WO2017030956A1 - Method of inducing a t-cell response to phosphopeptides using nucleic acids encoding phosphopeptide mimetics - Google Patents

Abstract

Provided are compositions and methods useful as therapeutic vaccines (e.g., cancer vaccines), and methods of producing such compositions. The compositions disclosed herein generally employ an RNA or DNA molecule, which may be a modified nucleotide, encoding a phosphopeptide mimetic of a phosphopeptide that occurs in at least one cancer cell.

Description

METHOD OF INDUCING A T-CELL RESPONSE TO PHOSPHOPEPTIDES USING NUCLEIC ACIDS

ENCODING PHOSPHOPEPTIDE MIMETICS

Related Applications

This application claims the benefit of U.S. Provisional Application No. 62/205,597, filed August 14, 2015, which is incorporated by reference herein in its entirety.

Field of the Invention

The invention relates to the field of cancer biology, and more specifical ly to the treatment and inhibition of recu rrence using anti-cancer vaccines.

Background

Cancer-specific antigen vaccines hold great promise for the treatment of cancer. These vaccines actively educate a patient's immune system to target and eradicate cancer cel ls containing cancer-specific antigens.

One approach to providing cancer-specific antigens for immunotherapy involves exploiting signal-transduction pathway deregulation that occurs in cancer (Nature, 2008. 456: pp 66-72; Science, 2008. 321: pp. 1801-1806; Science, 2011. 331: pp. 435-439; J. Clin. Oncol., 2005. 23: pp. 3886-3896; N. Engl. J. Med., 2011. 364: pp. 2305-2315). The dominant mechanism in signaling in oncogenesis is protein phosphorylation, and it has been shown that the phosphorylation status of a phosphoprotein is preserved du ring antigen processing for presentation by both major histocompatibility complex class I (M HC-I) and MHC-II molecules (Proc. Natl. Acad. Sci. USA, 2006. 103: pp. 14889-14894). Moreover, M HC-I restricted phosphopeptides derived from cancer samples have been characterized and demonstrated to be both cancer specific and to induce immu ne responses in healthy individuals (Sci. Transl. Med., 2013. 5(203): 203ral25). Accordingly, phosphopeptides have potential utility as cancer-specific antigens for use in immunotherapy (Nat. I mmunol., 2008. 9: pp. 1236-1243).

An alternative to ad ministering peptides to a subject to induce an immune response is to administer RNA or DNA molecules that encode a cancer-specific peptide. When these RNA or DNA molecules are taken up by a subject's cells, they can express the cancer-specific peptide in the cells and resu lt in the elicitation of an immune response to the cells expressing the peptide in the subject. Such nucleic acid vaccines hold great promise in the treatment of cancer; however, phosphoproteins per se cannot be directly encoded by nucleic acid molecules.

Accordingly, there is a need for methods of creating more effective nucleic acid- based anti-cancer vaccines that can deliver phosphopeptide mimetics into the cells of a subject with cancer.

Summary of the Invention

In a first aspect, the invention is directed to compositions comprising a

polynucleotide that encodes a first immunogenic peptide that is a su bstitution variant of a second immu nogenic peptide comprising at least one phosphorylated amino acid residue, wherein the second immunogenic peptide is a fragment of an aberrant protein that occurs in cancer cells of a subject having cancer, wherein the first immunogenic peptide comprises the amino acid sequence of the second immu nogenic peptide except that the first immunogenic peptide comprises at least one su bstituted amino acid residue at at least one amino acid position corresponding to the at least one phosphorylated amino acid residue in the second immunogenic peptide, and wherein the first immunogenic peptide does not comprise the entire amino acid sequence of a naturally-occurring protein. The

polynucleotide can be a DNA molecule or an RNA molecule, such as an mRNA molecule. The at least one phosphorylated amino acid residue in the second immunogenic peptide can be selected from the group consisting of phospho-Ser, phospho-Thr, phospho-Tyr, phospho- His, phospho-Arg, and phospho-Lys. The at least one substituted amino acid residue can be selected from the group consisting of Asp and Glu, such as Glu. In one embodiment, normal cel ls of the subject comprise a normal form of the aberrant protein, the normal form of the aberrant protein comprising the second immunogenic peptide except that at least one of the amino acid residues that is phosphorylated in the second immunogenic peptide is not phosphorylated in the normal form of the aberrant protein; in such cases, each amino acid residue that is phosphorylated in the second immunogenic peptide is not phosphorylated in the normal form of the aberrant protein. More than one amino acid residue can be phosphorylated in the second immunogenic peptide but not phosphorylated in the normal form of the aberrant protein. The first immunogenic peptide can comprise substituted amino acid residues at each amino acid position corresponding to the phosphorylated amino acid residues in the second immunogenic peptide; the substituted amino acid residues are each selected from the group consisting of Asp and G lu, and may be Glu. In one embodiment, exactly one amino acid residue is phosphorylated in the second immunogenic peptide but not phosphorylated in the normal form of the aberrant protein. The substituted amino acid residue is selected from the group consisting of Asp and Glu, such as Glu. In one embodiment, the polynucleotide encoding the first immunogenic peptide encodes a plurality of first immu nogenic peptides. In another embodiment, the polynucleotide encoding the first immunogenic peptide comprises a plurality of polynucleotides encoding a plurality of first immu nogenic peptides. The first immunogenic peptide can be 9-11 or 27-31 amino acids in length. The compositions of this first aspect can further comprise an adjuvant, which, e.g., can comprise an immunostimu latory nucleic acid and/or at least one cytokine, or is selected from the group consisting of poly IC, poly ICLC, and QS-21. The compositions can also further comprise a stress protein, such as one selected from the group consisting of hsc70, hsp70, hsp90, hspllO, grpl70, gp96, calreticulin, a mutant thereof, and combinations of two or more thereof, and may be hsc70. These compositions can fu rther comprise a cell penetration agent. The cancer can be multiple myeloma or glioblastoma. The compositions of this first aspect can be formulated in nanoparticles or poly(lactic-co-glycolic acid) (PLGA) microspheres. Finally, the compositions of this first aspect can be a pharmaceutical composition fu rther comprising a pharmaceutical ly acceptable excipient.

In a second aspect, the invention is directed to methods of treating a subject having or suspected of having a cancer, comprising administering to the subject any composition of any of the first aspect, or a pharmaceutical composition comprising those compositions. The cancer can be multiple myeloma or glioblastoma. Furthermore, the su bject can also be administered lenalidomide or dexamethasone or cyclophosphamide, or a combination of two or more selected from lenalidomide, dexamethasone, and cyclophosphamide. Or, the subject can be further administered a checkpoint antibody, such as one selected from the group consisting of anti-GITR, anti-OX40, anti-PD-1, anti-CTLA-4, anti-TIM-3, and anti-LAG-3 antibody. The checkpoint antibody can be a monoclonal antibody. Or, the subject can be fu rther ad ministered an inhibitor of indoleamine-2,3-dioxygenase (I DO), such as one selected from the group consisting of epacadostat, F001287, indoximod, and NLG919. In a third aspect, the invention is directed to methods of immunizing a subject recovering from a cancer, comprising administering to the su bject any composition of any of the first aspect, or a pharmaceutical composition comprising those compositions. The cancer can be mu ltiple myeloma or glioblastoma. Fu rthermore, the subject can also be administered lenalidomide or dexamethasone or cyclophosphamide, or a combination of two or more selected from lenalidomide, dexamethasone, and cyclophosphamide. Or, the subject can be further administered a checkpoint antibody, such as one selected from the group consisting of anti-GITR, anti-OX40, anti-PD-1, anti-CTLA-4, anti-TIM-3, and anti-LAG-3 antibody. The checkpoint antibody can be a monoclonal antibody. Or, the subject can be fu rther ad ministered an inhibitor of indoleamine-2,3-dioxygenase (I DO), such as one selected from the group consisting of epacadostat, F001287, indoximod, and NLG919.

In a fou rth aspect, the invention is directed to methods of treating a subject having or suspected of having a cancer, comprising: (a) contacting cells ex vivo with any composition of the first aspect; and (b) infusing the cel ls into the subject. The methods can comprise prior to step (a), isolating the cel ls from the subject. Step (a) can comprise transfecting or transducing the cells with the composition of any one of the first aspect. The cel ls can be antigen presenting cells such as dendritic cells. The methods can further comprise administering an adjuvant to the subject.

In a fifth aspect, the invention is directed to kits, comprising any composition of the first aspect, or a pharmaceutical composition thereof, and instructions for use. Such kits can fu rther comprise at least one selected from the group consisting of lenalidomide, dexamethasone, cyclophosphamide, and a checkpoint antibody. The checkpoint antibody can be selected from the group consisting of anti-GITR, anti-OX40, anti-PD-1, anti-CTLA-4, anti-TIM-3, and anti-LAG-3 antibody.

In a sixth aspect, the invention is directed to recombinant Listeria strains comprising the composition of any one of the polynucleotide compositions of the first aspect. The polynucleotide can be a DNA molecule. The recombinant Listeria strain can be a recombinant Listeria monocytogenes strain. The recombinant Listeria strains can be formulated into vaccines, which may comprise adjuvants. Such vaccines can be used in methods of treating a subject having or suspected of having a cancer, comprising administering to the subject a composition comprising the recombinant Listeria strain (including recombinant Listeria monocytogenes) or the vaccine formulated with a recombinant Listeria strain.

Brief description of the drawing

Figure 1 is a set of flow cytometry plots showing the staining of cells expressing a pCDC25b-specific TCR or a plRS-2-specific TCR using a PE-labeled anti-TC^ antibody together with APC-labeled HLA-A2 tetramers loaded with pCDC25b₃₈-4₆ GLLG[pS]PVRA, its non-phosphorylated counterpart CDC25b3g-46 GLLGSPVRA, or a phosphopeptide mimetic GLLGEPVRA or GLLGDPVRA. The percentage of tetramer+ TCR+ cel ls is indicated in each plot. Negative control represents cells without TCR expression.

Detailed Description

Cancers typically have 10-500 protein mutations, with over 98% of mutations in a tumor genome being specific to the tumor. In addition, some proteins are mis- phosphorylated, due to the signaling dysregulation that accompanies most cancers. For the immune system to "see" muteins (neo-antigens), many challenges need to be overcome, including the DNA mutation being realized as a protein (in the case of mutated proteins), which includes transcription into RNA and translation into a protein; the protein degraded by proteasomes into peptides that are transported to the endoplasmic reticulum, loaded onto mu ltiple histocompatibility complex (M HC) molecu les and transported to the cel l surface; and the peptides remaining on the subject's M HCs and being recognized as sufficiently "non-self" by T-cell receptors (TCRs) that have not been deleted or tolerized. In the case of phosphoproteins, the aberrant phosphorylated state must be maintained.

T cells constantly scan cell su rfaces seeking and eliminating cells presenting with "non-self" (neo-)antigens. Cells displaying neo-antigens that are recognized as non-self by T cel ls can be destroyed, the cells dying by apoptosis.

Disclosed herein are compositions (e.g., pharmaceutical compositions) comprising nucleic acid molecules that encode phosphopeptide mimetic of aberrantly phosphorylated proteins in cancer cells, and in some embodiments not in cognate healthy cells. Also disclosed herein are methods of treating a subject having cancer using these compositions.

The compositions disclosed herein generally comprise RNA or DNA molecules encoding the phosphoprotein amino acid sequence, or portions of the phosphoprotein sequence, wherein the aberrantly phosphorylated amino acid residue(s) is replaced with a phosphomimetic amino acid (e.g., Asp or Glu), such that the nucleic acids encode a phosphopeptide mimetic. Phospho amino acids that can be found in aberrantly

phosphorylated proteins include phospho-Ser, phospho-Thr, phospho-Tyr, phospho-His, phospho-Arg, and phospho-Lys. The term "aberra nt," in the context of a protein (or peptidic fragment thereof), refers to a protein (or peptidic fragment thereof) that contains an amino acid mutation (e.g., substitution, insertion, or deletion) in disease tissue (e.g., cancer cells); a protein (or peptidic fragment thereof) that contains an amino acid modification (e.g., a post- translational modification, such as phosphorylation ) that is found in a subject's disease tissue (e.g., cancer cells) but not in the subject's corresponding normal or healthy tissue, or vice versa; a protein (or peptidic fragment thereof) with different expression profiles in cancer cells as compared with normal or healthy cells (e.g., a protein that is expressed in cancer cells but not in normal cel ls); or a protein (or peptidic fragment thereof) that is processed differently in the antigen presentation pathway in the disease tissue (e.g., cancer cel ls) vs. normal cells, leading to different peptides presented by MHC molecules.

Alternatively, in certain embodiments, an aberrant protein is one that exhibits an elevated level of post-translational modification (e.g., phosphorylation) in cancer cel ls relative to normal tissue. For clarity, the term "amino acid sequence" means the sequence of amino acids of a polypeptide (i.e., the identity of the amino acid side chains as encoded at the mRNA level), irrespective of any post-translational modifications. A phosphopeptide mimetic is a peptide that mimics the action or activity of a phosphorylated peptide by, e.g., replacing a phosphorylated amino acid residue in the phosphorylated peptide with a phosphomimetic residue. In certain embodiments, the phosphopeptide mimetic substitutes Asp or Glu residues for the residue(s) that are phosphorylated in the phosphorylated peptide that is being mimicked.

Production ofRNA molecules

To introduce the phosphopeptide mimetic into cells of a subject to produce an immune response, DNA and RNA vaccines can be used. The DNA or RNA molecules express the immunogenic peptide (a peptide when administered in sufficient amounts elicits an immune response in a subject). RNA vaccine means a vaccine comprising at least one RNA molecu le comprising at least one open reading frame (ORF) coding for at least one antigen. The at least one RNA molecule comprised by the vaccine is preferably an isolated RNA molecu le. This at least one RNA is preferably mRNA. The RNA molecule may have at least one nucleoside modification.

An RNA vaccine can comprise more than one RNA molecu le encoding the antigen, preferably two, three, five, ten, etc. However, one RNA molecule can encode one or more antigens, such as two, three, five, ten, etc. different or identical antigens.

The RNA molecu le can further comprise an element, such as a replicase, β globin leader sequence, cap 0, cap 1, and a polyA tail.

The RNA sequence can be the "wild-type" sequence of the antigen (but modified to encode an Asp or Glu residue at the appropriate position(s)) (codons for Asp are GAC and GAT; codons for Glu are GAA and GAG), can be any variation thereof (e.g., due to the redundancy of the genetic code), or can be adapted with respect to its codon usage.

Adaptation of codon usage can increase translation efficacy and half-life of the RNA. A polyA tail at least 30 adenosines long can be attached to the 3' end of the RNA to increase RNA half-life. PolyA tails are further discussed in US20130244278 (incorporated by reference in its entirety). The 5' end of the RNA can be capped with a modified

ribonucleotide with the structure m7G(5')ppp(5')N (cap 0 structu re) or a derivative thereof, which can be incorporated du ring RNA synthesis or can be enzymatically engineered after RNA transcription by using Vaccinia virus capping enzyme (VCE, consisting of mRNA triphosphates, guanylyl-transferase and guanine-7-methyl transferase), which catalyzes the construction of N7-monomehtylated cap 0 structures. Cap 0 structu re plays an important role in maintaining the stability and translational efficacy of the RNA vaccine. The 5' cap of the RNA vaccine can be further modified by a 2'-0-methyltranserase that results in the generation of a cap 1 structu re (m7 Gppp[m2'-0]N), which further increases translational efficacy. Capping is discussed in more detail in US20130244278 and US20120195917 (each of which is herein incorporated by reference in its entirety).

Additionally, certain modified nucleosides, or combinations thereof, when introduced into the nucleic acid molecules, activate the innate immune response. Such activating molecules are useful as adjuvants when combined with polypeptides and/or other vaccines. I n some examples, the activating molecules contain a translatable region which encodes for a polypeptide sequence useful as a vaccine, thus providing the ability to be a self-adjuvant.

The nucleic acid molecules encoding an immunogen can be delivered to cells to trigger multiple innate response pathways (see WO2012006377 (incorporated herein by reference in its entirety)). As another non-limiting example, modified nucleic acid molecu les encoding an immunogen may be delivered to a vertebrate in a dose amount large enough to be immunogenic to the vertebrate (see WO2012006372 and WO2012006369, each of which is herein incorporated by reference in its entirety).

The RNA vaccine can be converted to a self-replicating vaccine. Such vectors include replication elements derived from al pha viruses and the substitution of the structural virus proteins with the gene of interest. Self-replicating RNA vaccines contain replicase RNA molecule derived from semliki forest virus (SFV), sindbis virus (SI N), Venezuelan equine encephalitis virus (VEE), Ross-River virus (RRV) or other viruses belonging to the alpha virus family. Downstream of the replicase is a su bgenomic promoter that controls replication of the neo-antigen RNA fol lowed by an artificial poly A tail consisting of at least 30 adenosine residues.

Modified nucleic acid synthesis

Nucleic acids may be prepared according to any available technique, including chemical synthesis, enzymatic synthesis (in vitro transcription), enzymatic or chemical cleavage of a longer precursor, etc. Methods of synthesizing RNA and DNA are known (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford (Oxfordshire), Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) "Oligonucleotide synthesis:

methods and applications," Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Hu mana Press, 2005; each of which is herein incorporated by reference in its entirety).

A method for production of an RNA transcript, e.g., mRNA, using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA transcript is also described in WO2014152027 (incorporated herein by reference in its entirety).

Modified nucleic acids need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structu res may exist at various positions in the nucleic acid. One of ordinary skil l in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification may also be a 5' or 3' terminal modification. The nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.

Modified nucleic acids have useful properties, including the lack of a substantial induction of the in nate immune response of a cell into which the RNA is introduced.

Modified nucleic acids can enhance the efficiency of protein production, intracellu lar retention of nucleic acids, and viability of contacted cells, as well as possess reduced immunogenicity (US Patent No. 8,822,663, incorporated by reference in its entirety).

The term "nucleic acid," in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary nucleic acid includes one or more of DNA, RNA, hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, and the like.

For fu rther discussion of modified nucleic acids and modified nucleosides, see WO2012045082 and WO2015034928, each of which is herein incorporated by reference in its entirety.

Modified nucleic acids contain a translatable region and one, two, or more than two different nucleoside modifications. The modified nucleic acid exhibits reduced degradation in a cel l into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid. Exemplary nucleic acids include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), th reose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), and locked nucleic acids (LNAs) or a hybrid thereof. In embodiments, the modified nucleic acid includes messenger RNAs (mRNAs).

See also US20150017211 (incorporated by reference in its entirety) for a fu rther discussion regarding combinations of nucleoside modifications.

Other components of the nucleic acids usefu l for making RNA vaccines are optional, but can be beneficial. For example, a 5' untranslated region (UTR) and/or a 3'UTR can be provided, wherein either or both may independently contain one or more different nucleoside modifications. In such cases, nucleoside modifications may also be present in the translatable region. Also provided are nucleic acids containing a Kozak sequence. The termini may be further modified, as described in WO2014081507 (incorporating translation enhancer elements), WO2014164253 (heterologous 5'UTRs) and WO2014152540

(incorporating 3'UTR microRNA binding sites to destabilize a construct), each of which is herein incorporated by reference in its entirety.

Modifying the UTR sequences can also stabilize the RNA and protein production. For example, introduction of 5' UTR of liver-expressed mRNA, such as albumin, serum amyloid A, apolipoprotein A/B/E, transferrin, alpha fetoprotein, eryth ropoietin, or Factor VIII, can be used to enhance expression of a modified nucleic acid molecu le, such as an RNA, in hepatic cel l lines or liver. Likewise, use of 5' UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin,

Hercu lin), for endothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AM LI, G-CSF, GM-CSF, CDllb, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and for lung epithelial cells (SP-A/B/C/D). See US20130236974 for fu rther details, incorporated herein by reference in its entirety.

Other non-UTR sequences may be incorporated into the 5' or 3' UTRs of modified nucleic acid molecules. For example, introns or portions of introns sequences may be incorporated into the flan king regions of the modified RNA. I ncorporation of intronic sequences may increase protein production as well as mRNA levels. See US20130236974 for fu rther details, incorporated herein by reference in its entirety.

3' UTRs have stretches of adenosines and uridines. These AU rich signatu res are prevalent in genes with high rates of turnover. Based on their sequence features and fu nctional properties, the AU rich elements (AREs) can be separated into three classes. Most proteins binding to AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been docu mented to increase the stability of mRNA. Hu R binds to AREs of al l th ree classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus stabilization of the message in vivo.

Introduction, removal, or modification of 3' UTR AU rich elements (AREs) can be used to modu late the stability of modified mRNA. When engineering specific mRNA, one or more copies of an ARE can be introduced to make modified mRNA less stable and thereby curtail translation and decrease production of the resu ltant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellu lar stability and thus increase translation and production of the resu ltant protein. Transfection experiments can be conducted in relevant cel l lines, using desired nucleic acid molecules, and protein production can be assayed at various time points post-transfection.

RNAs can be fu rther stabilized through 3' UTR modifications as provided by

US20100129877 and WO2013103659, each of which is herein incorporated by reference in its entirety.

Additionally, provided are nucleic acids containing one or more intronic nucleotide sequences capable of being excised from the nucleic acid.

Further, provided are nucleic acids containing an internal ribosome entry site (IRES).

An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites, of an mRNA. An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes ("multicistronic mRNA"). When nucleic acids are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences include those from picornavi ruses (e.g., FM DV), pest viruses (CFFV), polio viruses (PV),

encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FM DV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), mu rine leukemia virus (MLV), simian immune deficiency viruses (SIV), or cricket paralysis viruses (CrPV).

RNA structure

Exemplary RNA structures for vaccine production include those described in US Patent No. 9,089,604, incorporated herein by reference in its entirety. Briefly, a construct contains a first region of lin ked nucleotides that is flanked by a first flanking region and a second flaking region. The "first region" may be referred to as a "coding region" or "region encoding" or simply the "first region." This first region may include the encoded phosphopeptide mimetic of interest. The phosphopeptide mimetic of interest may comprise at its 5' terminus one or more signal sequences encoded by a signal sequence region. The first flan king region may comprise a region of linked nucleotides comprising one or more complete or incomplete 5' UTRs sequences. The first flan king region may also comprise a 5' terminal cap. The second flan king region may comprise a region of lin ked nucleotides comprising one or more complete or incomplete 3' UTRs. The second flan king region may also comprise a 3' tailing sequence.

Bridging the 5' terminus of the first region and the first flanking region is a first operational region. Traditionally this operational region comprises a start codon. The operational region may alternatively comprise any translation initiation sequence or signal including a start codon.

Bridging the 3' terminus of the first region and the second flanking region is a second operational region. Traditionally this operational region comprises a stop codon. The operational region may alternatively comprise any translation initiation sequence or signal including a stop codon. Multiple serial stop codons may also be used.

Where the tailing region is a polyA tail, the length may be determined in units of, or as a function of, polyA binding protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA binding protein. PolyA binding protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.

The capping region may comprise a single cap or a series of nucleotides forming the cap. In some embodiments, the cap is absent.

The first and second operational regions may comprise, in addition to a start and/or stop codon, one or more signal and/or restriction sequences.

Cyclic RNA

An RNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5'-end binding proteins. The mechanism of cyclization or concatemerization may occur through at least 3 different routes: (1) chemical-, (2) enzymatic-, and (3) ribozyme-catalyzed. The newly formed 5'-/3'- linkage may be intramolecular or intermolecular.

In the first route, the 5'-end and the 3'-end of the nucleic acid contain chemically reactive groups that, when close together, form a new covalent linkage between the 5'-end and the 3'-end of the molecu le. The 5'-end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a synthetic mRNA molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'-/3'-amide bond. In the second route, T4 RNA ligase may be used to enzymatical ly link a 5'- phosphorylated nucleic acid molecule to the 3'-hyd roxyl group of a nucleic acid forming a new phosphorodiester lin kage. The ligation reaction may occu r in the presence of a split oligonucleotide capable of base-pairing with both the 5'- and 3'-region in juxtaposition to assist the enzymatic ligation reaction.

In the third route, either the 5'- or 3'-end of the cDNA template encodes a ligase ribozyme sequence such that du ring in vitro transcription, the resu ltant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5'-end of a nucleic acid molecule to the 3'-end of a nucleic acid molecu le. The ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, or hairpin ribozyme, or may be selected by SELEX (systematic evolution of ligands by exponential en richment).

RNA Multimers

Multiple distinct RNA polynucleotides may be lin ked together th rough the 3'-end using nucleotides which are modified at the 3'-terminus. Chemical conjugation may be used to control the stoichiometry of delivery into cells. For example, the glyoxylate cycle enzymes, isocitrate lyase and malate synthase, may be supplied into HepG2 cel ls at a 1: 1 ratio to alter cel lular fatty acid metabolism. This ratio may be controlled by chemical ly linking polynucleotides, primary constructs or RNA using a 3'-azido terminated nucleotide on one polynucleotide, primary construct or RNA species and a C5-ethynyl or alkynyl- containing nucleotide on the opposite polynucleotide, primary construct or RNA species. The modified nucleotide is added post-transcriptional ly using terminal transferase. After the addition of the 3'-modified nucleotide, the two polynucleotide, primary construct or mmRNA species may be combined in an aqueous solution, in the presence or absence of copper, to form a new covalent linkage via a click chemistry mechanism.

In another example, more than two polynucleotides may be linked together using a fu nctionalized linker molecu le. For example, a functionalized saccharide molecule may be chemically modified to contain multiple chemical reactive groups (SH— , NH₂— , N₃, etc.) to react with the cognate moiety on a 3'-functionalized RNA molecu le (i.e., a 3'-maleimide ester, 3'-NHS-ester, alkynyl). The nu mber of reactive groups on the modified saccharide can be controlled in a stoichiometric fashion to directly control the stoichiometric ratio of conjugated polynucleotide, primary construct or RNA. Noncoding polynucleotides and primary constructsRNA polynucleotides may have a noncoding region. Such noncoding regions may be the "first region" of the RNA construct. Alternatively, the noncoding region may be a region other than the first region. Such can exert an effect on protein production by one or more of binding to and sequestering one or more translational machinery components, such as a ribosomal protein or a transfer RNA (tRNA), thereby effectively reducing protein expression in the cell or modulating one or more pathways or cascades in a cell which in turn alters protein levels. The polynucleotide or primary construct may contain or encode one or more long noncoding RNA (IncRNA, or lincRNA) or portion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA), or Piwi-interacting RNA (piRNA).

DNA vaccines

DNA vaccines can also be used in the compositions and methods disclosed herein, wherein the DNA molecule encodes a phosphopeptide mimetic.

The DNA construct can be operably incorporated in an expression vector such as the

BUDCE4.1 expression vector (Invitrogen, Inc., Carlsbad, CA). Other suitable expression vectors are commercial ly available, for example, from BD Biosciences Clonetech, Palo Alto, CA. Once incorporated in the expression vector, the DNA construct can be introduced into a host vector such as a live, attenuated bacterial vector, by transfecting the host cell with the expression vector to provide a vaccine of the present invention.

DNA constructs can include regu latory elements necessary for expression of nucleotides. Such elements include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for expression of a sequence that encodes the phosphopeptide mimetic. As is known in the art, these elements are preferably operably linked to the sequence that encodes the desired protein. Regu latory elements are preferably selected that are compatible with the species to which they are to be administered.

Initiation codons and stop codons are included as part of a nucleotide sequence that encodes the phosphopeptide mimetic. The initiation and termination codons must, of cou rse, be in frame with the coding sequences for the phosphopeptide mimetic.

Promoters and polyadenylation signals included in a vaccine are preferably selected to be functional within the cells of the subject to be immunized. Examples of usefu l promoters, especially in the context of hu mans, include promoters from Simian Virus 40 (SV40), Mouse Mammary Tu mor Virus (M MTV) promoter, Hu man I mmunodeficiency Virus (H IV), such as the H IV Long Terminal Repeat (LTR) promoter; Moloney virus, Cytomegalovirus (CMV), such as the CMV immediate early promoter; Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV); as well as promoters from human genes, such as human actin, hu man myosin, human hemoglobin, human muscle creatine, and human metalothionein.

Examples of usefu l polyadenylation signals, especially in the production of a vaccine for hu mans, include SV40 polyadenylation signals and LTR polyadenylation signals.

Other elements can be included in the DNA molecule, such as enhancers. The enhancer can be, for example, human actin, hu man myosin, human hemoglobin, hu man muscle creatine, and viral enhancers, such as those from CMV, RSV and EBV.

Regulatory sequences and codons are generally species-dependent. In order to maximize peptide production, the regu latory sequences and codons are selected to be effective in the species to be immunized.

The DNA constructs can be "naked" DNA as defined in Restifo et al. Gene Therapy 2000; 7:89-92. Preferably, the DNA is operably incorporated in a vector. Usefu l delivery vectors include biodegradable microcapsules, immuno-stimu lating complexes (ISCOMs), or liposomes; and genetically engineered attenuated live vectors, such as viruses or bacteria.

Examples of suitable attenuated live bacterial vectors include Salmonella typhimurium, Salmonella typhi, Shigella species, Bacillus species, Lactobacillus species, Bacille Calmette-Guerin (BCG), Escherichia coli, Vibrio cholerae, Campylobacter species, Listeria species, or any other suitable bacterial vector, as is known in the art.

Listeria can be exceptional ly useful. For example, recombinant Listeria can comprise a construct comprising a polynucleotide encoding at least one phosphopeptide mimetic. The Listeria can be L. monocytogenes and used to induce an immune response by administering the recombinant Listeria to a subject, as provided, for example, in

WO2006036550 (incorporated herein by reference in its entirety). See also WO2004084936 and WO2004110481, which are directed to specific strains of recombinant L.

monocytogenes. For example, WO2004084936 (incorporated by reference in its entirety) discloses vaccines comprising a free-living microbe, such as L. monocytogenes, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. In some cases, the microbial gene expression in the microbe is substantially unaffected by attenuation of the proliferation of the microbe. The microbe, such as L. monocytogenes, in the vaccine expresses an antigen at a sufficient level to induce an immu ne response to the antigen in a subject upon ad ministration. The microbe comprises a heterologous nucleic acid sequence encoding an antigen, such as a phosphopeptide mimetic.

Methods of transforming live bacterial vectors with an exogenous DNA construct are wel l described. See, for example, Joseph Sambrook and David W. Russell, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) (Sambrook and Russell).

Preferred viral vectors include bacteriophages, Herpes virus, adenovirus, polio virus,

Vaccinia virus, and Avipox. Methods of transforming viral vector with an exogenous DNA construct are also well described. See Sambrook and Russell, above.

Useful liposome vectors are unilamellar or mu ltilamel lar vesicles, having a membrane portion formed of lipophilic material and an interior aqueous portion. The aqueous portion is used to contain the polynucleotide material to be delivered to the target cel l. It is generally preferred that the liposome forming materials have a cationic group, such as a quaternary ammoniu m group, and one or more lipophilic groups, such as satu rated or unsatu rated alkyl groups having about 6 to about 30 carbon atoms. One group of suitable materials is described in EP 0187702, and further discussed in U.S. Pat. No. 6,228,844 (each of which is herein incorporated by reference in its entirety). Many other suitable liposome- forming cationic lipid compounds are described in the literatu re. See, e.g., L. Stamatatos, et al., Biochemistry 1988; 27:3917-3925; and H. Eibl, et al., Biophysical Chemistry 1979;

10:261-271, each of which is herein incorporated by reference in its entirety. Alternatively, a microsphere such as a polylactide-coglycolide biodegradable microsphere can be utilized. A nucleic acid construct is encapsulated or otherwise complexed with the liposome or microsphere for delivery of the nucleic acid to a tissue, as is known in the art.

Other usefu l vectors include polymeric microspheres comprising biodegradable poly(ortho ester) materials, as described by Wang et al., Nat. Mater., 2004; 3(3):190-6 (incorporated by reference in its entirety). Liposomes and microspheres, among other vectors, are described in more detail below.

In some embodiments, the DNA vaccines are ad ministered orally, intramuscularly, intranasally, intraperitoneal^, subcutaneously, intradermal^, or topically. In an embodiment, a DNA vaccine can be used to provide long-term inhibition of tumor growth in a patient treated with the vaccine. The DNA vaccine comprises a DNA polynucleotide construct operably encoding a phosphopeptide mimetic and a

pharmaceutically acceptable carrier. The vaccine is ad ministered to a mammal in need of inhibition cancer growth in an amount that is sufficient to elicit an immune response against tumor cells.

Cell penetrating peptides

Cell penetrating peptides can be included among the compositions that include RNA or DNA molecules encoding phosphopeptide mimetic. Cell penetrating peptides can be linked to cargo, such as nucleic acids (RNA or DNA), and, by way of the cell penetrating peptide, deliver this cargo to the interior of a cell. The cargo is linked covalently or non- covalently known in the art. See, for example, U.S. Patent Nos. 8,772,449, 8,673,313, and 8,372,951, each of which is herein incorporated by reference in its entirety.

Conjugates

Nucleic acids can include conjugates, such as being covalently linked to a carrier or targeting grou p, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting grou p and therapeutic protein or peptide).

Conjugates include a naturally occurring su bstance, such as a protein (e.g., hu man seru m albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an carbohyd rate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer).

Representative U.S. patents that teach the preparation of polynucleotide conjugates, particularly to RNA, include U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; and 7,037,646, each of which is herein incorporated by reference in its entirety.

Formulations for transfecting cells in vitro

A variety of methods are known in the art and are suitable for introduction of nucleic acids into a cell, including viral and non-viral techniques. Examples of non-viral tech niques include electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation (see US20100009424, incorporated by reference in its entirety), heat shock,

magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran,

polyethylenimine, and polyethylene glycol (PEG)) or cell fusion.

Formulations can include one or more cell penetration agents, e.g., transfection agents. I n one specific embodiment, nucleic acid molecules are mixed or admixed with a transfection agent (or mixtu re thereof), and the resulting mixtu re is used to transfect cells. Preferred transfection agents are cationic lipid compositions, particularly monovalent and polyvalent cationic lipid compositions, more particularly LIPOFECTIN^®, LIPOFECTACE^®, LIPOFECTAMIN E™, CELLFECT1N^®, DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER, and dend rimer compositions, particularly G5-G 10 dendrimers, including dense star dend rimers, PAMAM dendrimers, grafted dend rimers, and dendrimers known as dendrigrafts and SUPERFECT^®.

In a second specific transfection method, a ribonucleic acid is conjugated to a nucleic acid-binding group, for example a polyamine and more particu larly a spermine, which is then introduced into the cell or admixed with a transfection agent (or mixtu re thereof) and the resulting mixture is used to transfect cells. In a third example, a mixture of one or more transfection-enhancing peptides, proteins, or protein fragments, including fusagenic peptides or proteins, transport or trafficking peptides or proteins, receptor-ligand peptides or proteins, or nuclear localization peptides or proteins and/or their modified analogs (e.g., spermine modified peptides or proteins) or combinations thereof, are mixed and complexed with a ribonucleic acid to be introduced into a cell, optionally being admixed with transfection agent, and the resulting mixture is used to transfect cells. Further, a component of a transfection agent (e.g., lipids, cationic lipids or dend rimers) is covalently conjugated to selected peptides, proteins, or protein fragments directly or via a lin king or spacer grou p. Of particu lar interest are peptides or proteins that are fusagenic, membrane- permeabilizing, transport or trafficking, or which fu nction for cel l-targeting. The peptide- or protein-transfection agent complex is combined with a ribonucleic acid and used for transfection.

Ex vivo treatment

One embodiment that exploits in vitro cell transfection is an ex vivo approach for treating a su bject suffering from, suspected of suffering from, or who has suffered from, cancer. I n this approach, a construct that expresses a phosphopeptide mimetic is introduced into cells, such as antigen presenting cells (APCs) (e.g., dendritic cells), by, e.g., transfection or transduction. The cells can be autologous cells isolated from the subject before transfection or transduction. Alternatively, the cells can be allogeneic cel ls or universal donor cel ls. The cells are then returned to the subject by, e.g., infusion. In one

embodiment, the cel ls are infused with an adjuvant. Methods of isolating cells from a subject and returning them to a subject are well known in the art.

Delivery

The present disclosure encompasses the delivery of nucleic acid molecu les for any of therapeutic, pharmaceutical, diagnostic or imaging purpose by any appropriate route.

Delivery may be naked or formu lated.

The desired RNA or DNA molecules can be delivered to a cell naked, delivering nucleic acid molecules free from agents that promote transfection.

The desired RNA or DNA molecules can be formulated. The formulations may contain the desired RNA or DNA molecules, whether modified and/or un modified. The formulations may further include cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained- release delivery depot.

The compositions may also be formulated for direct delivery to an organ or tissue, including direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or d rops; by using substrates, such as fabric or biodegradable materials coated or impregnated with the compositions.

Vaccine adjuvants

In one embodiment, the pharmaceutical compositions of the invention comprise

RNA or DNA molecules encoding at least on phosphopeptide and a pharmaceutically acceptable carrier or excipient. Adjuvant(s) can be administered separately from the RNA or DNA molecu les.

RNA and DNA molecu les can be present in a composition in admixture with one or more adjuvants. The RNA or DNA molecules and adjuvant(s) may be mixed together in the same fluid volume, or the RNA or DNA molecu les and adjuvant(s) may be contained within a composition.

A systemic adjuvant is an adjuvant that can be delivered parenterally. Systemic adjuvants include adjuvants that create a depot effect, adjuvants that stimulate the immune system, and adjuvants that do both. An adjuvant that creates a depot effect is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cel ls to the antigen. This class of adjuvants includes alu m (e.g., aluminum hydroxide, alu minum phosphate); or emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water-in oil emu lsion, oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris,

France); M F-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC, Pharmaceuticals Corporation, San Diego, Calif.).

Other adjuvants include immunostimulatory nucleic acids, cytokines, saponins (e.g.,

QS-21), poly IC (a synthetic polyinosinic-polycytidylic acid double-stranded RNA), and Poly ICLC (a synthetic complex of carboxymethylcel lulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA). Heat shock (stress) proteins

Heat shock proteins, which are also referred to interchangeably as stress proteins, can be included in the compositions and pharmaceutical compositions of the invention to aid in nucleic acid delivery and/or stabilization (Henics, et al. 1999. J. Biol. Chem. 274:

17318-17324, incorporated herein by reference in its entirety). Such proteins can be selected from among any cellu lar protein that satisfies the following criteria. Stress proteins are capable of binding other proteins or peptides, capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or under acidic conditions; and show at least 35% homology with any protein having the above properties. Preferably, the intracellu lar concentration of such protein increases when a cell is exposed to a stressful stimulus. In addition to those heat shock proteins that are induced by stress, the HSP60, HSP70, HSP90, HSP100, sHSPs, and PDI families also include proteins that are related to stress-induced HSPs in sequence similarity, for exa mple, having greater than 35% amino acid identity, but whose expression levels are not altered by stress. Therefore, the definition of stress protein or heat shock protein (HSP) embraces other proteins, mutants, analogs, and variants thereof having at least 35% to 55%, preferably 55% to 75%, and most preferably 75% to 85% amino acid identity with members of these families whose expression levels in a cel l are enhanced in response to a stressful stimulus.

In addition to the major HSP families described supra, an endoplasmic reticulu m resident protein, calreticulin, has also been identified as yet another heat shock protein useful for eliciting an immune response when complexed to antigenic molecu les (Basu and Srivastava, 1999, J. Exp. Med. 189:797-202, incorporated herein by reference in its entirety). Other stress proteins that can be used in the invention include grp78 (or BiP), protein disulfide isomerase (PDI), hspllO, and grpl70 (Lin et al., 1993, Mol. Biol. Cell, 4:1109-1119; Wang et al., 2001, J. Immunol., 165:490-497, each of which is herein incorporated by reference in its entirety). Other stress proteins include hsc70, hsp70, hsp90, grpl70, and gp96.

Where HSPs are used, peptide-binding fragments of HSPs and functionally active derivatives, analogs, and variants of HSPs can also be used. The term "HSP peptide-binding fragment" is used to refer to a polypeptide that comprises a domain that is capable of becoming noncovalently associated with a peptide to form a complex, but that is not a ful l- length HSP. The term "variant of HSPs" refers to a polypeptide that is capable of becoming noncovalently associated with a peptide to form a complex, and that shares a high degree of sequence similarity with a HSP. Preparation of the pharmaceutical compositions

The invention encompasses pharmaceutical compositions comprising RNA or DNA molecules either by themselves as the active ingredient or in combination with one or more adjuvants, for the prevention and treatment of a cancer. In a specific embodiment, the invention encompasses pharmaceutical compositions comprising RNA or DNA molecu les encoding phosphopeptide mimetic. The vaccine formulation of the invention may be prepared by any method that results in a stable, sterile, preferably injectable formulation.

Dosage forms suitable for internal ad ministration preferably contain from about 0.1 μg to 100 μg of active ingredient per unit. The active ingredient may vary from 0.5 to 95% by weight based on the total weight of the composition. Those skilled in the art of immunotherapy will be able to adjust these doses.

The amou nt of phosphopeptide mimetic-encoding RNA or DNA and adjuvants used in the pharmaceutical compositions may vary depending on the chemical natu re and the potency of the RNA molecu les and adjuvants. Typically, the starting concentration of RNA or DNA molecules and adjuvants in the vaccine formu lation is the amount that is conventionally used for eliciting the desired immune response, using the conventional routes of administration. The concentration of the RNA or DNA molecu les and adjuvants is then adjusted, e.g., by dilution using a diluent, in the pharmaceutical compositions so that an effective protective immu ne response is achieved as assessed using standard methods known in the art.

Pharmaceutical compositions can be optional ly prepared as lyophilized product, which may then be formu lated for oral administration or reconstituted to a liquid form for parenteral administration.

Pharmaceutical compositions can additionally be formulated to contain other agents including bulking agents, stabilizing agents, buffering agents, sodiu m chloride, calciu m salts, surfactants, antioxidants, chelating agents, cel l penetration agents, other excipients, and combinations thereof.

Bu lking agents are preferred in the preparation of lyophilized formulations of the vaccine composition. Such bulking agents form the crystal line portion of the lyophilized product and may be selected from mannitol, glycine, alanine, and hydroxyethyl starch (HES).

Stabilizing agents may be selected from sucrose, trehalose, raffinose, and arginine. These agents are preferably present in amounts between 1-4%. Sodium chloride can be included in the present formu lations preferably in an amou nt of 100-300 mM, or if used without the aforementioned bul king agents, can be included in the formulations in an amount of between 300-500 mM NaCI. Calcium salts include calciu m ch loride, calcium gluconate, calciu m glu bionate, or calcium gluceptate.

Many formu lations known in the art can be used. For example, U.S. Pat. No.

5,763,401 (incorporated herein by reference in its entirety) describes a therapeutic formulation, comprising 15-60 mM sucrose, up to 50 mM NaCI, u p to 5 mM calciu m chloride, 65-400 mM glycine, and up to 50 mM histidine.

Formulations may comprise a pharmaceutically acceptable excipient, which includes any solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and tech niques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006). The use of a conventional excipient medium is contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

U.S. Pat. No. 5,733,873 (incorporated herein by reference in its entirety) discloses formulations which include between 0.01-1 mg/ml of a surfactant. This patent discloses formulations having the following ranges of excipients: polysorbate 20 or 80 in an amount of at least 0.01 mg/ml, preferably 0.02-1.0 mg/ml; at least 0.1 M NaCI; at least 0.5 mM calcium salt; and at least 1 mM histidine. More particularly, the following specific formulations are also disclosed: (1) 14.7-50-65 mM histidine, 0.31-0.6 M NaCI, 4 mM calcium ch loride, 0.001- 0.02-0.025% polysorbate 80, with or without 0.1% PEG 4000 or 19.9 mM sucrose; and (2) 20 mg/ml mannitol, 2.67 mg/ml histidine, 18 mg/ml NaCI, 3.7 mM calciu m chloride, and 0.23 mg/ml polysorbate 80.

The use of low or high concentrations of sodiu m ch loride has been described, for example U.S. Pat. No. 4,877,608 (incorporated herein by reference in its entirety) teaches formulations with relatively low concentrations of sodiu m chloride, such as formulations comprising 0.5 mM-15 mM NaCI, 5 mM calcium chloride, 0.2 mM-5 mM histidine, 0.01-10 mM lysine hydrochloride and up to 10% maltose, 10% sucrose, or 5% mannitol.

U.S. Pat. No. 5,605,884 (incorporated herein by reference in its entirety) teaches the use of formulations with relatively high concentrations of sodium chloride. These formulations include 0.35 M-1.2 M NaCI, 1.5-40 mM calciu m chloride, 1 mM-50 mM histidine, and up to 10% sugar such as man nitol, sucrose, or maltose. A formu lation comprising 0.45 M NaCI, 2.3 mM calciu m ch loride, and 1.4 mM histidine is exemplified.

WO 96/22107 (incorporated herein by reference in its entirety) describes formulations which include the sugar trehalose, for example formulations comprising: (1) 0.1 M NaCI, 15 mM calcium chloride, 15 mM histidine, and 1.27 M (48%) trehalose; or (2)

0.011% calcium chloride, 0.12% histidine, 0.002% TRIS, 0.002% Tween 80, 0.004% PEG 3350, 7.5% trehalose; and either 0.13% or 1.03% NaCI.

U.S. Pat. No. 5,328,694 (incorporated herein by reference in its entirety) describes a formulation which includes 100-650 mM disaccharide and 100 mM-1.0 M amino acid, for example (1) 0.9 M sucrose, 0.25 M glycine, 0.25 M lysine, and 3 mM calciu m chloride; and (2) 0.7 M sucrose, 0.5 M glycine, and 5 mM calcium chloride.

The pharmaceutical formu lation may include nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids, fibrin gel, fibrin hydrogel, fibrin glue, fibrin sealant, fibrinogen, thrombin, rapidly eliminated lipid nanoparticles (reLNPs) and combinations thereof.

Formulations using lipidoids

Pharmaceutical compositions include lipidoid— based formu lations allowing for localized and systemic delivery of RNA or DNA. The synthesis of lipidoids has been extensively described, and formu lations containing these compounds are particularly suited for delivery of polynucleotides (see, Mahon et al., Bioconjug Chem. 2010 21:1448-1454;

Sch roeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569;

Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; each of which is herein incorporated by reference in its entirety). Complexes, micel les, liposomes or particles can be prepared containing these lipidoids and therefore, resu lt in an effective delivery of RNA or DNA, as judged by the production of an encoded protein, following the injection of an RNA- or DNA-formulated lipidoids via localized and systemic routes of administration. RNA- or DNA- lipidoid complexes can be ad ministered by various means.

The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse th rough the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879, incorporated herein by reference in its entirety), use of lipidoid oligonucleotides to deliver the formulation to other cel ls types including endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited.

Effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including disteroyl phosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formu lation of the RNA or DNA molecu le for delivery to different cell types including hepatocytes, myeloid cel ls, muscle cel ls, etc. For example, the component molar ratio may include 50% lipid, 10% disteroyl phosphatidyl choline, 38.5% cholesterol, and 1.5% PEG. The lipid may be DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 (including variants and derivatives), DLin-MC3-DMA and analogs thereof. The use of lipidoid formulations for the localized delivery of nucleic acids to cel ls (such as adipose cells and muscle cel ls) via either subcutaneous or intramuscular delivery, may also not require all of the formulation components which may be required for systemic delivery, and as such may comprise the lipidoid and the RNA or DNA.

Combinations of different lipidoids may be used to improve the efficacy of RNA- or DNA-directed protein production.

RNA or DNA may be formulated by mixing the polynucleotide with the lipidoid at a set ratio prior to addition to cells. In vivo formulations may require the addition of extra ingredients to facilitate circulation throughout the body. To test the ability of these lipidoids to form particles suitable for in vivo work, a standard formulation process used for siRNA- lipidoid formulations may be used as a starting point. Initial polynucleotide-lipidoid formulations consist of particles composed of 42% lipidoid, 48% cholesterol and 10% PEG, with further optimization of ratios possible. After formation of the particle, polynucleotide is added and al lowed to integrate with the complex. The encapsulation efficiency is determined using a standard dye exclusion assays.

In vivo delivery of nucleic acids may be affected by many parameters, including the formulation composition, natu re of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as particle size (Akinc et al., Mol Ther. 2009 17:872-879, incorporated herein by reference in its entirety). As an example, smal l changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may resu lt in significant effects on in vivo efficacy. Formulations with the different lipidoids, including penta[3-(l- laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Mu rugaiah et al., Analytical Biochemistry, 401:61 (2010), incorporated herein by reference in its entirety), C12-200 (including derivatives and variants), MD1, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA and DLin-MC3-DMA, can be tested for in vivo activity.

The lipidoid referred to as "98N12-5" is disclosed by Akinc et al., Mol Ther. 2009

17:872-879 (incorporated herein by reference in its entirety). The lipidoid referred to as "C12-200" is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670, each of which is herein incorporated by reference in its entirety.

The lipidoid formu lations can include particles com prising either 3 or 4 or more components in addition to polynucleotide. As an example, formulations with certain lipidoids, include 98N12-5, and may contain 42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chain length). As another example, formulations with certain lipidoids, include C12- 200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.

Liposomes, lipoplexes, and lipid nanoparticles

Nucleic acid molecu les can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of a nucleic acid molecule include liposomes. Liposomes may be used as a delivery vehicle for the administration of pharmaceutical formulations. Liposomes can be of different sizes, such as a multilamellar vesicle (M LV) that may be hund reds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicel lular vesicle (SUV) that may be smaller than 50 n m in diameter, and a large u nilamel lar vesicle (LUV) that may be between 50 and 500 nm in diameter. Liposome design may include opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

The formation of liposomes may depend on the physicochemical characteristics such as the pharmaceutical formu lation entrapped and the liposomal ingredients, the natu re of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

For a thorough discussion of liposome, lipoplex and lipid nanoparticles constitution, see US20130244278; see also US20130245105 and US20130245107 (each of which is herein incorporated by reference in its entirety).

Polymers, biodegradable nanoparticles, and core-shell nanoparticles

Nucleic acid molecu les can be formulated using natu ral and/or synthetic polymers. Examples of polymers which can be used for delivery include DYNAM IC POLYCONJUGATE^® (Arrowhead Resea rch Corp., Pasadena,CA) formulations from MIRUS^® Bio (Madison,WI) and Roche Madison (Madison,WI), PHASERX™ polymer formulations such as SMARTT POLYMER TECHNOLOGYT™ (Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN^® adjuvant from Vical (San Diego,CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena,C A), dend rimers and poly(lactic-co-glycolic acid) (PLGA) polymers, RONDEL™

(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Resea rch Corporation, Pasadena, CA) and pH responsive co-block polymers such as, but not limited to, PHASERX™ (Seattle, WA.).

An example of chitosan formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (U.S. Pub. No. 20120258176, incorporated herein by reference in its entirety). Chitosan includes N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-pal mitoyl chitosan (N PCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.

The polymers used can undergo processing to reduce and/or inhibit the attachment of unwanted substances such as bacteria, to the surface of the polymer. The polymer may be processed by methods known and/or described in the art and/or described in

WO2012150467 (incorporated herein by reference in its entirety).

An example of PLGA formulations include PLGA injectable depots (e.g., ELIGARD^® formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the su bcutaneous space).

Many polymer approaches have demonstrated efficacy in delivering oligonucleotides in vivo into the cell cytoplasm (deFougerol les Hum Gene Ther. 2008 19:125-132, incorporated herein by reference in its entirety). Two polymer approaches that have yielded robust in vivo delivery of nucleic acids, in this case with small interfering RNA (siRNA), are dynamic polyconjugates and cyclodextrin-based nanoparticles. The first of these delivery approaches uses dynamic polyconjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887, incorporated herein by reference in its entirety). This particular approach is a multicomponent polymer system which key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and N- acetylgalactosamine (for hepatocyte targeting) groups are lin ked via pH-sensitive bonds (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887, incorporated herein by reference in its entirety). On binding to the hepatocyte and entry into the endosome, the polymer complex disassembles in the low-pH environment, with the polymer exposing its positive charge, leading to endosomal escape and cytoplasmic release of the siRNA from the polymer. Th rough replacement of the N-acetylgalactosamine group with a mannose grou p, it was shown one cou ld alter targeting from asialoglycoprotein receptor-expressing hepatocytes to sinusoidal endothelium and Kupffer cells. Another polymer approach involves using transferrin-targeted cyclodextrin-containing polycation nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI™ gene product in transferrin receptor-expressing Ewing's sarcoma tumor cel ls (Hu-Lieskovan et al., Cancer Res. 2005 65: 8984-8982) and siRNA formulated in these nanoparticles was well tolerated in non-human primates (Heidel et al., Proc Natl Acad Sci USA 2007 104:5715-21, incorporated herein by reference in its entirety). Both of these delivery strategies incorporate rational approaches using both targeted delivery and endosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release of nucleic acid molecules (e.g., following intramuscular or subcutaneous injection). The altered release profile for the nucleic acid molecule can result in, for example, translation of an encoded protein over an extended period of time. The polymer formulation may also be used to increase the stability of the nucleic acid molecule. Biodegradable polymers have been previously used to protect nucleic acids from degradation and been shown to resu lt in sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007

104: 12982-12887; Sullivan et al., Expert Opin Drug Deliv. 2010 7:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu et al., Acc Chem Res. 2012 Jan. 13; Manganiello et al., Biomaterials. 2012 33:2301-2309; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Singha et al., Nucleic Acid Ther. 2011 2:133-147; deFougerolles Hum Gene Ther. 2008

19:125-132; Schaffert and Wagner, Gene Ther. 2008 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 2011 8:1455-1468; Davis, Mol. Pharm. 2009 6:659-668; Davis, Natu re 2010 464: 1067-1070; each of which is herein incorporated by reference in its entirety).

The pharmaceutical compositions can be sustained release formulations. The sustained release formu lations can be for subcutaneous delivery. Sustained release formulations may include PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE^® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX^® (Halozyme Therapeutics, San Diego, CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL^® (Baxter I nternational, Inc Deerfield, IL), PEG-based sealants, and COSEAL^® (Baxter International, Inc).

Nucleic acids may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the nucleic acid in the PLGA microspheres while maintaining the integrity of the nucleic acid du ring the encapsulation process. EVAc are non-biodegradeable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications. Poloxamer F-407 N F is a hydrophilic, non-ionic su rfactant triblock copolymer of polyoxyethylene- polyoxypropylene-polyoxyethylene having a low viscosity at temperatures less than 5° C and forms a solid gel at temperatures greater than 15° C PEG-based su rgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE^® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.

Polymer formu lations can also be selectively targeted th rough expression of different ligands as exemplified by folate, transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887; Davis, Mol. Pharm. 2009 6:659-668; Davis, Natu re 2010 464: 1067-1070, each of which is herein incorporated by reference in its entirety).

Nucleic acid molecu les may be formulated with a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat. No. 8,236,330, each of which is herein incorporated by reference in its entirety) or PLGA-PEG-PLGA block copolymers (See U.S. Pat. No.

6,004,573, incorporated herein by reference in its entirety). Nucleic acid molecu les may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968, incorporated herein by reference in its entirety).

A polyamine derivative may be used to deliver nucleic acid molecules or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pu b. No. 20100260817, incorporated herein by reference in its entirety). A pharmaceutical composition may include the nucleic acid molecules and the polyamine derivative described in U.S. Pub. No. 20100260817 (incorporated herein by reference in its entirety). Nucleic acids may be delivered using a polyaminde polymer such as a polymer comprising a 1,3- dipolar addition polymer prepared by combining a carbohyd rate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280, incorporated herein by reference in its entirety).

Nucleic acids may be formulated with at least one polymer and/or derivatives thereof described in International Publication Nos. WO2011115862, WO2012082574 and WO2012068187 and U.S. Pub. No. 20120283427, each of which is herein incorporated by reference in its entirety. Nucleic acids may be formu lated with a polymer of formula Z as described in WO2011115862 (incorporated herein by reference in its entirety). Nucleic acids may be formulated with a polymer of formula Z, Z' or Z" as described in International Pub. Nos. WO2012082574 or WO2012068187, each of which is herein incorporated by reference in its entirety. The polymers formu lated with nucleic acids may be synthesized by the methods described in International Pub. Nos. WO2012082574 or WO2012068187, each of which is herein incorporated by reference in its entirety.

Formulations of nucleic acid molecules may include at least one amine-containing polymer such as polylysine, polyethylene imine, poly(amidoamine) dendrimers or combinations thereof.

Nucleic acid molecu les may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and described in US Pub. No.

20120269761 (incorporated herein by reference in its entirety).

The described polymers may be conjugated to a lipid-terminating PEG. PLGA may be conjugated to a lipid-terminating PEG forming PLGA-DSPE-PEG. PEG conjugates are described in International Pu blication No. WO2008103276 (incorporated herein by reference in its entirety). The polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No. 8,273,363 (incorporated herein by reference in its entirety).

Nucleic acid molecu les may be conjugated with another compound, such as those described in U.S. Pat. Nos. 7,964,578 and 7,833,992, each of which is herein incorporated by reference in its entirety. Nucleic acid molecules may be conjugated with conjugates of formula 1-122 as described in U.S. Pat. Nos. 7,964,578 and 7,833,992. Nucleic acid molecules may be conjugated with a metal such as gold. (See e.g., Giljohan n et al. Jou rn. Amer. Chem. Soc. 2009 131(6): 2072-2073, incorporated herein by reference in its entirety). In another example, nucleic acid molecules may be conjugated and/or encapsulated in gold- nanoparticles. (WO201216269 and US20120302940, each of which is herein incorporated by reference in its entirety).

As described in US20100004313 (incorporated herein by reference in its entirety), a gene delivery composition may include a nucleotide sequence and a poloxamer. For example, nucleic acid molecules may be used in a gene delivery composition with the poloxamer described in US20100004313.

Nucleic acid molecu les may be formulated in a polyplex of one or more polymers

(US20120237565 and US20120270927, each of which is herein incorporated by reference in its entirety). In one embodiment, the polyplex comprises two or more cationic polymers. The cationic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEL

Further details are provided in US20130244278 (incorporated herein by reference in its entirety).

Nanoparticles

Nucleic acid molecu les can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as calciu m phosphate.

Components may be combined in a core-shell, hybrid, and/or layer-by-layer architectu re, to allow for fine-tuning of the nanoparticle so to delivery of the nucleic acid molecu le may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fu ller et al., Biomaterials. 2008 29:1526- 1532; DeKoker et al., Adv Drug Deliv Rev. 2011 63:748-761; End res et al., Biomaterials. 2011 32:7721-7731; Su et al., Mol Pharm. 2011 Jun. 6; 8(3):774-87; each of which is herein incorporated by reference in its entirety). As a non-limiting example, the nanoparticle may comprise a plurality of polymers such as hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (WO20120225129, incorporated herein by reference in its entirety). The composition of nanoparticles is thorough ly discussed in US20130244278 and US20150086612 (incorporated by reference in their entireties).

Biodegradable calciu m phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver nucleic acid molecu les in vivo. In one embodiment, a lipid coated calcium phosphate nanoparticle, which may also contain a targeting ligand such as anisamide, may be used to deliver the nucleic acid molecu le. For example, to effectively deliver siRNA in a mouse metastatic lung model a lipid coated calcium phosphate nanoparticle was used (Li et al., J Contr Rel. 2010 142: 416-421; Li et al., J Contr Rel. 2012 158: 108-114; Yang et al., Mol Ther. 2012 20:609-615, each of which is herein incorporated by reference in its entirety). This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calciu m phosphate, in order to improve delivery of the siRNA.

Calciu m phosphate with a PEG-polyanion block copolymer may be used to deliver nucleic acid molecules (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel. 2006 111:368-370, each of which is herein incorporated by reference in its entirety). A PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011 32:3106-3114, incorporated herein by reference in its entirety) may be used to form a nanoparticle to deliver nucleic acid molecules. The PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.

In one example, the formu lation comprising nucleic acid molecules is a nanoparticle that may comprise at least one lipid. The lipid may be selected from DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids. In another aspect, the lipid may be a cationic lipid such as DLin-DMA, DLin- D-DMA, DLin-MC3-DMA, DLin-KC2-DMA and DODMA.

The lipid to nucleic acid molecu le ratio in the formulation may be between 10:1 and 30:10. The mean size of the nanoparticle formulation may comprise the nucleic acid molecules between 60 and 225 nm. The PDI of the nanoparticle formulation comprising the modified mRNA is between 0.03 and 0.15. The zeta potential of the lipid may be from -10 to +10 at a pH of 7.4.

The formulations of nucleic acid molecules may comprise a fusogenic lipid, cholesterol and a PEG lipid. The formu lation may have a molar ratio 50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipid may be, for example, PEG- c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC.

The formulation of nucleic acid molecu les may be a PLGA microsphere that may be between 4 and 20 μιτι. The nucleic acid molecules may be released from the formulation at less than 50% in a 48 hour time period. The PLGA microsphere formulation may be stable in seru m. Stability may be determined relative to unformulated modified mRNA in 90%.

The loading weight percent of the nucleic acid molecule PLGA microsphere may be at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4% or at least 0.5%. The encapsulation efficiency of the nucleic acid molecules in the PLGA microsphere may be at least 50%, at least 70%, at least 90% or at least 97%.

A lipid nanoparticle of the present invention may be formulated in a sealant such as, but not limited to, a fibrin sealant.

The use of core-shell nanoparticles has additional ly focused on a high-throughput approach to synthesize cationic cross-lin ked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-13001, incorporated herein by reference in its entirety). The complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle. For example, the core-shell nanoparticles may efficiently deliver siRNA to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.

A hol low lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG may be used to delivery of nucleic acid molecules. In mice bearing a luciferease-expressing tu mor, it was determined that the lipid-polymer-lipid hybrid nanoparticle significantly suppressed luciferase expression, as compared to a conventional lipoplex (Shi et a I, Angew Chem Int Ed. 2011 50:7027-7031, incorporated herein by reference in its entirety).

In one embodiment, the lipid nanoparticles may comprise a core of the nucleic acid molecules and a polymer shell. The polymer shell may be any of the polymers known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acids in the core.

Core-shell nanoparticles for use with the nucleic acid molecules are described and may be formed by the methods described in U.S. Pat. No. 8,313,777 (incorporated herein by reference in its entirety).

The core-shel l nanoparticles may comprise a core of the nucleic acid molecules and a polymer shell. The polymer shel l may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the nucleic acid molecules in the core.

Further details are provided in US20130244278 (incorporated herein by reference in its entirety).

Aberrant phosphoprotein identification

Aberrant phosphopeptides can be identified from a patient's cancer cells using any methods known in the art. For example, immunoaffinity isolation of HLA peptide complexes from, for example, primary cell tumors can be performed. In parallel, affinity isolation of HLA peptide complexes from cognate normal (healthy) tissue is also performed. The associated peptides can then be extracted (Proc. Natl. Acad. Sci. USA 2006. 103: pp. 14889- 14894, incorporated herein by reference in its entirety). The isolated peptides are then converted to, d₀- or d₃-methyl esters and subjected to Fe⁺³-im mobilized metal-affinity chromatography to isolate phosphopeptides (Proc. Natl. Acad. Sci. USA 2006. 103: pp. 14889-14894). The phosphopeptide methyl esters can then be analyzed by nanoflow H PLC, microelectrospray ionization, and collision activated dissociation on LTQ/FT or Orbitrap tandem mass spectrometers (Proc. Natl. Acad. Sci. USA 2006. 103: pp. 14889-14894). The samples can also be analyzed on a LTQ mass spectrometer to determine electron transfer dissociation spectra (Proc. Natl. Acad. Sci. USA, 2004. 101: pp. 9528-9533, incorporated herein by reference in its entirety). Peptide sequence can be determined by manual interpretation of CAD and ETD spectra recorded on the peptide esters (Sci. Transl. Med. 2013. 5(203): 203ral25, incorporated herein by reference in its entirety).

Additional methods include those set forth in Meyer et al. (J Proteome Res. 2009. 8: pp. 3666-367474, incorporated herein by reference in its entirety). See also US20130259883 (incorporated herein by reference in its entirety) for further details. Treatment of cancer

The pharmaceutical compositions can be used alone or in combination with other therapies for the treatment of cancer, such as multiple myeloma or glioblastoma.

The pharmaceutical compositions can be ad ministered when a cancer is detected, or prior to or du ring an episode of recu rrence.

Ad ministration can begin at the first sign of cancer or recurrence, followed by boosting doses u ntil at least symptoms are su bstantially abated and for a period thereafter.

Prevention of cancer recurrence

The pharmaceutical compositions can also be used for immunization against recu rrence of cancers, such as multiple myeloma or glioblastoma. Prophylactic

administration of a pharmaceutical composition to an individual can confer protection against a future recu rrence of a cancer.

Combination therapy

Combination therapy refers to the use of pharmaceutical compositions with another modality to prevent or treat the infectious disease. This approach is common ly termed combination therapy, adjunctive therapy or conjunctive therapy (the terms are used interchangeably). With combination therapy, additive potency or additive therapeutic effect can be observed. Synergistic outcomes where the therapeutic efficacy is greater than additive can also be expected. The use of combination therapy can also provide better therapeutic profiles than the administration of the treatment modality, or the

pharmaceutical compositions of the invention alone. The additive or synergistic effect may allow the dosage and/or dosing frequency of either or both modalities be adjusted to reduce or avoid u nwanted or adverse effects.

In various specific embodiments, the combination therapy comprises the

administration of pharmaceutical compositions to a su bject treated with a treatment modality wherein the treatment modality ad ministered alone is not clinical ly adequate to treat the subject such that the subject needs additional effective therapy, e.g., a subject is un responsive to a treatment modality without administering the pharmaceutical compositions. Included in such embodiments are methods comprising administering the pharmaceutical compositions to a subject receiving a treatment modality wherein said subject has responded to therapy yet suffers from side effects, relapse, develops resistance, etc. Such a subject might be non-responsive or refractory to treatment with the treatment modality alone. Administering the pharmaceutical compositions to a subject refractory to a treatment modality alone can improve the therapeutic effectiveness of the treatment modality. The determination of the effectiveness of a treatment modality can be assayed in vivo or in vitro using methods known in the art. In one embodiment, the pharmaceutical preparations are administered in combination with a second treatment modality comprising a different cancer vaccine.

In one embodiment, a lesser amount of the second treatment modality is required to produce a therapeutic benefit in a subject. In specific embodiments, a reduction of about 10%, 20%, 30%, 40% and 50% of the amount of second treatment modality can be achieved. The amou nt of second treatment modality to be used, including amou nts in a range that does not produce any observable therapeutic benefits, can be determined by dose-response experiments conducted in animal models by methods wel l known in the art.

In another embodiment, the pharmaceutical compositions are used in combination with one or more antibodies, including but not limited to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, antibody fragments, single chain antibodies, and the like. Exemplary antibodies include those that are immune checkpoint inhibitors, such as anti- GITR, anti-OX40, anti-PD-1, anti-CTLA-4, anti-TIM-3, and anti-LAG-3. Other immune checkpoint in hibitors include pazopanib, bevacizumab, nivolumab, pembrolizu mab/MK- 3475, pidilizumab, MEDI0680 (AMP-514), AM P-224; BMS-935559, M EDI4736, MPDL3280A, MSB0010718C, ipilimumab or tremelimumab.

In another embodiment, the pharmaceutical compositions are used in combination with one or more biological response modifiers. One group of biological response modifiers is the cytokines. In one such embodiment, a cytokine is ad ministered to a subject receiving the pharmaceutical compositions of the invention. In another such embodiment, the pharmaceutical compositions of the invention are ad ministered to a subject receiving a chemotherapeutic agent such as an antiviral agent, antibody, adjuvant, or biological response modifier, in combination with a cytokine.

In other embodiments, pharmaceutical compositions are used in combination with one or more biological response modifiers which are agonists or antagonists of various ligands, receptors and signal transduction molecu les of the immu ne system. These agonists and antagonists can be antibodies, antibody fragments, peptides, peptidomimetic compounds, polysaccharides, and small molecules.

In an embodiment, pharmaceutical compositions are used in combination with one or more additional adjuvants such as saponins and immunostimulatory nucleic acids. A saponin, such as QS-21, and the like, including those disclosed in U.S. Pat. Nos. 5,057,540; 5,273,965; 5,443,829; 5,650,398; 6,231,859; and 6,524,584 (each of which is herein incorporated by reference in its entirety) can be used.

Many immunostimulatory nucleic acids are oligonucleotides comprising an unmethylated CpG motif, are mitogenic to vertebrate lymphocytes, and are known to enhance the immune response. See Woolridge, et al., 1997, Blood 89:2994-2998. Such oligonucleotides are described in International Patent Publication Nos. WO 01/22972, WO 01/51083, WO 98/40100 and WO 99/61056, as wel l as U.S. Pat. Nos. 6,207,646, 6,194,388, 6,218,371, 6,239,116, 6,429,199, and 6,406,705, each of which is herein incorporated by reference in its entirety. Other kinds of immunostimulatory oligonucleotides such as phosphorothioate oligodeoxynucleotides containing YpG- and CpR-motifs have been described by Kandimalla et al. in "Effect of Chemical Modifications of Cytosine and Guanine in a CpG-Motif of Oligonucleotides: Structu re-lmmunostimulatory Activity Relationships." Bioorganic & Medicinal Chemistry 9:807-813 (2001). Also encompassed are immunostimulatory oligonucleotides that lack CpG dinucleotides which when administered by mucosal routes (including low dose ad ministration) or at high doses th rough parenteral routes, augment antibody responses, often as much as did the CpG nucleic acids, however the response was Th2-biased (lgGl»lgG2a). See United States Patent Publication No. 20010044416 (incorporated herein by reference in its entirety). Methods of determining the activity of immunostimulatory oligonucleotides can be performed as described in the aforementioned patents and publications. Moreover, immu nostimulatory oligonucleotides can be modified within the phosphate backbone, sugar, nucleobase and internucleotide linkages in order to modu late the activity. Such modifications are known to those of skill in the art.

Patient (subject) evaluation

Patients treated with the cancer vaccine may be tested for an anti-tumor immune response. In this regard, peripheral blood from patients may be obtained and assayed for markers of anti-tumor immunity. Using standard laboratory procedures, leukocytes may be obtained from the peripheral blood and assayed for frequency of different immune cell phenotypes, HLA subtype, and function of anti-tumor immune cells.

Kits

Kits are also provided for carrying out the prophylactic and therapeutic methods of the invention. The kits may optionally be accompanied by instructions on how to use the various components of the kits.

In a specific embodiment, a kit comprises a first container containing RNA or DNA molecules; and a second container containing an adjuvant or adjuvants that, when administered before, concurrently with, or after the administration of the RNA or DNA molecules in the first container, is effective to induce an immune response. In another embodiment, a kit comprises a first container containing RNA or DNA molecules; a second container containing an adjuvant or adjuvants; and a third container containing a second treatment modality. In yet another embodiment, the kit comprises a container containing both the RNA or DNA molecules and adjuvants in one container, and a second container containing a second treatment modality; or an additional adjuvant, such as a saponin, including QS-21. Additional containers may be present for additional treatment modalities that can be used in combination. Preferably, the RNA or DNA molecules and adjuvants in the container are present in pre-determined amounts effective to treat cancers or prevent their recu rrence. If desired, the pharmaceutical compositions can be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the RNA or DNA molecu les. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for ad ministration.

Examples

In this example, two phosphopeptide mimetics were generated based on an HLA-A2- restricted phosphopeptide derived from CDC25b: pCDC25b₃₈-46 G LLG[pS]PVRA (SEQ ID NO: 1). I n the two phosphopeptide mimetics, the phosphorylated serine in pCDC25b₃₈-46 was replaced with either a glutamic acid (GLLGEPVRA (SEQ I D NO: 3)) or an aspartic acid (GLLGDPVRA (SEQ ID NO: 4)). A mu rine cel l line that does not express TCR endogenously was engineered to express a TCR that is specific for pCDC25b₃₈-46/HLA-A2 and the resulting cel l line was examined by flow cytometry staining using HLA-A2 tetramers loaded with the phosphopeptide mimetics described above. As a negative control, the mu rine cel l line was also engineered to express an un related TCR that is specific for a phosphopeptide derived from insulin receptor substrate (IRS)-2: pi RS-21097-1105 RVA[pS]PTSGV (SEQ ID NO: 7) in the context of HLA-A2. Both the pCDC25b-specific TCR and the plRS-2-specific TCR were described in International Pu blication No. WO2015160928 and Zarling ef al., Cancer Res. 2014 Dec l;74(23):6784-95, herein incorporated by reference in their entireties. The a and β chains of the pCDC25b-specific TCR comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively (Table 1). The a and β chains of the plRS-2-specific TCR comprise the amino acid sequences of SEQ ID NOs: 8 and 9, respectively (Table 1).

Table 1. TCR amino acid sequences

Briefly, 1.0 X 10^s cells expressing the pCDC25b-specific TCR or the plRS-2-specific TCR were stained using PE-labeled anti-mouse TCRβ chain clone H57-597 (BD Pharmingen, Cat. No. : 553172) or APC-label led HLA-A2 tetramers loaded with one of the following peptides: pCDC25b₃₈-4₆ GLLG[pS] PVRA (SEQ ID NO: 1), its non-phosphorylated cou nterpart CDC25b₃₈-₄₆ GLLGSPVRA (SEQ ID NO: 2), and the phosphopeptide mimetic GLLGEPVRA (SEQ ID NO: 3) or GLLGDPVRA (SEQ ID NO: 4). Fc receptor blocker (BD Biosciences, Cat. No.: 553172) was used to eliminate backgrou nd binding of the Fc region of the antibody to cells. Flow cytometry was performed using BD FACSCanto with FACSDiva software (Becton-Dickinson).

As shown in Figu re 1, the pCDC25b-specific TCR bound specifical ly to its

phosphorylated target pCDC25b₃₈-46 but not the non-phosphorylated counterpart CDC25b₃₈. 46. Weaker binding was detected with the phosphopeptide mimetic GLLGEPVRA (SEQ ID NO: 3), in which the phosphorylated serine was replaced with a glutamic acid, suggesting that the glutamic acid substitution may be able to retain binding to the phosphopeptide-specific TCR. The negative control cell line expressing plRS-2-specific TCR did not exhibit detectable binding to any of the tetramers tested. The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Al l references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all pu rposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all pu rposes. Other embodiments are within the following claims.

Claims

We claim:

1. A composition comprising a polynucleotide that encodes a first immunogenic peptide that is a substitution variant of a second immunogenic peptide comprising at least one phosphorylated amino acid residue, wherein the second immunogenic peptide is a fragment of an aberrant protein that occurs in cancer cells of a subject having cancer, wherein the first immunogenic peptide comprises the amino acid sequence of the second immunogenic peptide except that the first immunogenic peptide comprises at least one substituted amino acid residue at at least one amino acid position corresponding to the at least one phosphorylated amino acid residue in the second immunogenic peptide, and wherein the first immunogenic peptide does not comprise the entire amino acid sequence of a naturally-occurring protein.

2. The composition of claim 1, wherein the polynucleotide is an RNA molecule.

3. The composition of claim 2, wherein the polynucleotide is an mRNA molecule.

4. The composition of claim 1, wherein the polynucleotide is a DNA molecule.

5. The composition of any one of claims 1-4, wherein the at least one

phosphorylated amino acid residue in the second immunogenic peptide is selected from the group consisting of phospho-Ser, phospho-Thr, phospho-Tyr, phospho-His, phospho-Arg, and phospho-Lys.

6. The composition of any one of claims 1-5, wherein the at least one substituted amino acid residue is selected from the group consisting of Asp and Glu.

7. The composition of claim 6, wherein the at least one substituted amino acid residue is Glu.

8. The composition of any one of claims 1-7, wherein normal cells of the subject comprise a normal form of the aberrant protein, the normal form of the aberrant protein comprising the second immunogenic peptide except that at least one of the amino acid residues that is phosphorylated in the second immunogenic peptide is not phosphorylated in the normal form of the aberrant protein.

9. The composition of claim 8, wherein each amino acid residue that is phosphorylated in the second immunogenic peptide is not phosphorylated in the normal form of the aberrant protein.

10. The composition of claim 9, wherein more than one amino acid residue is phosphorylated in the second immunogenic peptide and is not phosphorylated in the normal form of the aberrant protein.

11. The composition of claim 10, wherein the first immunogenic peptide comprises substituted amino acid residues at each amino acid position corresponding to the

phosphorylated amino acid residues in the second immunogenic peptide.

12. The composition of claim 11, wherein the substituted amino acid residues are each selected from the group consisting of Asp and Glu.

13. The composition of claim 12, wherein the substituted amino acid residues are each Glu.

14. The composition of claim 8, wherein exactly one amino acid residue is phosphorylated in the second immunogenic peptide and is not phosphorylated in the normal form of the aberrant protein.

15. The composition of claim 14, wherein the substituted amino acid residue is selected from the group consisting of Asp and Glu.

16. The composition of claim 15, wherein the substituted amino acid residue is Glu.

17. The composition of any one of claims 1-16, wherein the polynucleotide encoding the first immunogenic peptide encodes a plurality of first immunogenic peptides.

18. The composition of any one of claims 1-16, comprising a plurality of

polynucleotides encoding a plurality of first immunogenic peptides.

19. The composition of any one of claims 1-18, wherein the first immunogenic peptide is 9-11 amino acids in length.

20. The composition of any one of claims 1-18, wherein the first immunogenic peptide is 27-31 amino acids in length.

21. The composition of any one of claims 1-20, further comprising an adjuvant.

22. The composition of claim 21, wherein the adjuvant comprises an

immunostimulatory nucleic acid and/or at least one cytokine.

23. The composition of claim 21, wherein the adjuvant is selected from the group consisting of poly IC, poly ICLC, and QS-21.

24. The composition of any one of claims 1-23, further comprising a stress protein.

25. The composition of claim 24, wherein the stress protein is selected from the group consisting of hsc70, hsp70, hsp90, hspllO, grpl70, gp96, calreticulin, a mutant thereof, and combinations of two or more thereof.

26. The composition of claim 25, wherein the stress protein is hsc70.

27. The composition of any one of claims 1-23, further comprising a cell penetration agent.

28. The composition of any one of claims 1-27, wherein the cancer is multiple myeloma or glioblastoma.

29. The composition of any one of claims 1-20, wherein the composition is formulated in nanoparticles or poly(lactic-co-glycolic acid) (PLGA) microspheres.

30. A pharmaceutical composition comprising the composition of any one of claims 1-29 and a pharmaceutically acceptable excipient.

31. A method of treating a subject having or suspected of having a cancer, comprising administering to the subject the composition of any one of claims 1-29 or the pharmaceutical composition of claim 30.

32. The method of claim 31, wherein the cancer is multiple myeloma or

glioblastoma.

33. The method of claim 32, further comprising administering lenalidomide or dexamethasone.

34. The method of claim 32 or 33, further comprising administering

cyclophosphamide.

35. The method of claim 31 or 32, further comprising administering a checkpoint antibody.

36. The method of claim 35, wherein the checkpoint antibody is selected from the group consisting of anti-GITR, anti-OX40, anti-PD-1, anti-CTLA-4, anti-TIM-3, and anti-LAG-3 antibody.

37. The method of claim 36, wherein the checkpoint antibody is a monoclonal antibody.

38. The method of claim 31 or 32, further comprising administering an inhibitor of indoleamine-2,3-dioxygenase (I DO).

39. The method of claim 38, wherein the inhibitor is selected from the group consisting of epacadostat, F001287, indoximod, and NLG919.

40. A method of immunizing a subject recovering from a cancer, comprising administering to the subject the composition of any one of claims 1-29 or the pharmaceutical composition of claim 30.

41. The method of claim 40, wherein the cancer is multiple myeloma or glioblastoma.

42. The method of claim 41, further comprising administering lenalidomide or dexamethasone.

43. The method of claim 41 or 42, further comprising administering

cyclophosphamide.

44. The method of claim 40 or 41, further comprising administering a checkpoint antibody.

45. The method of claim 44, wherein the checkpoint antibody is selected from th group consisting of anti-GITR, anti-OX40, anti-PD-1, anti-CTLA-4, anti-TIM-3, and anti-LAG-3 antibody.

46. The method of claim 45, wherein the checkpoint antibody is a monoclonal antibody.

47. A method of treating a subject having or suspected of having a cancer, comprising:

a. contacting cells ex vivo with the composition of any one of claims 1-20; and b. infusing the cells into the subject.

48. The method of claim 47, further comprising prior to step (a) isolating the cell from the subject.

49. The method of claim 47 or 48, wherein step (a) comprises transfecting or transducing the cells with the composition of any one of claims 1-20.

50. The method of any one of claims 47-49, wherein the cells are antigen present cells.

51. The method of claim 50, wherein the cells are dendritic cells.

52. The method of any one of claims 47-51, further comprising administering an adjuvant to the subject.

53. A kit, comprising the composition of any one of claims 1-29 or the

pharmaceutical composition of claim 30 and instructions for use.

54. The kit of claim 53, further comprising at least one selected from the group consisting of lenalidomide, dexamethasone, cyclophosphamide, and a checkpoint antibody.

55. The kit of claim 54, wherein the checkpoint antibody is selected from the group consisting of anti-GITR, anti-OX40, anti-PD-1, anti-CTLA-4, anti-TIM-3, and anti-LAG-3 antibody.

56. A recombinant Listeria strain comprising the composition of any one of claims 1-

20.

57. The recombinant Listeria strain of claim 56, wherein the recombinant Listeria strain is a recombinant Listeria monocytogenes strain.

58. The recombinant Listeria strain of claim 56 or 57, wherein the polynucleotide is a DNA molecule.

59. A vaccine comprising the recombinant Listeria strain of any one of claims 56-58 and an adjuvant.

60. A method of treating a subject having or suspected of having a cancer, comprising administering to the subject a composition comprising the recombinant Listeria strain of any one of claims 56-58 or the vaccine of claim 59.