lmmunol 32: 13-20. 33. S. Kreiter, M. Diken, A. Selmi, J. Diekmann, S. Attig, Y. Husemann, M. Koslowski, C. Huber, 0. Tureci, and U. Sahin (2011). FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines. Cancer Res 71: 6132-6142.

67 167460060.1 34. F. S. Gao, Y. T. Zhan, X. D. Wang, and C. Zhang (2018). Enhancement of anti-tumor effect of plasmid DNA-carrying MUCl by the adjuvanticity ofFLT3L in mouse model. Immunopharmacol Immunotoxicol 40: 353-357. 35. S. M. Sumida, P. F. McKay, D. M. Truitt, M. G. Kishko, J.C. Arthur, M. S. Seaman, S. S. Jackson, D. A. Gorgone, M.A. Lifton, N. L. Letvin, et al. (2004). Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines. J Clin Invest 114: 1334-1342. 36. S. N. Robinson, J.M. Chavez, V. M. Pisarev, R. L. Mosley, G. J. Rosenthal, J.M. Blonder, and J.E. Talmadge (2003). Delivery ofFlt3 ligand (Flt3L) using a poloxamer-based formulation increases biological activity in mice. Bone Marrow Transplant 31: 361-369. 37. J.C. Solheim, A. J. Reber, A. E. Ashour, S. Robinson, M. Futakuchi, S. G. Kurz, K. Hood, R. R. Fields, L. R. Shafer, D. Cornell, et al. (2007). Spleen but not tumor infiltration by dendritic and T cells is increased by intravenous adenovirus-Flt3 ligand injection. Cancer Gene Ther 14: 364-371. 38. K. Furumoto, L. Soares, E.G. Engleman, and M. Merad (2004). Induction of potent antitumor immunity by in situ targeting of intratumoral DCs. J Clin Invest 113: 774- 783. 39. C. F. Hung, K. F. Hsu, W. F. Cheng, C. Y. Chai, L. He, M. Ling, and T. C. Wu (2001). Enhancement of DNA vaccine potency by linkage of antigen gene to a gene encoding the extracellular domain ofFms-like tyrosine kinase 3-ligand. Cancer Res 61: 1080- 1088. 40. Y. M. Chuang, L. He, M. L. Pinn, Y. C. Tsai, M.A. Cheng, E. Farmer, P. C. Karakousis, and C. F. Hung (2021). Albumin fusion with granulocyte-macrophage colony-stimulating factor acts as an immunotherapy against chronic tuberculosis. Cell Mol Immunol 18: 2393-2401. 41. E.G. W. Schmidt, M. L. Hvam, F. Antunes, J. Cameron, D. Viuff, B. Andersen, N. N. Kristensen, and K. A. Howard (2017). Direct demonstration of a neonatal Fe receptor (FcRn)- driven endosomal sorting pathway for cellular recycling of albumin. J Biol Chem 292: 13312- 13322. 42. M. T. Larsen, M. Kuhlmann, M. L. Hvam, and K. A. Howard (2016). Albumin-based drug delivery: harnessing nature to cure disease. Mol Cell Ther 4: 3. 43. N. Desai, V. Trieu, Z. Yao, L. Louie, S. Ci, A. Yang, C. Tao, T. De, B. Beals, D. Dykes, et al. (2006). Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, albumin-bound paclitaxel, ABI-007, compared with cremophor- based paclitaxel. Clin Cancer Res 12: 1317-1324. 44. R. L. Sabado, S. Balan, and N. Bhardwaj (2017). Dendritic cell-based immunotherapy. Cell Res 27: 74-95. 45. H. Salmon, J. Idoyaga, A. Rahman, M. Leboeuf, R. Remark, S. Jordan, M. Casanova- Acebes, M. Khudoynazarova, J. Agudo, N. Tung, et al. (2016). Expansion and Activation ofCD103(+) Dendritic Cell Progenitors at the Tumor Site Enhances Tumor Responses to Therapeutic PD-LI and BRAF Inhibition. Immunity 44: 924-938. 46. T. Oba, M. D. Long, T. Keler, H. C. Marsh, H. Minderman, S. I. Abrams, S. Liu, and F. Ito (2020). Overcoming primary and acquired resistance to anti-PD-LI therapy by induction and

68 167460060.1 activation of tumor-residing cDC1s. Nat Commun 11: 5415. 47. H. M. Horton, P.A. Lalor, and A. P. Rolland (2008). IL-2 plasmid electroporation: from preclinical studies to phase I clinical trial. Methods Mol Biol 423: 361-372. 48. A. I. Daud, R. C. DeConti, S. Andrews, P. Urbas, A. I. Riker, V. K. Sondak, P. N. Munster, D. M. Sullivan, K. E. Ugen, J. L. Messina, et al. (2008). Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 26: 5896-5903. 49. K. E. Ugen, M.A. Kutzler, B. Marrero, J. Westover, D. Coppola, D. B. Weiner, and R. Heller (2006). Regression of subcutaneous B16 melanoma tumors after intratumoral delivery of an IL-15-expressing plasmid followed by in vivo electroporation. Cancer Gene Ther 13: 969- 974. 50. N. Bhardwaj, P.A. Friedlander, A. C. Pavlick, M. S. Ernstoff, B. R. Gastman, B. A. Hanks, B. D. Curti, M. R. Albertini, J. J. Luke, A. B. Blazquez, et al. (2020). Flt3 ligand augments immune responses to anti-DEC-205-NY-ESO-1 vaccine through expansion of dendritic cell subsets. Nat Cancer 1: 1204-1217. 51. M.A. Cheng, E. Farmer, C. Huang, J. Lin, C. F. Hung, and T. C. Wu (2018). Therapeutic DNA Vaccines for Human Papillomavirus and Associated Diseases. Hum Gene Ther 29: 971- 996. 52. G. Widera, M. Austin, D. Rabussay, C. Goldbeck, S. W. Barnett, M. Chen, L. Leung, G. R. Otten, K. Thudium, M. J. Selby, et al. (2000). Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol 164: 4635-4640. 53. E. Neumann, and K. Rosenheck (1972). Permeability changes induced by electric impulses in vesicular membranes. J Membr Biol 10: 279-290. 54. P. Chiarella, E. Massi, M. De Robertis, A. Sibilio, P. Parrella, V. M. Fazio, and E. Signori (2008). Electroporation of skeletal muscle induces danger signal release and antigen- presenting cell recruitment independently of DNA vaccine administration. Expert Opin Biol Ther 8: 1645-1657. 55. N. Y. Sardesai, and D. B. Weiner (2011). Electroporation delivery of DNA vaccines: prospects for success. Curr Opin Immunol 23: 421-429. 56. J. L. Young, and D. A. Dean (2015). Electroporation-mediated gene delivery. Adv Genet 89: 49-88. 57. R. Girelli, S. Prejano, I. Cataldo, V. Corbo, L. Martini, A. Scarpa, and B. Claudio (2015). Feasibility and safety of electrochemotherapy (ECT) in the pancreas: a pre-clinical investigation. Radiol Oncol 49: 147-154. 58. T. Komel, M. Bosnjak, S. Kranjc Brezar, M. De Robertis, M. Mastrodonato, G. Scillitani, G. Pesole, E. Signori, G. Sersa, and M. Cemazar (2021). Gene electrotransfer ofIL-2 and IL-12 plasmids effectively eradicated murine B16.Fl0 melanoma. Bioelectrochemistry 141: 107843. 59. T. A. Waldmann (2018). Cytokines in Cancer Immunotherapy. Cold Spring Harb Perspect Biol 10. 60. I. Melero, E. Castanon, M. Alvarez, S. Champiat, and A. Marabelle (2021). Intratumoural

69 167460060.1 administration and tumour tissue targeting of cancer immunotherapies. Nat Rev Clin Oncol 18: 558-576. 61. M. Chehelgerdi, and M. Chehelgerdi (2023). The use of RNA-based treatments in the field of cancer immunotherapy. Mol Cancer 22: 106. 62. H. K. Lyerly (2023). Self-replicating messenger RNA based cancer immunotherapy. Cancer Gene Ther 30: 769-770. OTHER EMBODIMENTS [000248] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. [000249] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their disclosure.

70 167460060.1

Claims

What is claimed: 1. A composition comprising 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.

2. The composition of claim 1, wherein the Flt3L is human.

3. The composition of claim 1 or 2, wherein the albumin is human.

4. The composition of claim 1, wherein the Flt3L is murine.

5. The composition of claim 1 or 4, wherein the albumin is murine.

6. The composition of any one of claims 1-5, further comprising a pharmaceutically acceptable carrier.

7. The composition of any one of claims 1-6, further comprising at least one chemotherapeutic agent.

8. The composition of any one of claims 1-7, further comprising at least one immunotherapeutic agent.

9. The composition of any one of claims 1-3 or 6-8 wherein the expression vector or nucleic acid encodes a polypeptide having at least 70% sequence identity to SEQ ID. NO.1.

10. The composition of any one of claims 1-3 or 6-8 wherein the expression vector or nucleic acid encodes a polypeptide having at least 80% sequence identity to SEQ ID. NO.1.

11. The composition of any one of claims 1-3 or 6-8 wherein the expression vector or nucleic acid encodes a polypeptide having at least 90% sequence identity to SEQ ID. NO.1.

12. The composition of any one of claims 1-3 or 6-8 wherein the expression vector or nucleic acid encodes a polypeptide having at least 95% sequence identity to SEQ ID. NO.1.

13. A method of treating cancer in a subject, comprising administering a composition of any one of claims 1-12 to a subject, thereby treating the subject.

71 167460060.1

14. The method of claim 13 further comprising applying electroporation to the subject in conjunction with administering the composition.

15. The method of claim 14 wherein a tumor of the subject tis treated by electroporation.

16. 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 80% identity to the amino acid sequence of SEQ ID NO: 1 or a nucleic acid encoding a polypeptide comprising a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 to a tumor, thereby treating the subject.

17. The method of claim 16 further comprising applying electroporation to the subject in conjunction with administering the composition.

18. The method of claim 17 wherein a tumor of the subject tis treated by electroporation.

19. The method of any one of claims 16-18 wherein the polypeptide has at least 90% sequence identity to SEQ ID. NO.1.

20. The method of any one of claims 16-19 wherein the polypeptide has at least 95% sequence identity to SEQ ID. NO.1.

21. The method of any one of claims 13-18, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 1.

22 The method of any one of claims 13-21, further comprising administering one or more chemotherapeutics.

23. The method of claim 22, wherein the chemotherapeutics comprise chemotherapeutic agents, radiation, surgery or combinations thereof.

24. The method of any one of claims 13-23, wherein the expression vector comprises: a plasmid, viral vector, phage or a bacterial vector.

25. The method of claim 24, wherein the expression vector is a plasmid.

26. The method of any one of claims 13-25, wherein the composition induces an anti-tumor immune response.

27. The method of claim 26, wherein the composition stimulates expansion of circulating

72 167460060.1 dendritic cells (DCs) as compared to the subject’s baseline circulating dendritic cells.

28. The method of claim 27, wherein 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.

29. The method of claim 28, wherein the circulating dendritic cells comprise conventional dendritic cells (cDC1).

30. The method of any one of claims 13-29, wherein 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.

31. The method of claim 30, wherein the composition is administered intratumorally.

32. A method of treating tumors in a subject, comprising 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, 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 subject and/or tumor to electroporation; thereby treating the subject.

33. The method of claim 32 wherein electroporation is applied to the site of administration of the expression vector or nucleic acid to the subject.

34. The method of claim 32 or 33, wherein the protein comprises albumin or variants thereof.

35. The method of any one of claims 32-34, further comprising administering one or more chemotherapeutics.

36. The method of claim 35, wherein the chemotherapeutics comprise chemotherapeutic agents, radiation, surgery or combinations thereof.

37. The method of any one of claims 32-36, wherein the expression vector comprises: a plasmid, viral vector, phage or a bacterial vector.

73 167460060.1

38. The method of claim 37, wherein the expression vector is a plasmid.

39. The method of any one of claims 32-38, wherein the composition induces an anti-tumor immune response.

40. The method of claim 39, wherein the composition stimulates expansion of circulating dendritic cells (DCs) as compared to the subject’s baseline circulating dendritic cells.

41. The method of claim 40, wherein 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.

42. The method of claim 41, wherein the circulating dendritic cells comprise conventional dendritic cells (cDC1).

43. The method of any one of claims 32-42, wherein 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.

44. The method of any one of claims 32-43, wherein the composition is administered intratumorally.

45. An expression vector encoding a fusion polypeptide comprising FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof, and albumin.

46. The expression vector of claim 45, wherein the expression vector is a plasmid.

47. A nucleic acid encoding a fusion polypeptide comprising FMS-like tyrosine kinase receptor 3 ligand (Flt3L) or variants thereof, and albumin.

48. A host cell expressing the expression vector of claim 47.

49. 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.

74 167460060.1

50. The method of claim 49, wherein the expression vector comprises: a plasmid, viral vector, phage or a bacterial vector.

51. The method of claim 50, wherein the expression vector is a plasmid.

52. The method of any one of claims 49-51, wherein the composition stimulates expansion of circulating dendritic cells (DCs) as compared to the subject’s baseline circulating dendritic cells.

53. The method of claim 52, wherein 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.

54. The method of claim 53, wherein the circulating dendritic cells comprise conventional dendritic cells (cDC1).

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