# Patent

## Title
Chimeric nucleic acid molecules with non-AUG translation initiation sequences and uses thereof

## Abstract

The present disclosure relates to nucleic acid vaccine compositions and methods for preventing or treating pathological conditions, such as cancer or infectious disease. Further, the disclosure provides methods for more efficient production of antigens via mRNA containing one or more non-conventional start codons to promote multiplex initiation of translation in eukaryotic cells.

## Background
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 890079_401C2_SEQUENCE_LISTING.txt. The text file is 210 KB, was created on Sep. 1, 2016, and is being submitted electronically via EFS-Web.
BACKGROUND
The immune system can be categorized into innate immunity, which involves numerous cellular and soluble factors that respond to all foreign challenges, and adaptive immunity, which responds specifically to precise epitopes from foreign or abnormal agents. The adaptive immune response includes a humoral arm, which involves the production of antibodies by B lymphocytes, and a cellular arm, which involves the killer activity of cytotoxic T lymphocytes (CTLs). A key mechanism for detecting and eliminating abnormal cells by the adaptive immune response is surveillance by CTLs. Abnormal cells may be those infected with a virus, parasite or bacteria, or those that have undergone a tumorigenic transformation.
Cells naturally produce a repertoire of peptides from essentially any cellular translation product that has been marked for elimination (e.g., ubiquitination), which results in presentation of peptide/major histocompatibility complex (MHC; in humans known as human leukocyte antigen or HLA) class I complexes on their surface. A ubiquitinated protein is targeted to the proteasome for proteolysis, producing smaller peptides that may be recognized by transporter associated with antigen presentation (TAP) proteins that are localized in the endoplasmic reticulum. TAP is a heterodimer that moves small peptides from the cytosol into the endoplasmic reticulum where they bind to HLA/MHC molecules to form a peptide/HLA complex. The peptide/HLA complex is then trafficked to the cell surface.
T cell receptors (TCRs) on the surface of circulating CTLs probe the peptide/MHC complexes for the presence of foreign peptides, such as viral proteins or tumor specific proteins, which will trigger a T cell directed immune response. Cells can present tens of thousands of distinct peptides in the context of WIC molecules as potential ligands for the TCR, although the quantity of each peptide will be very low. Nonetheless, CTLs are very sensitive probes for peptides displayed by WIC class I. By some estimates, only three copies of an antigenic peptide are sufficient to target cells for lysis (Purbhoo et al., Nat. Immunol. 5:524, 2004).
Vaccines have had a profound and long lasting effect on world health. Smallpox has been eradicated, polio is near elimination, and diseases such as diphtheria, measles, mumps, pertussis, and tetanus are contained. Gene therapy and nucleic acid immunization are promising approaches for the treatment and prevention of both acquired and inherited diseases (Li et al., J. Biotechnol. 162:171, 2012). These techniques involve the administration of a desired nucleic acid vaccine directly into a subject in vivo, or by transfecting a subject's cells or tissues ex vivo and reintroducing the transformed material into the subject. Each of these techniques requires efficient expression of a nucleic acid molecule in the transfected cell, which may be affected by several factors, to provide a sufficient amount of a therapeutic or antigenic gene product. Alternatively, antigenic peptides that are defined T cell epitopes may be administered directly to form productive peptide/MHC complexes and stimulate a T cell response.
Current vaccines, however, address only a handful of the infections or cancers suffered by people and domesticated animals. Common infectious diseases for which there are no vaccines cost the United States alone about $120 billion per year (Robinson et al., American Acad. Microbiol., 1996). In first world countries, emerging infections such as immunodeficiency viruses, as well as reemerging diseases like drug resistant forms of tuberculosis, pose new threats and challenges for vaccine development. The need for both new and improved vaccines is even more pronounced in third world countries where effective vaccines are often unavailable or cost-prohibitive.
In view of the limitations associated with current vaccines, there is a need in the art for alternative compositions and methods useful for more efficient and manageable vaccines and vaccinations. The present disclosure meets such needs, and further provides other related advantages.
BRIEF SUMMARY
In some embodiments, the present disclosure provides a chimeric nucleic acid molecule, comprising a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding an antigen, an antigenic epitope, or a combination thereof, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the antigen, antigenic epitope, or combination thereof.
In certain embodiments, the present disclosure provides a vector, comprising a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding an antigen, an antigenic epitope, or a combination thereof, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the antigen, antigenic epitope, or combination thereof.
In some embodiments, the present disclosure provides a cell, comprising a chimeric nucleic comprising a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding an antigen, an antigenic epitope, or a combination thereof, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the antigen, antigenic epitope, or combination thereof.
In further embodiments, the present disclosure provides a cell comprising a vector, comprising a chimeric nucleic comprising a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding an antigen, an antigenic epitope, or a combination thereof, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the antigen, antigenic epitope, or combination thereof.
In some embodiments, the present disclosure provides a method of eliciting a cellular immune response, comprising administering to a subject an effective amount of an immunization composition comprising a nucleic acid molecule according as described in embodiments herein, an antigen encoded by a nucleic acid molecule as described in embodiments herein, or both, thereby eliciting a cellular immune response.
In some embodiments, the present disclosure provides a method of eliciting a cellular immune response, comprising administering to a subject an effective amount of a cell comprising a nucleic acid molecule as described in embodiments herein, or a vector as described in embodiments herein, thereby eliciting a cellular immune response, thereby eliciting a cellular immune response.
In further embodiments, the present disclosure provides a method of eliciting a cellular immune response, comprising (a) administering to a subject an effective amount of an antigen immunization composition comprising one or more antigens encoded by any one of the nucleic acid molecules as described in embodiments herein, and (b) administering to the subject an effective amount of a nucleic acid molecule immunization composition comprising a nucleic acid molecule as described in embodiments herein or a vector as described in embodiments herein.
In in yet further embodiments, the present disclosure provides a method of eliciting an cellular immune response, comprising (a) administering to a subject an effective amount of a nucleic acid molecule immunization composition comprising a nucleic acid molecule as described in embodiments herein, (b) allowing a time sufficient to generate an initial immune response, and (c) administering to the subject a second effective amount of a nucleic acid molecule immunization composition comprising a nucleic acid molecule as described in embodiments herein, or an effective amount of an antigen immunization composition.
In some embodiments, the present disclosure provides a method of treating breast cancer, comprising administering to a subject an effective amount of nucleic acid molecule immunization composition comprising a comprising a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding an antigen, an antigenic epitope, or a combination thereof, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the antigen, antigenic epitope, or combination thereof, and the antigenic epitope comprises a peptide from folate receptor alpha, HER2/neu, or any combination thereof.
In some embodiments, the present disclosure provides a method of treating cancer, comprising administering to a subject an effective amount of a nucleic acid immunization composition comprising a nucleic acid molecule as described in embodiments herein, wherein the one or more antigens encoded the nucleic acid molecule comprise an antigen having an oncogenic mutation identified in the subject.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts an exemplary DNA vaccine expression vector. A) The vaccine vector contains full-length, codon-optimized TAP 1 and TAP 2 driven by an SV40 promoter and the MTI-peptide antigen array (PAA) under the control of the CMV promoter. B) Overexpression of the PAA is achieved through the application of three non-classical translation initiation sites (CUG) that can be found in the MTI. These CUG and AUG sites mediate nuclear and cytosolic expression respectively, and can be altered as needed. The PAA is flanked by Xba1 sites facilitating insertion and excision of various PAAs. C) Peptide sequences in the A*0201 PAA are depicted (SEQ ID NO.: 204). A2 binding peptides are presented in bold font. Each peptide is flanked by 3-4 amino acid residues at the NH2 and COOH ends to retain the natural proteasome processing sites for each peptide. Each peptide is also separated by the (G4S)2 spacer. This specific PAA also includes an Influenza M1 protein as a control, and a C-terminal VSVG protein epitope tag (SEQ ID NO.: 14) for evaluating epitope expression.
FIG. 2 depicts immunoprecipitation detection of TAP1 and MTI-PAA. A) Cell extracts from TAP1-transfected COS cells were immunoprecipitated with goat anti-V5 antibody (the NH2-terminal tag on recombinant TAP1) and protein G agarose, separated by 12% SDS-PAGE, transferred to PVDF, and immunoblotted with rabbit anti-V5 antibody. B) Cell extracts from MTI-PAA transfected COS cell were heparin sepharose (HS) purified, separated by 12% PAGE, transferred, and immunoblotted with goat anti-FGF2 antibodies.
FIG. 3 depicts immunofluorescence micrographs of expression of TAP1 in COS cells using anti-V5 and anti-TAP1 antibodies.
FIG. 4 depicts immunofluorescence micrographs of expression of MTI-PAA. Staining was performed using an anti-VSV antibody directed against the C-terminal portion of the MTI-PAA.
FIG. 5 depicts the immunoprecipitation detection of PAA products in the presence or absence of proteasome activity. HEK cells were transfected with pcDNA3PAA or control. Immunoprecipitation of expression products in the presence of the proteasome inhibitor MG132 showed the expression of the predicted translation products of MTI-PAA.
FIG. 6 depicts T cell recognition of PAA-expressing targets. Splenocytes from peptide-vaccinated (#22, #25, #28, #30, HBV core Ag: emulsified in incomplete Freund's adjuvant (IFA)) mice were incubated with THP-1 cells (column 1), THP-1 cells pulsed with 1 ug/ml of each peptide (p22, p25, p28, p30) (column 2), or were transfected with MTI-PAA expression vector for 18 hours (column 3).
FIG. 7 depicts T cell reactivity, measured by ELISPOT, against a series of stably transfected HEK cell lines. The cell lines were super-transfected as described. The first pair of bars correspond to HEK cells stably transfected with a clone expressing Tap1 (white/clear bar). These cells were super-transfected with an expression vector encoding a small pox PAA (diagonal line bar). Moving left to right, the next four pairs of bars represent four independent stably transfected HEK cell lines expressing Tap1. Each Tap1 transfected cell line was super-transfected with a PolyStartTM-PAA encoding small pox antigenic peptides. The diagonal lined bars show elevated T-cell reactivity, indicating proteasome processing of a small pox PAA. The two right most bars are negative controls (clear/white is non-transfected normal HEK cells and HEK cells pulsed with small pox peptides only).
FIG. 8 depicts results from IFNγ ELISPOT assays measuring the immune response in HLA-A2 mice after a two-dose vaccination regimen. Peptide responses are reported as the fold increase (in IFNγ spot forming units) in response to target cells expressing peptide immunogen over response to target cells expressing an unrelated peptide. All responses shown are significant (p<0.05) except p19 and p25.
FIG. 9 depicts an immune response after peptide vaccination. HLA-A2 mice were vaccinated twice with peptides #22, #25, #28 emulsified in IFA. Immune responses were measured against syngeneic splenocytes pulsed with pooled peptide cocktail (peptides #22, #25, #28: 1 uM each) (cross-hatched bars) or unpulsed (white bars).
FIG. 10 depicts the effect of peptide-vaccine on survival after lethal intranasal challenge with VACV-WR. Kaplan-Meier survival curve for unvaccinated mice (broken line) and peptide-vaccinated (#22, #25, #28, #30) mice (solid line) following lethal intranasal challenge with VACV-WR (1×106 pfu). Significance was assessed using the log-rank test.



DETAILED DESCRIPTION
In some aspects, the present disclosure provides a chimeric nucleic acid molecule comprising a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding one or more antigens, antigenic epitopes, or a combination thereof (e.g., polyantigen array or PAA), wherein the multiplex translation sequence comprises at least one non-AUG translation initiation site that mediates translation initiation of the one or more antigens, antigenic epitopes, or a combination thereof.
In certain embodiments, the present disclosure provides a method for prime and boost or multiple antigenic challenges to elicit a robust immune response that results in the production memory T cells. For example, an immune response is elicited against a cancer or infectious disease by (a) contacting a subject with an antigenic peptide immunization composition, (b) optionally allowing a time sufficient to generate an initial immune response, (c) contacting the subject with a nucleic acid molecule immunization composition as described herein, wherein the nucleic acid molecule of the nucleic acid molecule immunization composition encode one or more of the same antigenic peptides as used in step (a).
Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.
In the present description, any concentration range, percentage range, ratio range, or integer range includes the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth or one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature (such as polymer subunits, size or thickness) include any integer within the recited range, unless otherwise indicated. As used herein, the term “about” means (1) ±20% of the indicated range, value or structure; (2) a value that includes the inherent variation of error for the method being employed to determine the value; or (3) a value that includes the variation that exists among replicate experiments, unless otherwise indicated. As used herein, the terms “a” and “an” refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) means either one, both, or any combination thereof of the alternatives or enumerated components. As used herein, the terms “include,” “have” and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.
The term “multiplex translation leader sequence,” “multiplex translation initiation sequence,” or “MTI” refers to a nucleic acid molecule comprising at least one non-conventional CUG start codon in addition to the one standard ATG start codon. In some embodiments, an MTI allows the production of more than one mole of protein per mole of mRNA. In certain embodiments, an MTI nucleic acid molecule corresponds to a 5′-portion of an FGF2 gene (e.g., a human FGF2 gene as set forth in GenBank Accession No. NM_002006.4) containing (a) the ATG start codon of FGF2 and (b) a sequence comprising about 123 nucleotides to about 385 nucleotides upstream (5′) of the ATG start codon of FGF2, wherein this portion upstream of the ATG start codon comprises from one to three translationally active non-conventional (e.g., CUG) start codons. In certain embodiments, an MTI further comprises (c) about 15 nucleotides (encoding about 5 amino acids) to about 45 nucleotides (encoding about 15 amino acids) downstream (3′) of the ATG start codon of FGF2. In some embodiments, an MTI comprises (d) at least one to two nuclear localization domains located upstream of the AUG start codon and downstream of at least one non-conventional CUG start codon.
For example, a multiplex translation initiation sequence comprising four FGF2 translation initiation sites (three non-conventional CUG start codons and one standard ATG start codon) linked upstream of a nucleic acid molecule encoding a fusion protein (e.g., a plurality of antigen peptides linked in a linear array) can yield four moles of a multi-peptide fusion protein translation product for every one mole of transcribed mRNA. In certain embodiments, a nucleic acid molecule encoding a fusion protein is introduced into a host cell and expressed, wherein the nucleic acid molecule contains a multiplex translation leader sequence comprising at least one non-AUG (e.g., CUG) translation initiation site that mediates translation initiation at the non-AUG (e.g., CUG) translation initiation site. In some embodiments, the MTI comprises at least five translation initiation sites (four non-conventional CUG start codons and one standard ATG start codon).
Non-canonical translation initiation start site have been described by Florkiewicz et al., BBRC 409 (3):494-499, 2011; Ivanov et al., Nucleic Acids Res 39 (10), 2011; Peabody D S, J Biological Chem 264 (9):5031-5035, 1989; Starck et al., Science 336 (6089):1719-23, 2012; Hann et al., Genes and Dev 6:1229-1240, 1992; Touriol et al., Biol Cell 95 (3-4):169-78, 2003; Schwab et al., Science 301 (5638):1367-71, 2003; Malarkannan et al., Immunity 10 (6):681-90, 1999, all of which are incorporated by reference in entirety. In certain situations, classical AUG mediated translation may be inhibited while translation initiation from non-AUG initiation codons may continue or may even be enhanced.
As used herein, “tumor associated antigen” or “TAA” refers to a protein, peptide, or variant thereof that is preferentially expressed on cancer cells or pre-cancer cells that exhibit deregulated growth. “Preferentially expressed” refers to expression of detectable levels of the protein or peptide in a cell or on the surface of a cell, wherein the protein or peptide is not expressed on normal cells of the same type. Preferably, the TAA is not expressed on non-cancer cells. Exemplary, TAAs include human epidermal growth factor receptor 2 (also known as ERBB2, EGFR2, Neu (neuro/glioblastoma derived oncogene homolog), HER2, or HER2/neu), BRAF, BRCA1/2, folate receptor-α, WT1, PI3K, NY-ES01, GNRH1, CTAG1A, CEA, IGFBP2, Cyclin D1, and MIF. The terms “tumor associated antigen,” “TAA,” “biomarker,” “cancer marker,” and “marker” are used interchangeably throughout.
A “fusion protein” or “chimeric protein,” as used herein, refers to a linear single chain protein that includes polypeptide components based on one or more parental proteins, polypeptides, or fragments thereof (e.g., antigenic peptides) and does not naturally occur in a host cell. A fusion protein can contain two or more naturally-arising amino acid sequences that are linked together in a way that does not occur naturally. For example, a fusion protein may have two or more portions from the same protein or a fragment thereof (e.g., antigenic fragment) linked in a way not normally found in a cell or a protein, or a fusion protein may have portions (e.g., antigenic portions) from two, three, four, five or more different proteins linked in a way not normally found in a cell. Also, a fusion protein may have two or more copies of the same portion of a protein or a fragment thereof (e.g., antigenic fragment). A fusion protein can be encoded by a nucleic acid molecule wherein a polynucleotide sequence encoding one protein or a portion thereof (e.g., antigen) is appended in frame with a nucleic acid molecule that encodes one or more proteins or a portion thereof (e.g., same or different antigens), which two or more proteins or portions thereof are optionally separated by nucleotides that encode a linker, spacer, cleavage site, junction amino acids, or a combination thereof. The valency of any one or more antigenic peptide epitope of the compositions herein, for example of a fusion protein comprising antigenic peptides, may be increased by duplicating, tripling, quadrupling, or further expanding the number of individual antigenic peptide epitopes contained therein.
A “spacer” refers to an amino acid sequence that connects two proteins, polypeptides, peptides, or domains and may provide a spacer function compatible with cleavage of a linear antigen array into individual antigenic peptides capable of associating with an MHC (HLA) molecule. A spacer can promote proteolytic processing into antigenic peptides by enhancing or promoting a disordered conformation of a primary translation product and thereby promoting its ubiquitin modification.
“Junction amino acids” or “junction amino acid residues” refer to one or more (e.g., about 2-10) amino acid residues between two adjacent motifs, regions or domains of a polypeptide, such as between a antigenic peptides or between an antigenic peptide and an adjacent peptide encoded by a multiplex translation leader sequence or between an antigenic peptide and a spacer or cleavage site. Junction amino acids may result from the construct design of a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein). Junction amino acids may be derived from a sequence authentic or native to a T-cell antigenic epitope sequence by extending the NH2 and/or COOH-terminus of a HLA Class 1 or HLA Class II peptide identified by computer algorithm or other means such as mass spectrometry analysis of peptides eluted from HLA Class I or HLA Class II restricted protein complexes.
As used herein, a molecule or compound “consists essentially of” one or more domains or encodes one or more domains “consisting essentially of” (e.g., an antigen, a linker or spacer, a proteolytic cleavage site, a nuclear localization signal, a multiplex translation initiation sequence) when the portions outside of the one or more domains (e.g., amino acids at the amino- or carboxy-terminus or between domains), in combination, contribute to no more than 20% (e.g., no more than 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the molecule or compound and do not substantially affect (i.e., do not alter the activity by more than 50% (e.g., no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) of the activities of one or more of the various domains (e.g., the immunogenicity of an antigen or antigenic epitope, the capability of localizing to the nucleus, the capability of forming a polypeptide complex (such as an HLA-peptide complex), the capability of promoting translation initiation from non-AUG codons). In certain embodiments, a nucleic acid molecule consists essentially of a multiplex translation leader sequence and a sequence encoding one or more antigens, antigenic epitopes or combination thereof, wherein an encoded antigen may comprise junction amino acids at the amino- and/or carboxy-terminus or between antigens. In certain embodiments, such junction amino acids between antigens or antigenic epitopes form proteolytic cleavage sites such that the antigens or antigenic epitopes are separated in vivo and can associate, for example, with a corresponding mammalian class I or class II HLA or MHC molecule.
As used herein, “nucleic acid” or “nucleic acid molecule” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any one or more of ligation, scission, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids may be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-enantiomeric forms of naturally-occurring nucleotides), or a combination thereof. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, morpholino, or the like. The term “nucleic acid molecule” also includes “peptide nucleic acids” (PNAs), which comprise naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acid molecules can be either single stranded or double stranded.
The term “construct” refers to any polynucleotide that contains a recombinant nucleic acid. A construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector, or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. Exemplary vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors).
Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
“Lentiviral vector,” as used herein, means HIV-based lentiviral vectors that are useful for gene delivery because of their relatively large packaging capacity, reduced immunogenicity and their ability to stably transduce with high efficiency a large range of different cell types. Lentiviral vectors are usually generated following transient transfection of three or more plasmids (e.g., packaging, envelope, and transfer) into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration in the DNA of infected cells.
The term “operably-linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). “Unlinked” means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
As used herein, “expression vector” refers to a DNA construct containing a nucleic acid molecule that is operably-linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. A viral vector may be DNA (e.g., an Adenovirus or Vaccinia virus) or RNA-based including an oncolytic virus vector (e.g., VSV), replication competent or incompetent. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid,” “expression plasmid,” “virus” and “vector” are often used interchangeably.
The term “expression,” as used herein, refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation. Translation may initiate from a non-conventional start codon, such as a CUG codon, or translation may initiate from several start codons (standard AUG and non-conventional) to produce more protein (on a per mole amount) than mRNA produced.
The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
As used herein, “contacting,” “contacting a cell,” “contacting a subject” or variants thereof, in the context of contacting with a nucleic acid molecule or composition refers to introducing the nucleic acid into the cell such that the at least some of the encoded content of the nucleic acid molecule is expressed in the cell. By “introduced” is meant transformation, transfection, transduction, or other methods known in the art for the introduction of nucleic acid molecules such as by a gene gun or nanoparticles.
Expression of recombinant proteins may be inefficient outside their original host since codon usage bias has been observed across different species of bacteria (Sharp et al., Nucl. Acids Res. 33:1141, 2005). Even over-expression of recombinant proteins within their native host may be difficult. In certain embodiments, nucleic acid molecules (e.g., nucleic acids encoding antigenic peptides) to be introduced into a host as described herein may be subjected to codon optimization prior to introduction into the host to ensure protein expression is enhanced. “Codon optimization” refers to the alteration of codons in genes or coding regions of nucleic acids before transformation to reflect the typical codon usage of the host without altering the polypeptide encoded by the DNA molecule. Codon optimization methods for optimum gene expression in heterologous hosts have been previously described (see, e.g., Welch et al., PLoS One 4:e7002, 2009; Gustafsson et al., Trends Biotechnol. 22:346, 2004; Wu et al., Nucl. Acids Res. 35:D76, 2007; Villalobos et al., BMC Bioinformatics 7:285, 2006; U.S. Patent Publication Nos. 2011/0111413 and 2008/0292918; disclosure of which are incorporated herein by reference, in their entirety). In certain embodiments, the multiplex translation leader sequence of this disclosure is human and is not codon optimized. Codon optimized recombinant nucleic acids may be distinguished from corresponding endogenous genes based on the use of PCR primers designed to recognize a codon optimized portion that is consequently distinct from a non-codon optimized portion of a nucleic acid.
The terms “identical” or “percent identity,” in the context of two or more polypeptide or nucleic acid molecule sequences, means two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using methods known in the art, such as a sequence comparison algorithm, by manual alignment, or by visual inspection. For example, a preferred algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403, 1990, with the parameters set to default values.
As used herein, TAP (Transporter Associated with Antigen Processing), refers to a heterodimer comprising a TAP1 and a TAP2 protein. Heterodimers of TAP are located in the endoplasmic reticulum (ER) where it functions to move selected peptides from the cytosol into the lumen of the ER where the peptide binds to a HLA Class I protein. Another intracellular TAP protein is TAPL (transporter associated with antigen processing like protein) localized to intracellular vesicular compartments such as the endosome and lysosome. TAP1, TAP2, and TAPL are members of the ATP binding cassette (ABC) transporter family. However, unlike ER localized TAP1/2 heterodimers, TAPL functions as a homodimer. Examples of TAP include TAP1 (GenBank No. NM_000593.5), TAP2 (GenBank No. NM_000544.3), and TAPL (GenBank No. NM_019624.3).
As used herein, “mutation” refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of changes in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s). In other embodiments, a mutation is a substitution of one or more nucleotides or residues. In certain embodiments, an altered or mutated protein or polypeptide only contains conservative amino acid substitutions as compared to the reference molecule. In certain other embodiments, an altered or mutated protein or polypeptide only contains non-conservative amino acid substitutions as compared to the reference molecule. In yet other embodiments, an altered or mutated protein or polypeptide contains both conservative and non-conservative amino acid substitutions. In any of these embodiments, an alteration or mutation does not alter or eliminate an antigenic epitope of a protein or peptide and the altered or mutated peptide is still recognized by its cognate MHC (HLA) molecule.
A “conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published Mar. 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY: N.Y. (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, Mass. (1990), p. 8). In certain embodiments, a conservative substitution includes, for example, a leucine to serine substitution.
As used herein, “recombinant” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that has at least one engineered genetic alteration or has been modified by the introduction of a heterologous nucleic acid molecule, or refers to a cell that has been altered such that the expression of an endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from a non-natural cell or is progeny of a non-natural cell having one or more such modifications. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, or other nucleic acid molecule additions, deletions, substitutions or other functional alteration of a cell's genetic material. For example, recombinant cells may express genes or other nucleic acid molecules that are not found in identical or homologous form within a native (wild-type) cell (e.g., a fusion or chimeric protein), or may provide an altered expression pattern of endogenous genes, such as being over-expressed, under-expressed, minimally expressed, or not expressed at all.
Recombinant methods for expression of exogenous or heterologous nucleic acids in cells are well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999). Exemplary exogenous proteins or enzymes to be expressed include TAP1, TAP2, antigens, cytokines, or any combination thereof. Genetic modifications to nucleic acid molecules encoding fusion proteins can confer a biochemical or metabolic capability to a recombinant or non-natural cell that is altered from its naturally occurring state.
As used herein, the term “endogenous” or “native” refers to a gene, protein, compound or activity that is normally present in a host cell. The term “homologous” or “homolog” refers to a molecule or activity from an exogenous (non-native) source that is the same or similar molecule or activity as that found in or derived from a host or host cell.
As used herein, “heterologous” nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule sequence that is not native to a cell in which it is expressed, a nucleic acid molecule or portion of a nucleic acid molecule native to a host cell that has been altered or mutated, or a nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous control sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule that is normally expressed in nature or culture. In certain embodiments, a heterologous nucleic acid molecule may be homologous to a native host cell gene, but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous or exogenous nucleic acid molecules may not be endogenous to a host cell or host genome (e.g., fusion protein), but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).
In certain embodiments, more than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof, and still be considered as more than one heterologous or exogenous nucleic acid. When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, as single or multiple mRNA molecules, integrated into the host chromosome at a single site or multiple sites, and each of these embodiments is still to be considered two or more exogenous nucleic acid molecules. Thus, the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.
For example, a cell can be modified to express two or more heterologous or exogenous nucleic acid molecules, which may be the same or different, that encode one or more fusion proteins, as disclosed herein. In certain embodiments, a host cell will contain a first nucleic acid molecule encoding a first fusion protein and a separate second nucleic acid molecule encoding a second fusion protein, or a host cell will contain a single polycistronic nucleic acid molecule that encodes a first fusion protein and second fusion protein, or single nucleic acid molecule that encodes a first fusion protein, a cleavable amino acid sequence (e.g., trypsin, pepsin, proteasome site) or a self-cleaving amino acid sequence (e.g., 2A protein), and a second fusion protein.
“T cell receptor” (TCR) is a molecule found on the surface of T cells that, along with CD3, is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. A TCR consists of a disulfide-linked heterodimer of the highly variable α and β chains in most T cells. In other T cells, an alternative receptor made up of variable γ and δ chains is expressed. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one amino-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see, Abbas and Lichtman, Cellular and Molecular Immunology (5th Ed.), Editor: Saunders, Philadelphia, 2003; Janeway et al., Immunobiology: The Immune System in Health and Disease, 4th Ed., Current Biology Publications, p 148, 149, and 172, 1999).
HLA Class I binding CD8+ T-cell are T-cells that kill tumor cells or virally infected cells through interactions between an antigenic peptide bound cell surface localized HLA (also known as MHC). Cells that present HLA class I antigenic peptide epitopes are referred to as antigen presenting cells.
HLA Class II presentation is different than HLA Class I in that peptide antigens are bound to HLA class II complexes that are already on the cell surface or that are present in certain intracellular vesicles, such as endosomes or lysosomes. Class II antigen presentation thus does not a priori require the activity of TAP. However, the protein referred to as TAPL, which is found localized to certain intracellular vesicles such as an endosome or lysosome, may function to recognize cytosol localized class II peptides and consequently transfer a 12-18 amino acid peptide from the cytosol into a intracellular vesicle containing a Class II HLA that then traffics back onto the cell surface where it can then interact with and consequently expand a population of CD4+ T-cells. A host's T-cells that recognized cell surface localized HLA class II peptide complexes are called CD4+ T-cells, which are principally responsible for preserving the function of CD8+ killer T-cells mediated by the release of a set of CD8+ cytokines.
The term “antigen specific T-cell response” refers to an immune response mediated by T-cells directed at a cell expressing a specific antigen. In some embodiments, the T-cell response is a CD8+ T-cell response, a CD4+ T-cell response, or a combination thereof.
The term “antigen, “antigenic peptide” or variants thereof refers to a polypeptide that can stimulate a cellular immune response. In some embodiments, an antigen is an HLA Class I, an HLA Class II peptide, or HLA Class II peptide having an embedded HLA Class I peptide.
The term “immunization composition” refers to a composition that can stimulate or elicit an immune response. Preferably, the immune response is a cellular immune response, such as an adaptive immune response mediated by T-cells (e.g., CD8+ T-cells or CD4+ T-cells). In some embodiments, an immunization composition is a pharmaceutical formulation. In further embodiments, an immunization composition is an antigenic peptide immunization composition, a nucleic acid immunization composition, a cell immunization composition, or a combination thereof.
The term “antigen immunization composition” or “peptide immunization composition” refers to an immunization composition that includes one or more antigens that are capable of promoting or stimulating a cellular immune response. In some embodiments, an antigen immunization composition comprises an HLA Class I peptide, HLA Class II peptide, HLA Class II peptide having an embedded HLA Class I peptide, or combinations thereof.
The term “nucleic acid immunization composition” refers to an immunization composition that includes a nucleic acid molecule that encodes one or more antigens or antigenic epitopes, and that can be contained in a vector (e.g., plasmid, virus). A nucleic acid immunization composition can be introduced into a host cell ex vivo, or in vivo for expression of the one or more antigenic peptides in a subject. In certain embodiments, a nucleic acid immunization composition encodes an HLA Class I peptide, HLA Class II peptide, HLA Class II peptide having an embedded HLA Class I peptide, or combinations thereof.
The term “nucleic acid molecule immunization” or “DNA immunization” as used herein refers to a nucleic acid molecule encoding one or more antigens introduced into a host or host cell in order to express the one or more antigens in vivo. A nucleic acid molecule immunization can be by direct administration into a host, such as by standard injection (e.g., intramuscular, intradermal), transdermal particle delivery, inhalation, topically, orally, intranasally, or mucosally. Alternatively, a nucleic acid molecule can be introduced ex vivo into host cells (e.g., host cells or cells from a donor HLA matched to the host) and the transfected host cells can be administered into the host such that an immune response can be mounted against the one or more antigens encoded by the nucleic acid molecule.
The term “nucleic acid molecule vaccine” or “DNA vaccine” as used herein refers to a nucleic acid molecule encoding one or more antigens or antigenic epitopes that is used in a nucleic acid molecule immunization as defined herein.
“Treatment,” “treating” or “ameliorating” refers to medical management of a disease, disorder, or condition of a subject (e.g., patient), which may be therapeutic, prophylactic/preventative, or a combination treatment thereof. A treatment may improve or decrease the severity at least one symptom of a disease, delay worsening or progression of a disease, or delay or prevent onset of additional associated diseases. “Reducing the risk of developing a disease” refers to preventing or delaying onset of a disease (e.g., cancer) or reoccurrence of one or more symptoms of the disease.
A “therapeutically effective amount (or dose)” or “effective amount (or dose)” of a compound or composition refers to that amount of compound sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner. The precise amount will depend upon numerous factors, e.g., the activity of the composition, the method of delivery employed, the immune stimulating ability of the composition, the intended patient and patient considerations, or the like, and can readily be determined by one of ordinary skill in the art.
A therapeutic effect may include, directly or indirectly, the reduction of one or more symptoms of a disease (e.g., reduction in tumor burden or reduction in pathogen load). A therapeutic effect may also include, directly or indirectly, the stimulation of a cellular immune response.
A “matched” vaccination strategy is one in which a peptide vaccine and a DNA vaccine are administered to a subject wherein the peptides of the peptide vaccine and peptides encoded by the DNA vaccine are derived from the same protein (e.g., same cancer related TAA or pathogen derived antigen). In some embodiments, the peptide vaccine peptides are HLA class II antigens. In some embodiments, the peptides encoded by the DNA vaccine are HLA class I peptides. A matched vaccination strategy can include administering respective compositions as a prime-and-boost.
As used herein, “prime-and-boost” or “immunogenic challenge” refers to sequentially or simultaneously delivering one or more of a series of peptide vaccines followed by one or more in a series of DNA vaccines, or, in the alternative, sequentially or simultaneously delivering one or more of a series of DNA vaccines followed by one or more in a series of peptide vaccines.
Cancer cells may aberrantly express certain polypeptides, either by inappropriate expression or overexpression. As such, inappropriately expressed polypeptides have been identified as tumor associated antigens (TAAs). Tumor-associated antigens however may be functionally non-immunogenic or are ineffectively or weakly immunogenic. This may be referred to immune tolerance. Compositions and methods of the instant disclosure are designed to enhance or further stimulate a patient's immune system so that it is capable of functioning more effectively to kill cancer cells or to kill pathogen infected cells.
As used herein, a “subject,” may be any organism capable of developing a cellular immune response, such as humans, pets, livestock, show animals, zoo specimens, or other animals. For example, a subject may be a human, a non-human primate, dog, cat, rabbit, rat, mouse, guinea pig, horse, cow, sheep, goat, pig, or the like. Subjects in need of administration of therapeutic agents as described herein include subjects at high risk for developing a cancer or infectious disease as well as subjects presenting with an existing cancer or infectious disease. A subject may be at high risk for developing a cancer if the subject has experienced an injury, such as exposure to carcinogens or radiation, or has a genetic predisposition, such as a mutation in the BRCA1/2, folate receptor-α, or p53 genes. Subjects suffering from or suspected of having an infectious disease or a cancer can be identified using methods as described herein and known in the art.
A “subject in need” refers to a subject at high risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a compound or a composition thereof provided herein. In certain embodiments, a subject in need is a human.
Accordingly, in some embodiments the composition of the instant disclosure provides a chimeric nucleic acid molecule, comprising a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding an antigen, an antigenic epitope, or a combination thereof, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the antigen, antigenic epitope, or combination thereof.
A nucleic acid based plasmid or virus delivery system as described herein provides a transcribed mRNA that is recognized by eukaryotic cell translation components, which is translated into a protein following the initiation of translation that occurs at the first or appropriate translation initiation codon. The canonical mechanism of translation initiation starts at an AUG codon. However, as used herein, translation initiation can also begin at a non-AUG codon, such as a CUG codon. The human gene encoding FGF2 is an example of a gene with translation that is mediated via three CUG start codons, in addition to one AUG codon. In some embodiments, the MTI nucleic acid molecule set forth herein comprises a portion of the FGF2 multiple translation initiation domain of the gene/mRNA.
In some embodiments, an MTI of the chimeric nucleic acid molecule corresponds to a 5′-portion of an FGF2 gene (e.g., a human FGF2 gene as set forth in GenBank Accession No. NM_002006.4) containing the ATG start codon of FGF2 and a sequence comprising about 123 nucleotides to about 385 nucleotides upstream (5′) of the ATG start codon of FGF2, wherein this portion upstream of the ATG start codon comprises from one to three translationally active non-conventional (e.g., CUG) start codons. In certain embodiments, the MTI further comprises about 15 nucleotides (encoding about 5 amino acids) to about 45 nucleotides (encoding about 15 amino acids) downstream (3′) of the ATG start codon of FGF2. In some embodiments, the MTI comprises at least one to two nuclear localization domains located upstream of the AUG start codon and downstream of at least one non-conventional CUG start codon. In certain embodiments, an MTI sequence comprising four FGF2 translation initiation sites (three non-conventional CUG start codons and one standard ATG start codon) linked upstream of a nucleic acid molecule encoding a fusion protein (e.g., a plurality of antigen peptides linked in a linear array) can yield four moles of a multi-peptide fusion protein translation product for every one mole of transcribed mRNA. In certain embodiments, a nucleic acid molecule encoding a fusion protein is introduced into a host cell and expressed, wherein the nucleic acid molecule contains a multiplex translation leader sequence comprising at least one non-AUG (e.g., CUG) translation initiation site that mediates translation initiation at the non-AUG (e.g., CUG) translation initiation site. In some embodiments, an MTI sequence is a nucleic acid molecule having a sequence as set forth in any one of SEQ ID NOS.: 1-6, 95, or 96. In certain embodiments, an MTI sequence is nucleic acid molecule having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOS.:1-6.
In certain embodiments, a nucleic acid molecule encoding a fusion protein comprises a plurality of class I MHC (HLA) antigenic peptides, a plurality of class II MHC (HLA) antigenic peptides, or a combination thereof. In further embodiments, a plurality of MHC (HLA) class I antigenic peptides, class II antigenic peptides, or a combination thereof are expressed as a linear single-chain polypeptide antigen array, wherein each antigen is separated from the adjacent antigen by a spacer, a cleavable site (e.g., enzyme recognition site or self-cleaving), or both, and wherein the polypeptide antigen array is processed by the cellular machinery (e.g., proteasome or nuclear proteasome) into individual antigen peptides capable of forming a complex with their cognate MHC molecules.
In accordance with the disclosure set forth herein, a translated polypeptide or protein comprises, for example, a linear peptide antigen array (PAA) that is or may then be modified intracellularly by the addition of a ubiquitin moiety. The ubiquitin-modified PAA is recognized by and proteolytically processed through the actions of the proteasome. The results of proteolytic processing are small peptides approximately 8-12 amino acids in length that are then recognized by the intracellular ER localized protein termed TAP.
Accordingly, in some embodiments, the chimeric nucleic acid molecule includes a nucleic acid molecule that encodes one or more antigens, antigenic epitopes, or a combination thereof (referred to collectively as antigens), which is referred to herein as a polyantigen array (PAA). In some embodiments, a PAA encodes one or more antigens from the same protein. In other embodiments, a PAA encodes one or more antigens from more than one protein (i.e., comprises a chimeric polypeptide). For example, a PAA can encode one or more antigens from folate receptor-α and one or more antigens from Her2/neu. In some embodiments, a nucleic acid molecule encodes a plurality of antigens ranging from about 2 to about 20, from about 2 to about 15, from about 2 to about 10, from about 2 to about 9, from about 2 to about 8, or a nucleic acid molecule encodes 2, 4, 5, 6, 7, or 8 antigens.
In some embodiments, a PAA encodes a plurality of antigens wherein two or more of the plurality of antigens are separated by a spacer. In certain embodiments, a spacer is comprised of about 2 to about 35 amino acids, or about 5 to about 25 amino acids or about 8 to about 20 amino acids or about 10 to about 15 amino acids. In some embodiments, a spacer may have a particular sequence, such as a (G4S)n repeat, wherein n is an integer from 1-20, from 1-15, from 1-10, from 1-5, from 1-3, or the like. In particular embodiments, a spacer is a (G4S)2 peptide.
In some embodiments, a chimeric nucleic acid encodes a PAA wherein two or more of the plurality of antigens are separated by a cleavage site. In certain embodiments, a cleavage site comprises from about 2 to about 20 amino acids amino-terminal to the antigenic peptide as found in the reference protein, from about 2 to about 20 amino acids carboxy-terminal to the antigenic peptide as found in the reference protein, a self-cleaving amino acid sequence, or a combination thereof. In certain embodiments, the cleavage site comprises from about 2 to about 15, about 2 to about 10, or about 2 to about 5 amino acids at the amino-terminal or the carboxy-terminal end of the antigen. In some embodiments, the cleavage site is a self-cleaving amino acid sequence comprising a 2A peptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot-and-mouth disease virus (F2A), or any combination thereof (see, e.g., Kim et al., PLOS One 6:e18556, 2011, which 2A nucleic acid and amino acid sequences are incorporated herein by reference in their entirety).
In some embodiments, an antigen or antigenic epitope is an HLA Class I antigenic peptide, an HLA Class II antigenic peptide, an HLA class II antigenic peptide with an embedded HLA Class I antigenic peptide, or any combination thereof. In certain embodiments, an antigen or antigenic epitope is an HLA class II antigenic peptide comprising an embedded HLA Class I antigenic peptide. An “embedded antigen” is an antigenic sequence or epitope that is contained within a larger antigenic sequence or epitope. For example, a sequence corresponding to an antigenic HLA Class II peptide antigen may contain within it a sequence representative of an HLA Class I antigenic peptide. In some embodiments, an extended antigenic peptide sequence may contain multiple overlapping (i.e., embedded) antigens.
Cancer cells may be distinguished from normal cells by the de novo expression of one or more marker proteins or tumor-associated antigens (TAA). A marker protein or a TAA may comprise one or more antigenic peptides. For example, antigenic peptides may represent either HLA Class I or HLA Class II restricted antigenic peptide epitopes. A TAA or cancer marker protein or antigenic peptides thereof may be used in a vaccine composition capable of eliciting an immune response (e.g., a cellular immune response, such as an antigen-specific T cell response) targeting the unwanted cancer cell for destruction by the patients' immune system.
In some embodiments, an antigen or antigenic epitope is a tumor-associated antigen (TAA). An antigenic peptide of a PAA disclosed herein may be derived from one or more TAAs. A TAA can be an antigen associated with breast cancer, triple negative breast cancer, inflammatory breast cancer, ovarian cancer, uterine cancer, colorectal cancer, colon cancer, primary peritoneal cancer, testicular cancer, renal cancer, melanoma, glioblastoma, lung cancer, or prostate cancer. In certain embodiments, a TAA derived antigenic T-cell epitope (either Class I or Class II) is from a HER2/neu, folate receptor alpha, Cyclin D1, IGFBP2, macrophage migration inhibitory factor (MIF), human carcinoembryonic antigen (CEA), gonadotropin releasing hormone (GnRH), melanoma related gp100 as well as MAGE-2 and MAGE-3, a testis cancer antigen (e.g., NY-ESO-1), cancer/testis antigen 1A (CTAG1A), Wilms tumor protein 1 (WT1), p53, BRCA1, BRCA2, PI3K, BRAF, insulin-like growth factor binding protein 2, or PD-1 antagonists. In some embodiments, a PAA as used herein is a combination of T-cell antigenic epitopes from more than one TAA (e.g., a combination of a HER2/neu and folate receptor alpha antigenic T-cell epitopes). In certain embodiments, a TAA comprises a HER2/neu antigen, a folate receptor-α antigen, or a combination thereof. Exemplary folate receptor-α antigenic peptides are presented in U.S. Pat. No. 8,486,412, which peptides are herein incorporated by reference. Exemplary HER2/neu, Cyclin D1, IGFBP2, and CEA antigenic peptides are described in Table I and II of U.S. Application Publication No. US 2010/0310640, which antigenic peptides are herein incorporated by reference in their entirety.
In certain embodiments, a TAA has an amino acid sequence derived from HER2 as set forth in SEQ ID NOS.:117-135, or any combination thereof. In some embodiments, the TAA has an amino acid sequence derived from folate receptor-α as set forth in SEQ ID NOS.:69-93, or any combination thereof. Accordingly, in some embodiments, the TAA has an amino acid sequence as set forth in any one of SEQ ID NOS.:69-93 or 117-135, or any combination thereof. In certain embodiments, the nucleic acid molecule has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS.:97, 98, 136, or any combination thereof. In certain embodiments, the nucleic acid molecule encodes an antigen having an amino acid sequence as set forth in any one of SEQ ID NOS.:67, 68, 115, 116, or any combination thereof. In some embodiments, the acid molecule has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOS.:52, 57, 58, 100, 137, 139, 141, 145, or 149. In some embodiments, the TAA has an amino acid sequence derived from IGFB2 as set forth in SEQ ID NOS.:103-112, or any combination thereof. In some embodiments, the TAA has an amino acid sequence derived from CEA as set forth in SEQ ID NOS.:186-202, or any combination thereof. In some embodiments, the TAA has an amino acid sequence derived from Cyclin D1 as set forth in SEQ ID NOS.:152-183, or any combination thereof. In certain embodiments, the TAA has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOS.:69-93, 103-112, 117-135, 152-183, 186-202, or any combination thereof. As noted herein, an antigenic T-cell epitope may be identified using a patient's sample.
In certain embodiments, the instant disclosure provides a chimeric nucleic acid molecule, comprising a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding a fusion protein comprising from two to about ten human folate receptor-alpha (FRα) antigenic peptides, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the fusion protein and allows the production of more than one mole of fusion protein per mole of mRNA. In some embodiments, the MTI sequence has at least 90% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOS.:1-6, 95, or 96. In further embodiments, the fusion protein comprises from two to about five antigenic peptides or comprises five antigenic peptides. In further embodiments, two or more of the FRα antigenic peptides of the fusion protein are separated by a spacer comprising a (G4S)n, wherein n is an integer from 1 to 5. In further embodiments, two or more of the FRα antigenic peptides of the fusion protein are separated by a natural cleavage site comprising from about two to about ten amino acids, a self-cleaving amino acid sequence, or combinations thereof. In further embodiments, each FRα antigenic peptide of the fusion protein has at least 90% sequence identity to any one of SEQ ID NOS.:69-93. In further embodiments, the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:49, 67, or 68. In further embodiments, the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:53-55 or 59-61. In further embodiments, one or more of the FRα antigenic peptides are an HLA Class I antigenic peptide, an HLA Class II antigenic peptide, an HLA Class II antigenic peptide comprising an embedded HLA Class I antigenic peptide, or any combination thereof.
In certain aspects, this disclosure provides a method of eliciting a cellular immune response, comprising administering to a human subject a therapeutically effective amount of a chimeric nucleic acid molecule, wherein the chimeric nucleic acid molecule comprises a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding a fusion protein comprising from two to about ten human folate receptor-alpha (FRα) antigenic peptides, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the fusion protein and allows the production of more than one mole of fusion protein per mole of mRNA, thereby eliciting a cellular immune response. In further embodiments, the MTI sequence has at least 90% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOS.:1-6, 95, or 96. In further embodiments, each FRα antigenic peptide of the fusion protein has at least 90% sequence identity to any one of SEQ ID NOS.: 69-93. In further embodiments, the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:49, 67, or 68. In further embodiments, the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:53-55 or 59-61. In further embodiments, the chimeric nucleic acid molecule is formulated as a composition comprising a therapeutically acceptable carrier or excipient. In further embodiments, the method comprises contacting the chimeric nucleic acid molecule with an immune cell ex vivo before administration, and administering to the human subject a population of immune cells containing the chimeric nucleic acid molecule. In further embodiments, the elicited immune response treats FRα-associated cancer.
In other aspects, this disclosure provides a method of eliciting a cellular immune response, comprising (a) administering to a human subject an effective amount of an antigenic peptide immunization composition comprising at least one FRα antigenic peptide, and (b) administering to the human subject an effective amount of a chimeric nucleic acid molecule, wherein the chimeric nucleic acid molecule comprises a multiplex translation initiation (MTI) sequence and a nucleic acid molecule encoding a fusion protein comprising from two to about ten human folate receptor-alpha (FRα) antigenic peptides, wherein the MTI comprises at least one non-AUG translation initiation site that mediates translation initiation of the fusion protein and allows the production of more than one mole of fusion protein per mole of mRNA, thereby eliciting a cellular immune response. In further embodiments, the chimeric nucleic acid molecule encodes one or more of the same antigenic peptides as used in step (a). In further embodiments, step (b) is performed simultaneously with step (a), or step (b) is performed from 1 hour to 8 weeks after step (a), or wherein step (a) is performed from 1 hour to 8 weeks after step (b). In further embodiments, the method further comprises (c) administering to the human subject an effective amount of a second antigenic peptide immunization composition, wherein the second antigenic peptide immunization composition comprises the same antigenic peptide immunization composition as used in (a). In further embodiments, the method further comprises administering an adjunctive therapy selected from surgery, chemotherapy, radiation therapy, antibody therapy, immunosuppressive therapy, or any combination thereof, such as cyclophosphamide, trastuzumab, anti-PD1, anti-PDL1, anti-CTLA4, or any combination thereof. In further embodiments, the the elicited immune response treats FRα-associated cancer.
Accordingly, cancers that may be treated using the compositions and methods disclosed herein include breast cancer, triple negative breast cancer, inflammatory breast cancer, ovarian cancer, uterine cancer, colorectal cancer, colon cancer, primary peritoneal cancer, testicular cancer, renal cancer, melanoma, glioblastoma, lung cancer, or prostate cancer.
Effective countermeasures to biological pathogens are a critical component of biodefense and national security (Altmann, Expert Rev Vaccines 4, 275-279, 2005), and additional products are needed to meet biodefense needs (Matheny, J., Mair, M. & Smith, B. Nat Biotechnol 26, 981-983, 2008; Cohen, J. Science 333, 1216-1218, 2011; Artenstein, A. W. & Grabenstein, J. D. Expert Rev Vaccines 7, 1225-1237, 2008). Vaccines provide not only preventive or therapeutic countermeasures, but can also serve as actual deterrents (Poland et al., Vaccine 27, D23-D27, 2009) in that they can be rapidly deployed to negate the primary outcomes of biological terrorism and thereby removing the motivation to use a bioweapon.
For a vaccine composition incorporating nucleic acid molecules as described herein, the desired end result is a safe product capable of inducing long-lasting, protective immunity with minimal side effects, and as compared to other strategies (e.g., whole live or attenuated pathogens), is inexpensive to produce, will minimize or eliminate contraindications that have otherwise (typically) been associated with the use of whole or attenuated virus vaccine compositions, and have an extended shelf-life because it is nucleic acid and/or synthetic peptide-based. The ability to rapidly respond to infectious disease emergencies (natural outbreaks, pandemics, or bioterrorism) is a benefit of an effective use of the embodiments disclosed herein, whether in context of biodefense or cancer immunotherapies or technologies. The instant disclosure sets forth the rapid identification and selection of relevant pathogen-related or cancer-related antigenic epitopes for peptide-based vaccines and nucleic acid-based vaccines and is easily adaptable to multiple Category A-C agents, as well as new and emerging pathogens.
In other embodiments, a chimeric nucleic acid molecule encodes an antigen or antigenic epitope from a pathogen. In certain embodiments, the pathogen is a virus, parasite, or bacteria. In further embodiments, a chimeric nucleic acid molecule encodes a PAA from one or more pathogens.
In certain embodiments, a PAA comprises an antigen from a virus, wherein the virus is a small pox virus and other related pox viruses, lentiviruses such as HIV, influenza virus, Arenaviruses such as Junin, Machupo, Guanarito, Chapare, Lassa, and Lujo, Bunyaviruses such as Hantaviruses, Rift Valley Fever virus, and Crimean Congo Hemorrhagic Fever virus, Flaviruses such as Dengue fever virus, Filoviruses such as Ebola and Marburg virus, retrovirus, adenovirus, parvovirus, coronavirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, adenovirus, herpesvirus, Norwalk virus, togavirus, reovirus, papovarius, hepadnavirus, hepatitis virus, avian leucosis-sarcoma, mammalian C-type viruses, B-type viruses, D-type viruses, HTLV-BLV group viruses, or spumavirus. In some embodiments, the influenza virus is influenza strain H7N9 (bird flu), H5N1, H3N8, H2N2, H3N2, H3N3, H9N2, H7N7, H1N1, or a combination thereof.
In other embodiments, a PAA comprises an antigen from a bacteria. In certain embodiments, the bacteria is a Mycobacterium tuberculosis, pathogenic Escherichia coli, Yersinia pestis, Listeria monocytogenes, Clostridium botulinum, Bacillus anthracis, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, or Salmonella enterica. The pathogenic E. coli can be E. coli strain 0157:H7, ETEC, EPEC, EIEC, EHEC, EAEC, or a combination thereof.
In certain embodiments, a PAA comprises an antigen from a parasite. In some embodiments, the parasite is a Protozoa such as Plasmodium sp., Entamoeba, Giardia Trypanosoma brucei, Toxoplasma gondii, Acanthamoeba, Leishmania, Babesia, Balamuthia mandrillaris, Cryptosporidium, Cyclospora, or Naegleria fowleri, a Parasitic worms such as Guinea worm (Dracunculus), Ascaris lumbricoides, Pinworm, Strongyloides stercoralis, Toxocara, Guinea Worm, Hookworm, Tapeworm, or Whipworm, or a Parasitic fluke such as Schistosoma, Gnathostoma, Paragonimus, Fasciola hepatica, or Trichobilharzia regent.
In any of the embodiments disclosed herein, a nucleic acid sequence encoding the antigen can be codon optimized for expression in the appropriate subject (e.g., human). In certain embodiments, a smallpox nucleic acid sequence comprises a smallpox MTI-PAA encoding an antigen set forth in SEQ ID NOS.:24-30, or any combination thereof. In some embodiments, a nucleic acid sequence encoding a smallpox MTI-PAA corresponds to the sequence set forth in SEQ ID NO.:7. In further embodiments, a nucleic acid sequence encoding a smallpox MTI-PAA corresponds to a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO.: 7.
In certain embodiments, a nucleic acid vector containing a multiplex translation leader sequence portion (or PolyStartTM) may also contain a nucleic acid sequence encoding a TAP protein. An encoded TAP may be as a heterodimer (TAP1 plus TAP2) or only TAP1 or only TAP2. A TAP may be codon optimized so that it can be distinguished from an endogenous TAP. In addition, a TAP translation product may incorporate a peptide tag detection moiety/portion such as a VSV G protein epitope tag, an HA epitope tag, a Myc epitope tag or any other detection portion.
Accordingly, in addition to the MTI-PAA, the chimeric nucleic acid molecule disclosed herein can further include a nucleic acid molecule encoding a TAP protein. In some embodiments the TAP protein is TAP1, TAP2, TAPL, or any combination thereof. In certain embodiments, the TAP protein is a heterodimer of TAP1 and TAP2. In some embodiments, the nucleic acid molecule encoding the TAP protein has a polynucleotide sequence as set forth in any one of SEQ ID NOS.:206-210, or any combination thereof.
In some embodiments, the chimeric nucleic acid disclosed herein is included in a nucleic acid vector. In some embodiments, the vector is an expression vector. Examples of an expression vector include a plasmid, a cosmid, a viral vector, an RNA vector, or a linear or circular DNA or RNA molecule. In some embodiments, the vector is a plasmid and comprises pcDNA3, pSG5, pJ603, or pCMV. In some embodiments, the vector is a viral vector selected from a retrovirus, adenovirus, parvovirus, coronavirus, influenza virus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, adenovirus, herpesvirus, poxvirus, Norwalk virus, togavirus, flavivirus, reovirus, papovarius, hepadnavirus, hepatitis virus, avian leucosis-sarcoma, mammalian C-type viruses, B-type viruses, D-type viruses, HTLV-BLV group viruses, lentivirus, or spumavirus. In the case of a viral vector incorporating a nucleic acid molecule of this disclosure, a virus may be a DNA virus (e.g., vaccinia, adenovirus) or an RNA virus (e.g., vesicular stomatitis virus). In certain embodiments, a virus is oncolytic.
In certain embodiments, a multiplex vector of the instant disclosure has a nucleic acid molecule that encodes an MTI-PAA fusion operably linked to a CMV promoter and the fusion protein contains a carboxy-terminal VSV-g epitope tag (SEQ ID NO.:13). In further embodiments, a multiplex vector of the instant disclosure includes a Tap1 gene expressed by an SV40 promoter and the TAP1 includes an amino-terminal V5 epitope tag (SEQ ID NO.:14), while a nucleic acid encoding an MTI-PAA is operably linked to a CMV promoter and a carboxy-terminal VSV-g epitope tag (SEQ ID NO.:13). In still further embodiments, a multiplex vector of the instant disclosure includes a Tap2 gene expressed by an SV40 promoter and the TAP2 includes an amino-terminal AU5 epitope tag (SEQ ID NO.:15), while a nucleic acid molecule encoding an MTI-PAA is operably linked to a CMV promoter and the PAA included a carboxy-terminal VSV-g epitope tag (SEQ ID NO.:13). In yet further embodiments, a multiplex vector of the instant disclosure includes a Tap1 and Tap2 gene separated by an IRES and operably linked to an SV40 promoter, wherein the TAP1 includes an amino-terminal V5 epitope tag (SEQ ID NO.:14) and the TAP2 includes an amino-terminal AU5 epitope tag (SEQ ID NO.:15), while a nucleic acid molecule encoding an MTI-PAA is operably linked to a CMV promoter and the PAA included a carboxy-terminal VSV-g epitope tag (SEQ ID NO.:13).
A class I peptide vaccines incorporated into a nucleic acid-based immunization composition can elicit protective immunity, and the inclusion of HLA class II peptides and the generation of T helper responses is expected to stimulate optimal cellular immunity and augment the protection provided by CTL alone. In some embodiments, a nucleic acid of the instant disclosure encoding one or more Class II ‘reporter epitope’ PAA and class II epitope-containing PAAs may be represented as a MTI-VSVG-PAA, wherein the MTI portion is mutated to remain cytosolic. As designed, a MTI-VSVG-PAA results in a primary translation product containing what is referred to herein as an internalized VSVG signal sequence (e.g., SEQ ID NO.: 42). The internalized signal sequence is processed and the chimeric protein secreted or membrane localized, thereby facilitating entry of the PAA into the endocytic compartment and class II processing pathway. VSVG processing and signaling sequences are discussed in greater detail in Rose et al, J. Virol. 39: 519-528, 1981; Gallione et al. J. Virol. 54:374-382, 1985; Machamer et al., Mol. Cell. Biol. 5:3074-3083, 1985; Rottier et al. J. Biol. Chem. 262:8889-8895, 1987, all of which are herein incorporated by reference in their entirety.
Accordingly, in some embodiments is a chimeric nucleic acid disclosed in any of the embodiments herein further comprises an internalization VSVG signal sequence, a VSVG secretion signal sequence, a trafficking sequence, a dendritic cell targeting sequence, a membrane localization sequence, or any combination thereof. In some embodiments, the chimeric nucleic acid comprises an internalization VSVG signal sequence as set for in SEQ ID NO.: 43. In some embodiments, the nucleic acid comprises a VSVG secretion signal sequence as set for SEQ ID NOS.:47 or 48. The VSVG secretion signal sequence promotes trafficking through ER/Golgi to the cell surface (i.e., plasma membrane). In some embodiments, the nucleic acid comprises a dendritic cell targeting sequence as set for in SEQ ID NO 45. The DC targeting sequence is optional, and may be placed at the COOH terminus. In some embodiments, the nucleic acid comprises a VSVG membrane localization sequence as set for in SEQ ID NO.:57. In some embodiments, the nucleic acid comprises a PAA including sequences encoding folate receptor-α Class II antigens and is a secreted chimera. In some embodiments, the nucleic acid comprises a sequence as set forth in SEQ ID NO.:52. In some embodiments, the nucleic acid comprises a PAA including sequences encoding folate receptor-α Class II antigens and is a membrane localized polypeptide. In some embodiments, nucleic acid comprises a PAA including sequences encoding folate receptor-α Class II antigens and is a membrane localized polypeptide such as the fusion protein encoded by the nucleic acid sequence as set forth in SEQ ID NO.:58.
The chimeric nucleic acid and vectors described herein can be introduced into a cell. Methods of introducing nucleic acid molecules are well known in the art and include transformation and transduction. Accordingly, some embodiments comprise a cell that includes a nucleic acid molecule as described herein. In addition, certain embodiments comprise a cell that includes a vector according to any of the embodiments described herein. In some embodiments, the cell is an autologous cell obtained from a first subject or an allogeneic cell from a subject different from the first subject. The cell can be located in vitro or in vivo. In some embodiments, the cell is an antigen presenting cell, such as a professional or non-professional antigen presenting cell. The antigen presenting cell can be a professional antigen presenting cell such as a dendritic cell or a macrophage. In a preferred embodiment, the antigen presenting cell is a dendritic cell.
The term dendritic cells (DCs) was described by Steinman et al in 1973 (Steinman R M and Cohn Z A J Exp Med 137:1142-62, 1973). Dendritic cells are derived from myeloid bone marrow progenitor cells and have the potential to be used as a viable cell-based anti-cancer therapy (Vacchelli et al., Oncolmmunology 2:10, 2013; Slingluff et al., Clin Cancer Res 12 (7 Suppl):2342s-2345s, 2006; Steinman, Immunity 29: 319-324, 2008). DCs localize to lymphoid tissues, skin (e.g., epidermal Langerhans cells) and various mucosae. When mature, DCs are potent professional antigen presenting cells for both HLA Class II as well as HLA Class I restricted systems (Santambrogio et al., PNAS 96 (26):15050-55, 1999). Mature DCs express elevated levels of HLA Class II proteins on the cell surface, migrate to lymph nodes and secrete high levels of cytokines/chemokines. T-cell activating antigenic peptides bound to MHC Class II proteins presented on the cell surface of an activated DC stimulate (activate) both cognate CD4+ helper T-cells and CD8+ cytotoxic T-cells, while at the same time secreting a number of cytokines and other growth promoting factors. Subdermal administration (a site rich in DCs) of a peptide or nucleic acid-based composition set forth herein results the uptake and immune system presentation of antigenic T-cell peptides (including naturally processed antigenic peptides) in context with DC expressed HLA Class I or Class II proteins.
The affinity of a TAA-derived peptide or nucleic acid-based antigenic T-cell peptide composition, as disclosed herein, for a DC may be enhanced by inclusion of one or more DC targeting motif such as a polypeptide, small molecule, or antibody-based technology such as taught in Diebold et al., Gene Therapy 8:487-493, 2001; Bonifaz et al., J Exp Med 196 (12):1627-1638, 2002; Birkholz et al., Blood 116 (13):2277-2284, 2010; Apostolopoulos et al., J. Drug Delivery, p 1-22, 2013; Lewis et al., Biomaterials 33 (29):7221-7232, 2012; Gieseler et al., Scandinavian J Immunol. 59: 415-424, 2004, all of which are herein incorporated by reference in their entirety. For example, DC affinity may be enhanced by including one or more antibodies or other molecules with affinity to DC surface markers such as DEC-205, DC-SIGN, CLEC4A, and may also include a maturation signal, e.g., IL-15.
In contrast to conventional polypeptide based vaccines, DNA vaccines may comprise a nucleic acid in the form of a plasmid (Li et al., J. Biotechnol. 162:171, 2012) but may also be incorporated in the form of RNA or incorporated into the nucleic acid of a virus vector for delivery. The plasmid DNA includes a promoter driving expression of one or more transcription units set forth herein, as would be appreciated by one of ordinary skill in the art. A nucleic acid based vaccine can be administered by, for example, intramuscular injection, subcutaneously, intranasally, via mucosal presentation, intravenously or by intradermal or subcutaneous administration.
A nucleic acid based vaccine is administered to a patient (either using a DNA plasmid, a RNA, or a viral vector) whereby the nucleic acid is taken up into a cell's cytoplasm and/or nucleus where it is transcribed into mRNA and then translated into a polypeptide or protein. Vaccine modalities set forth herein may be administered individually or sequentially as a prime-and-boost platform. The priming vaccine composition may be peptide-based followed by a vaccine boost comprising a nucleic acid delivered, for example, as a plasmid DNA or as a viral delivery system. Alternately, the priming vaccine composition may be nucleic acid delivered followed by a peptide-based vaccine. Either the vaccine prime or boost, or both the prime and boost, may be administered once or multiple times to a patient in need thereof.
Accordingly, in some embodiments are methods of eliciting a cellular immune response, comprising administering to a subject an effective amount of an immunization composition comprising a nucleic acid molecules including an MTI-PAA as described in any of the embodiments herein, an antigen encoded by a nucleic acid molecule according to any of the embodiments herein, or both, thereby eliciting a cellular immune response. In some embodiments, the immunization composition comprises a nucleic acid immunization composition. In some embodiments, the immunization composition comprises an antigen or polyantigen immunization composition. In some embodiments, the nucleic acid immunization composition and the antigen immunization composition are both administered. In certain embodiments, the antigen immunization composition is administered first and the nucleic acid immunization composition is administered second. In other embodiments, the nucleic acid immunization composition is administered first and the antigen immunization composition is administered second. In yet further embodiments, the nucleic acid immunization composition and the antigen immunization composition are administered concurrently. In some embodiments, the immunization composition comprises a cell comprising a nucleic acid according to the embodiments disclosed herein. In some embodiments, the cellular immune response is an antigen-specific T-cell response. In some embodiments, the subject is human. In certain embodiments, the nucleic acid immunization composition is taken up by an antigen presenting cell. The antigen presenting cell can be a professional or non-professional antigen presenting cell. In some embodiments, the antigen presenting cell is a dendritic cell. In certain embodiments, the method comprises administering a nucleic acid immunization composition to a subject wherein the nucleic acid immunization composition comprises a chimeric nucleic acid molecule including an MTI as described herein (e.g., any of SEQ ID NOS.:1-6, 95, or 96) and including a PAA wherein the PAA comprises antigens from folate receptor-α (e.g., any of SEQ ID NOS.:69-93), Her2/neu (e.g., any of SEQ ID NOS.:117-135), or any combination thereof. Exemplary folate receptor-α PAA and MTI-PAA sequences are provided in SEQ ID NOS.:49, 50, 52, 53-55, 58-64, 66-68, and 97-100. Exemplary HER2/neu PAA and MTI-PAA sequences are provided in 115, 116, 136-150.
The MTI technology described herein may also be used to modify a patient's cells ex vivo. For example, isolated or enriched preparations of a patient's T-cells, dendritic cells, or other antigen presenting cell population may be transfected with a nucleic acid molecule as disclosed herein. Transfected cells may be expanded and then reintroduced into the patient as effector T-cells.
Therefore, in certain embodiments, the method of eliciting a cellular immune response includes contacting a cell with a nucleic acid immunization composition comprising the chimeric nucleic acid molecule as described in any of the embodiments herein, wherein the cell is contacted ex vivo and then administered to a subject. Methods of isolating cells from a subject and introducing nucleic acid molecules are known in the art. In some embodiments, the cell is autologous or allogeneic. In certain embodiments, the cell is an antigen presenting cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the subject is a human.
In certain embodiments, the instant disclosure provides a method of eliciting a cellular immune response, comprising administering to a subject an effective amount of a cell comprising a nucleic acid molecule or a vector of any one of the embodiments disclosed herein, thereby eliciting a cellular immune response. In some embodiments, a cell is autologous or allogeneic. In some embodiments, a cell is an antigen presenting cell. In certain embodiments, the antigen presenting cell is a dendritic cell. In some embodiments, the subject is a human.
In some embodiments, the present disclosure provides a method for prime and boost to elicit a robust immune response that results in the production of memory T cells. For example, an immune response is elicited against a cancer or infectious disease by (a) contacting the subject with a nucleic acid molecule immunization composition as described herein, wherein the nucleic acid molecule of the nucleic acid molecule immunization composition encodes one or more antigenic peptides, (b) optionally allowing a time sufficient to generate an initial immune response, (c) contacting a subject with an antigenic peptide immunization composition. The peptide immunization composition and the nucleic acid immunization composition can correspond to the same one or more HLA Class I peptide antigen(s). Alternatively, the peptide immunization composition and the nucleic acid immunization composition can correspond to the same cancer marker protein or TAA.
In certain embodiments, provided herein is a method of eliciting a cellular immune response, comprising (a) administering to a subject an effective amount of an antigen immunization composition comprising one or more antigens encoded by any one of the nucleic acid molecules disclosed herein, and (b) administering to the subject an effective amount of a nucleic acid molecule immunization composition comprising a nucleic acid molecule or vector of any one of the embodiments described herein. In some embodiments, the nucleic acid molecule immunization composition of step (b) encodes a plurality of antigens. In some embodiments, the antigen immunization composition of step (a) comprises a plurality of antigens. In certain embodiments, the nucleic acid molecule encodes one or more antigens from the same protein as the one or more antigens used in step (a). In some embodiments, the nucleic acid molecule of the nucleic acid molecule immunization composition encodes one or more of the same antigens as the one or more antigens used in step (a). In certain embodiments, step (b) is performed simultaneously with step (a). In other embodiments, step (b) is performed sequentially to step (a). In some embodiments, step (b) is performed from 1 hour to 5 months, from 1 hour to 4 months, from 1 hour to 3 months, from 1 hour to 8 weeks, from 1 hour to 6 weeks, from 1 hour to 4 weeks, from 1 hour to 3 weeks, from 1 hour to 2 weeks, from 1 hour to 1 week, from 1 hour to 72 hours, from 1 hour to 48 hours, or from 1 hour to 24 hours after step (a). In other embodiments, step (a) is performed 1 hour to 5 months, from 1 hour to 4 months, from 1 hour to 3 months, from 1 hour to 8 weeks, from 1 hour to 6 weeks, from 1 hour to 4 weeks, from 1 hour to 3 weeks, from 1 hour to 2 weeks, from 1 hour to 1 week, from 1 hour to 72 hours, from 1 hour to 48 hours, or from 1 hour to 24 hours after step (b). In some embodiments, the method further comprising (c) administering to the subject a second effective amount of an antigen immunization composition, wherein the antigen immunization composition comprises a peptide that is derived from the same protein as the antigenic peptide used in (a). For example, in some embodiments the antigen immunization composition of step (c) is the same as the antigen immunization composition used in (a).
In other embodiments, provided herein is a method of eliciting an cellular immune response, comprising (a) administering to a subject an effective amount of a nucleic acid molecule immunization composition comprising the nucleic acid molecule of any of the embodiments described herein, (b) allowing a time sufficient to generate an initial immune response, and (c) administering to the subject a second effective amount of a nucleic acid molecule immunization composition comprising a nucleic acid molecule according to any of the embodiments described herein, or an effective amount of an antigenic peptide immunization composition. In some embodiments, the antigen immunization composition comprises one or more antigens that are from the same protein as the antigen encoded by the nucleic acid molecule of step (a). In some embodiments, the nucleic acid molecule of the nucleic acid molecule immunization composition encodes one or more of the same antigenic peptides as used in (c). In certain embodiments, the time sufficient to generate an initial immune response is from 1 hour to 5 months, from 1 hour to 4 months, from 1 hour to 3 months, from 1 hour to 8 weeks, from 1 hour to 6 weeks, from 1 hour to 4 weeks, from 1 hour to 3 weeks, from 1 hour to 2 weeks, from 1 hour to 1 week, from 1 hour to 72 hours, from 1 hour to 48 hours, or from 1 hour to 24 hours after step (a). In certain embodiments, the method further comprising (d) administering to a subject an effective amount of an antigen immunization composition, wherein the antigen immunization composition comprises one or more antigens that are from the same protein as the antigen used in (c). In some embodiments, the antigen immunization composition of step (d) comprises the same antigen used in (c).
In certain embodiments, a nucleic acid molecule immunization composition and a antigen immunization composition encode a different peptide antigen class, for example peptide(s) of a peptide immunization complex may comprise one or more HLA Class II antigenic peptide epitopes derived from a cancer marker protein, while the nucleic acid molecule may encode one or more HLA Class I antigenic peptides derived from the same cancer marker protein (e.g., Her2/neu or folate receptor alpha).
Antigenic peptide epitopes contained within a nucleic acid immunization composition and antigenic peptide epitopes contained within a peptide immunization composition may be derived from more than one cancer marker protein (e.g., Her2/neu and folate receptor alpha).
Typically, a patient having cancer is incapable of eliciting a strong enough immune response that can destroy sufficient numbers of cancer cells to either eliminate the tumor entirely or sufficient to reduce tumor burden to a manageable level and thereby provide an improved standard of living, with a long time to recurrence. The health care options to patients with cancer have historically focused on surgical resection of the tumor mass, chemotherapy or radiation therapy. In each of these historical options, the treatment is particularly invasive or particularly indiscreet in that chemotherapies also kill a patient's good cell as well as unwanted cancer cells.
An advantage of stimulating a patient's own immune system to destroy cancer cells is that, in general accordance with the embodiments set forth herein, such immune stimulation is long lived and consequently should prevent or extend the time to recurrence. In contrast, radiation therapy or chemotherapy requires continued repetitive treatment in order to keep killing unwanted cancer cells. Stimulating a patient's own immune system cells may be mediated in vivo or ex vivo. For an ex vivo administration, a patient's cells (e.g., dendritic cells and/or T-cells) are removed from the patient then contacted with a peptide, nucleic acid or viral composition of the instant invention. After contacting, the patient's cells are administered back to the patient.
A vaccine approach as described herein is designed to stimulate a patient's immune cells to function for a long time and therefore if a new cancer cell comes into existence it will be destroyed. In one embodiment, long term immune function is accomplished using a multi-antigenic peptide composition capable of stimulating both cytotoxic CD8+ T-cells (HLA Class I restricted) and helper CD4+ T-cells (HLA Class II restricted). Thereby, the patient does not need to repeatedly go to their health care provider (doctor, hospital) for another round of chemotherapy or radiation therapy. In many situations in which the treatment with chemotherapy, radiation therapy or surgery, the treatment can be an obstacle to success, indeed, often the patient feels worse as a consequence of treatment rather than the disease (cancer) itself.
In some embodiments, the methods of eliciting a cellular immune response described above are to elicit an immune response against a TAA as described herein. In other embodiments, the methods of eliciting a cellular immune response described above are to elicit an immune response against a pathogen as described herein. In any of the methods of eliciting a cellular immune response described above, the subject can be a human.
In any of the methods of eliciting a cellular immune response described above, the method can further comprise administering an adjunctive therapy. In some embodiments, the adjunctive therapy is surgery, chemotherapy, radiation therapy, antibody therapy, or a combination thereof. In some embodiments, the adjunctive therapy is cyclophosphamide.
Cancer, for example breast cancer, is diagnosed in approximately 210,000 women each year. Conventional standards of care such as surgery, chemotherapy and radiation therapy are successful treatments at least initially however recurrence is a common problem and is frequently the main source of morbidity and mortality. Monoclonal antibodies have been advanced in the treatment of some cancers, for example trastuzumab for HER2/neu+ breast cancer. Although the combination of trastuzumab extends survival time for women with advanced HER2/neu+ cancer, a majority of women develop resistance within one year of the beginning of treatment. The development of additional or alternative strategies may provide patients with new treatment options and improve the current standard of care. A vaccine that delays the time to disease recurrence or prevents disease recurrence has significant clinical and commercial potential. In addition, a cancer vaccine described herein may be used to boost immunity against tumor antigenic T-cell epitopes that are known or expected to generate pre-existent immunity towards a TAA detected in a cancer patient.
In other embodiments, provided herein is a method of treating breast cancer, comprising administering an effective amount of nucleic acid molecule immunization composition comprising a nucleic acid molecule immunization composition comprising a breast cancer TAA. In some embodiments, the breast cancer TAA is HER2/neu, folate receptor-α, or a combination thereof. In some embodiments, the nucleic acid immunization composition comprises a nucleic acid sequence corresponding SEQ ID NOS.:49, 50, 52-55, 58-64, 66-93, 97-100, 115-150, or any combination thereof. In certain embodiments, the method of treating breast cancer further comprises administering an adjunctive therapy. The adjunctive therapy can be surgery, chemotherapy, radiation therapy, antibody therapy, or any combination thereof. In some embodiments, an antibody therapy comprises trastuzumab, pertuzumab, anti-CTLA4, anti-PD1, anti-PDL1, anti-VEGF (e.g., bevacizumab), anti-Folate Receptor alpha (e.g., farletuzumab); as well as small molecule inhibitors of kinase domain function (e.g., laptinib, gefitinib, erlotinib, or the like). In some embodiments, the adjunctive therapy is cyclophosphamide. In some embodiment, the adjunctive therapy is therapy that inhibits immunosuppression. In certain embodiments, the therapy that inhibits immunosuppression, is an inhibitor of an immunosuppression signal is an antibody, fusion protein, or siRNA specific for PD-1, PD-L1, PD-L2, CTLA4, HVEM, BTLA, KIR, LAG3, GAL9, TIM3, TGFβ, IL-10, IL-35, or any combination thereof. In some embodiments, the anti-CTL4 antibody is an CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof. In some embodiments, the anti-PD-1 antibody is a PD-1 specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MK-3475, AMP-224, or any combination thereof. In some embodiments, the anti-PD-L1 antibody is a PD-L1 specific antibody or binding fragment thereof, such as MDX-1105, BMS-936559, MEDI4736, MPDL3280A, MSB0010718C, or any combination thereof.
In some embodiments, the methods of eliciting a cellular immune response as described herein further comprise identifying patient specific TAA antigens. Accordingly a population of a patient's T-cells can be screened against a collection of Class I and/or Class II T-cell peptides in order to identify antigenic epitopes to which the patient already has a detectable T-cell immune response. A patient's cells are isolated, incubated with peptides displayed using a multi-well plate and cytokine responses are measured, such as gamma interferon. One or more of the antigenic peptides so identified can then be formulated into a peptide-based vaccine composition. The detected antigenic peptides may be Class I or Class II restricted. In addition, the same Class I and/or Class II peptides may be incorporated into a nucleic acid-based delivery system disclosed herein. A patient may then be administered the tailored peptide or corresponding nucleic acid based compositions as standalone medicines or in sequential combination as a prime-and-boost modality as described in embodiments above. In this regard, the prime may be nucleic acid based and the boost peptides-based, or vice versa. Accordingly, in some embodiments is a method of treating cancer, comprising administering to a subject an effective amount of a nucleic acid immunization composition comprising a nucleic acid molecule of any of the embodiments described herein, wherein the one or more antigens encoded by the nucleic acid molecule comprise an antigen having an oncogenic mutation identified in the subject.
Supporting of the instant disclosure, compositions and methods described herein combine mass spectrometry to aid in the identification of novel peptide epitopes presented by HLA class I and HLA class II alleles. After administration, one of ordinary skill in the art can follow and characterize immune responses using cell culture and small animal or non-human primate model systems. Peptide sequences are converted into a nucleic acid sequence (which may be fully or partially codon optimized) and designed for inclusion into nucleic acid-based expression/delivery vaccine systems. Exemplary peptides may be from cancer (e.g., HER2/neu peptides such as those disclosed in US Patent Pub. No. 2010/0310640, which peptides are incorporated herein by reference; folate receptor-α peptides; NY-ES01 testes specific antigens; WT1 peptides, or the like) or other infectious diseases (e.g., other viruses, parasites, bacteria).
The compositions and methods of the instant disclosure may include a molecular adjuvant mechanism (TAP1/TAP2) that is suited to enhance HLA class I peptide presentation and subsequent CD8 T cell responses and combines it with a vaccine vector designed to maximize antigen expression (FIG. 1) incorporating features that enhance translation efficiency; induces production of an array of antigenic peptides; and marks peptides for proteasome targeting/trafficking and proteolytic processing. The compositions and methods of the instant disclosure may incorporate multiple translation initiation sites for increased expression (for example, one mRNA initiates and synthesizes four translation products); incorporate cytosolic and/or nuclear targeting and subsequent processing; distinguish between endogenous and recombinant protein; distinguish between endogenous and recombinant nucleic acid encoding a TAP1 and/or a TAP2; and allow for nuclear and cytosolic antigen targeting. The vectors may also be constructed such that each feature can be independently manipulated for characterization and testing.
Additional immune stimulatory or modulating agents, including negative immune modulators such as regulatory T-cells and related check point inhibitors (anti-PD1, Yervoy®, cyclophosphamide), may be included in any composition or method described herein. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF) may function as a vaccine adjuvant as previously described (see Mohebtash et al., Clin. Cancer Res. 17:7164, 2011). Other adjuvants such as alum, MF59, CpG, R848 and the like may be included in the compositions or methods described herein.
As an example, one infectious disease treatable by administering the instant invention is smallpox as this disease has claimed hundreds of millions of lives in the last two centuries alone (Dixon, C. W. Smallpox. (J. & A. Churchill, 1962); Fenner et al., Smallpox and its Eradication, Vol. 6, World Health Organization, 1988), and although considered eradicated since 1980 (Fenner, 1988), biodefense research into poxviruses remains vitally important because of the following: (1) concerns about the use of smallpox as a biological weapon (Henderson et al., Working Group on Civilian Biodefense. JAMA 281:2127-2137, 1999; Kennedy et al., Vaccine 27 (Suppl 4):D73-79, 2009; Whitley, Antiviral Res 57:7-12, 2003; Bossi et al., Cell Mol Life Sci 63:2196-2212, 2006; Mayr, Comp. Immunol. Microbiol. Infect. Dis. 26:423-430, 2003; Wiser et al., Vaccine 25:976-984, 2007); (2) emerging zoonotic poxviruses, such as monkeypox in the US and Africa (Hutson et al., Am. J. Trop. Med. Hyg. 76:757-768, 2007; Kile et al., Arch. Ped. Adolesc. Med. 159:1022-1025, 2005; Di Giulio and Eckburg, The Lancet Infect. Dis. 4:15-25, 2004; Edghill-Smith et al., J. Infect. Dis. 191:372-381, 2005; Larkin, The Lancet Infect. Dis. 3:461, 2003; Jezek et al., Am. J. Epidemiol. 123:1004, 1986), vaccinia-like viruses in South America (Leite et al., Emerging Infect. Dis. 11:1935-1938, 2005; Silva-Fernandes et al., J. Clin. Virol. 44:308-313, 2009; Trindade et al., Clin. Infect. Dis. 48:e37-40, 2009), and buffalopox in India (Singh et al., Animal Health Res. Rev./Conference of Research Workers in Animal Diseases 8:105-114, 2007); (3) numerous vaccine contraindications (Neff et al., Clin. Infect. Dis. 46 (Suppl 3):S258-270, 2008; Poland et al., Vaccine 23:2078-2081, 2005; Bonilla-Guerrero & Poland, J. Lab. Clin. Med. 142:252-257, 2003); (4) concerns with VACV transmission (Fulginiti et al., Clin. Infect. Dis. 37:241-250, 2003; Wharton et al., MMWR Recomm. Rep. 52:1-16, 2003; Redfield et al., N. Engl. J. Med. 316:673-676, 1987); and (5) safety issues inherent to live virus smallpox vaccines resulting in rare but serious adverse events (Fulginiti et al., Clin. Infect. Dis. 37:251-271, 2003; Fulginiti, JAMA 290:1452; author reply 1452, 2003; Lane et al., JAMA 212:441-444, 1970; Lane et al., N. Engl. J. Med. 281:1201-1208, 1969; Morgan et al., Clin. Infect. Dis. 46 (Suppl 3):S242-250, 2008; Vellozzi et al., Clin. Infect. Dis. 39:1660-1666, 2004; Halsell et al., JAMA 289:3283-3289, 2003; Poland and Neff, Immunol. Allergy Clin. North Am. 23:731-743, 2003; Chapman et al., Clin. Infect. Dis. 46 (Suppl 3):S271-293, 2008). Recently introduced second-generation (live) tissue culture-based vaccines (ACAM2000) have replaced Dryvax®, but retain nearly all of the drawbacks of the first-generation vaccines (Kennedy et al., Curr. Opin. Immunol. 21:314-320, 2009; Artenstein et al., Vaccine 23:3301-3309, 2005; Greenberg and Kennedy, Expert Opin. Investig. Drugs 17:555-564, 2008; Poland, Lancet 365:362-363, 2005; Greenberg et al., Lancet 365:398-409, 2005). Attenuated vaccines based on MVA (IMVAMUNE) may have improved safety profiles (Kennedy and Greenberg, Expert Rev Vaccines 8:13-24, 2009; Frey et al., Vaccine 25:8562-8573, 2007; Vollmar et al., Vaccine 24:2065-2070, 2006; Edghill-Smith et al., J. Infect. Dis. 188:1181, 2003), but remain live viral vaccines and there are some concerns regarding both safety and immunogenicity.
The preparation and characterization of expression vectors (plasmid and vaccinia virus based) encoding peptide antigen arrays (PAAs) consisting of multiple HLA Class I and Class II-derived peptides together with TAP1 and/or TAP2 is set forth herein. As one example of the instant disclosure, a plasmid expression vector encoding various combinations of the features outlined in FIG. 1: (1) a vaccinia-peptide antigen array and (2) an expression cassette containing TAP1 and/or TAP2. These expression vectors direct the synthesis of a chimeric protein containing an amino-terminal portion of FGF2 (MTI) followed by an interchangeable peptide antigen array containing a selection of peptides associated with infectious disease or cancer.
In order to promote efficient proteasome processing of the encoded peptide sequences and thereby preserve complete protease processing sites, the NH2 and COOH ends can be extended by three or four or more native/naturally occurring amino acids (FIG. 1, lower case). Each antigenic peptide portion may be further separated from the next antigenic peptide by a spacer (e.g., G4S). The four MTI translation initiation sites will yield four moles of a multi-peptide PAA translation product for every one mole of transcribed mRNA (FIG. 1) (Florkiewicz and Sommer, PNAS 86:3978-3981, 1989; Florkiewicz et al., Growth Factors 4:265-275, 1991).
In accordance with the instant disclosure, nucleic acid molecule-based expression vectors have been designed so that a PAA portion of a MTI-PAA is a cassette that is easily excised and replaced. This enables one of ordinary skill to rapidly incorporate additional VACV/VARV epitopes, create a ‘reporter epitope’ PAA, or design PAAs specific to other pathogens or cancer targets, consistent with a modular recombinant strategy.
The COOH-terminal VSVG epitope-tag can be used for detection by immunoprecipitation, immunoblotting and indirect immunofluorescence (and can also be used to design PCR primer pairs for detection of transcribed mRNA). In order to enhance expression of antigenic peptides/proteins along with TAP1 and/or TAP2, nucleic acid molecule encoding TAP1, TAP2, and PAA (not MTI) are prepared synthetically and in context with a preferred codon utilization algorithm, thereby providing enhanced translation efficiency consistent with the preferred codon bias of Homo sapiens, while at the same time minimizing potential cis-acting mRNA sequences that could negatively impact translation efficiency.
Furthermore, nucleic acids of this disclosure may incorporate common epitope tags for the differential immune detection of vector-encoded, as opposed to endogenous Tap1 and/or Tap2, protein. The NH2-terminal Tap1 tag is V5 and the NH2-terminal tag for Tap2 is AU5.
It is appreciated that proteasomes differ in composition and processing ability (Bedford et al., Trends Cell Biol. 20:391, 2010; de Graaf et al., Eur J Immunol 41:926-935, 2011; Khan et al., J Immunol 167:6859, 2001; Sijts & Kloetzel, Cellular and Molecular Life Sciences: CMLS 68, 1491-1502, 2011; Wilk et al., Arch Biochem Biophys 383, 265, 2000; Xie, Y. J Mol Cell Biol 2, 308-317, 2010). Furthermore, it is recognized, although not well characterized, that nuclear proteasomes appear different from cytosolic proteasomes. In accordance with the invention, MTI translation products initiated with CUG codons are targeted to the nucleus while the translation product initiated at the AUG codon remain cytosolic. By adjusting the MTI sequence (site specific or deletion mutagenesis remove multiple GR repeats), the invention disclosed can exclusively express either cytosolic or nuclear localized MTI-PAA.
The invention disclosed herein may include a molecular adjuvant, such as the proteins termed Tap1 and/or Tap2, and can be used to demonstrate that inclusion of TAP1 can further enhance presentation of a PAA derived peptide.
In some embodiments, a ‘reporter epitope’ PAA consisting of epitopes for which there are peptide/MHC complex-specific antibodies may be incorporated into a PAA. Such a reporter epitope may allow one of ordinary skill to use these antibodies to directly measure surface expression of the presented peptides in the context of their restricting MHC/HLA allele.
Immunogenicity testing includes a series of dose-ranging and vaccination-schedule-testing experiments designed to characterize vaccine-induced immune responses. Different HLA class I and HLA class II transgenic animal strains with matching HLA restriction to the antigenic epitopes may be used within the context of the disclosed invention.
Vaccine efficacy testing includes evaluation of the PAA/TAP vector vaccination dose/schedule followed by survival studies using, for example, the intranasal infection model. Intranasal inoculation of Vaccinia Western Reserve (WR) into mice results in a lethal infection characterized by weight loss, ruffled fur, lethargy, and death by day six or seven post infection (Turner, J Gen Virol 1:399, 1967; Reading & Smith J. Gen. Virol. 84:1973, 2003; Williamson et al., J. Gen. Virol. 71:2761, 1990).
Prior to the vaccine efficacy experiments, the VACV-WR LD50 for each transgenic strain may be determined experimentally as per the Reed-Muench method, and 5-10 LD50 will be used for survival experiments, and to develop correlates of protection/immunity to derive levels of protection in a human vaccinated population (Reed & Muench, Am. J. Epidemiol. 27:493, 1938).
CTL function: as appreciated by one of ordinary skill in the relevant art, percent specific lysis is calculated for each effector:target ratio according to established protocols (Latchman, Y. E. et al. PNAS 101, 10691-10696, 2004) and compared between groups using ANOVA.
T helper function: Antigen-specific T cell activity is calculated by subtracting the median IFNγ ELISPOT response value of the unstimulated wells from that of the stimulated wells. Values may be calculated for individual mice and differences between immunized and control groups will be compared using Student's t test. CFSE a tracking reagent used in flow cytometry is used to determine proliferation rate differences between groups and compared using established models (Banks et al., J. Immunol. Methods 373:143-160, 2011; Banks et al. Bull. Math Biol. 73:116-150, 2011).
Survival studies: Power calculations (assuming a type I error rate of 0.05 and a two-sided test of hypothesis) indicate that, with 20 mice/group, about an 80% power of detection for a difference in survival will be measurable if the true survival rates at seven days are 50% for the immunized group and 10% for the unimmunized group. For all survival experiments, Kaplan-Meier survival curves will be plotted and compared using standard statistical tests for survival, such as the log rank test.
Proteasome processing of a PAA modified to include a consensus protein ubiquitination motif (e.g., KEEE (SEQ ID NO.: 203) or EKE) (Catic et al., Bioinformatics 20:3302-3307, 2004; Chen, Z. et al. PLoS One 6, e22930, 2011; Jadhav, T. & Wooten, M. W. J Proteomics Bioinform 2, 316, 2009; Sadowski, M. & Sarcevic, B. Cell Div 5, 19, 2010) may be included in the embodiments listed herein along with fluorescently-labeled antibodies specific for peptide/MHC complexes in order to directly measure epitope presentation.
In certain embodiments, TAP is selected as a molecular adjuvant to enhance class I antigen presentation. By way of background, there is considerable cross-talk between the HLA class I and class II processing and presentation pathways (Chicz, R. M. et al. The Journal of experimental medicine 178, 27-47, 1993; Lechler, R., Aichinger, G. & Lightstone, L. Immunological reviews 151, 51-79, 1996; Rudensky, A., et al., Nature 353, 622-627, 1991). In addition, intracellular peptides can be loaded onto HLA class II peptides through multiple mechanisms (Dengjel et al., PNAS 102:7922-7927, 2005; Dongre, A. R. et al. Eur J Immunol 31, 1485-1494, 2001; Nedjic, J., et al., Curr Opin Immunol 21, 92-97, 2009). There is also increasing evidence that the class I antigen processing machinery affects this endogenous pathway for class II processing and presentation (Jaraquemada et al., J. Exp. Med. 172:947-954, 1990; Loss et al., J. Exp. Med. 178:73-85, 1993; Oxenius, A. et al., Euro. J. Immunol. 25:3402-3411, 1995). Tewari et al. have identified two I-Ed restricted epitopes from the influenza HA and NA proteins that are processed by TAP and the proteasomal machinery and loaded onto recycling MHC class II molecules (Nature Immunol. 6:287-294, 2005).
Plasmid expression vectors: Detection of overexpressed TAPs or MTI-PAA is determined by PCR, immunoprecipitation, Western blot, immunofluorescence and/or IHC. Immuno-detection makes use of commercially available antibodies (see Examples). Intracellular localization is evaluated by indirect immunofluorescence and subcellular fractionation using commercially available methodologies (e.g., NE-PER, Pierce, Inc.). Preparation of VACV vectors may be accomplished by the ordinary skill artisan in accordance with standard methodologies.
Levels of antigen presentation are assessed by several methods: flow cytometry using purified p/MHC specific antibodies isolated from hybridomas and conjugated to PE or APC (Molecular Probes Antibody Staining Kits); co-incubation of transfected APCs with peptide-specific T cells and assessing immune recognition by IFNγ ELISPOT assay or intracellular cytokine staining.
One or more epitope tags may be used to aid in the detection of a fusion polypeptide encoded by a nucleic acid molecule of this disclosure when contained in a plasmid or viral vector, wherein the nucleic acid molecule is operably linked to an expression control sequence.
Antigenic peptides (Class I or II restricted) arise when proteins or fragments thereof undergo proteasomal processing. The peptides generated can then bind to MHC (HLA) molecules and can elicit protective immunity if various additional parameters are met (Gilchuk et al., J. Clin. Investig. 123:1976-1987, 2013), such as (1) HLA binding affinity; (2) peptide/HLA stability; (3) interactions with molecular chaperones (TAP/HLA-DM) (Yin et al., J. Immunol. 189:3983-3994, 2012); (4) strength of the TCR:peptide:MHC complex interaction; (5) antigen expression level and kinetics; (6) cellular localization; and (7) T cell recognition/peptide immunogenicity. In certain embodiments, peptides elicit multi-functional T cell responses and generate long-lived memory cells that protect the host from infectious disease or from developing cancer.
EXAMPLES
Example 1
Nucleic Acid Molecules Comprising a Multiplex Translation Initiation Sequence
FIG. 1 depicts an exemplary construction of a nucleic acid-based expression vector incorporating the MTI (based on FGF2 upstream multiplex translation initiation from CUG start codons) technology as disclosed herein. Expression vectors may comprise a plasmid vector or may comprise a nucleic acid molecule encoding peptide antigenic epitopes incorporated into one or more viral delivery systems. A first series of plasmid expression vectors encoding (a) MTI-PAA (PAA=Peptide Antigen Array; see FIG. 1C), (b) codon-optimized and epitope tagged TAP1, and (c) codon-optimized TAP2. These MTI-PAA nucleic acid molecule-based systems may further include combinations of TAP1 and/or TAP2. The vector design allows the PAA portion to be easily excised as a cassette and replaced with the sequence corresponding to any series of T-cell epitopes, enabling incorporation of additional epitopes from other pathogens, cancer genes or HLA supertypes into the expression vector—enhancing its utility as a “plug-and-play” platform.
In the context of MHC Class I peptides, expression vectors direct the synthesis of a chimeric protein containing an NH2 portion of hFGF2 (MTI) followed by a specific peptide antigen array (PAA) representative of the aforementioned antigenic peptides. We assembled a selection of peptides into a linear peptide antigen Array (PAA) and converted the same into a nucleic acid expression vector. However, we hypothesized that protease processing requires the presentation of a complete protease processing site. Therefore, in order to encourage or otherwise recapitulate efficient proteasome processing of the peptide sequences, we extended the amino acid sequence at their NH2 and COOH ends by three or four or more native/naturally occurring amino acids.
The expression vectors incorporate features that will enhance the amount of selected PAA/peptides (T cell epitopes) synthesized. Particular features include (1) codon optimized DNAs, and (2) the ability to synthesize four moles of MTI-PAA for every one mole of mRNA transcribed. Codon optimization of the PAA portion of the expression vectors used herein will increase the translational efficiency of transcribed mRNAs. In some cases, codon optimization also provides a means by which to molecularly distinguish (e.g., by PCR) vector encoded genes (e.g., TAP1 and TAP2) from corresponding endogenous genes in preclinical as well as clinical evaluation.
Regarding above mentioned ability to synthesize four moles of MTI-PAA for every one mole of mRNA transcribed, expression system takes advantage of the fact the human FGF2 gene is capable of directing the synthesis of four polypeptides from one mRNA via the recognition of three unconventional CUG translation initiation codons and one classical AUG codon. The three CUG codons initiate translation of three co-linear amino terminally extended versions of 18 kDa FGF2 that initiates translation at a downstream AUG codon. The portion of FGF2 included in the vector described above retains the three CUG codons as well as the one AUG codon. Therefore, the vector provides four moles of a particular PAA for every one mole of mRNA.
An additional feature of the FGF2 portion of the expression vectors is the ability to evaluate the efficiency of cytosolic versus nuclear proteasome processing. It is well established that proteasome processing provides peptide substrates for MHC Class I mediated cell surface presentation. It is also appreciated that proteasomes differ in composition, where, for example, immune proteasomes are distinct from non-immune (normal) proteasomes. Further, it is recognized, although not well characterized, that nuclear proteasomes appear different from cytosolic proteasomes.
FGF2 translation products initiated with CUG codons are targeted to the nucleus while the translation product initiated at the AUG codon is cytosolic (Data not shown). Accordingly, FGF2 sequences are modified (site specific or deletion mutagenesis) to exclusively express either cytosolic or nuclear localized MTI-PAA chimera.
Example 2
Expression of Poly-Antigen Array Epitopes
TAP1, TAP2, and PolyStartTM MTI-PAA gene expression in transfected COS-1 cells have been observed by PCR (data not shown). The pJ603 expression plasmid was selected for these experiments. The CMV and/or SV40 late promoter contained in the plasmid drives transcription of downstream coding sequence and (in some cases) sequence deemed important for maintaining mRNA structure and appropriate recognition of CUG/AUG translation initiation codons. Accordingly, we have designed/prepared a first series of plasmid expression vectors encoding (a) MTI-PAA (smallpox peptides), (b) Tap1 only, (c) Tap2 only, (d) Tap1-IRES-Tap2, (e) Tap1 plus MTI-PAA, and (f) Tap2 plus MTI-PAA. A VSVG peptide-tag epitope tag was included at the COOH-terminal end of the MTI-PAA.
The expression vectors have been designed so that the PAA portion of the MTI-PAA can be easily excised and replaced with any codon-optimized DNA sequence encoding any PAA corresponding to any series of T-cell peptide epitopes (or full length/truncated protein), enabling us to rapidly incorporate additional peptide epitopes such as VEEV E3 or E2, cancer epitopes such as breast cancer related epitopes (e.g., Her2/neu, folate receptor alpha, NY-ESO-1, MIF, GnRh or GnRHR), or peptide epitopes specific to any pathogen(s) or targeted cancer cell protein into our expression vector.
In addition, the nucleic acid sequences encoding TAP1, TAP2, and PAA, were prepared synthetically and in context with a preferred codon utilization algorithm. Codon optimization provides enhanced translation efficiency consistent with the preferred codon bias of Homo sapiens while simultaneously minimizing potential cis-acting mRNA sequences that could also negatively impact translation efficiency. In this example, the FGF2 (MTI) portion was not codon optimized to preserve sequence dependent mRNA structure that may be needed to mediate translation from unconventional (i.e., CUG) translation initiation codons in a manner mechanistically consistent with internal ribosome entry. This strategy will support higher levels of expression and thereby provide a higher level of corresponding antigenic polypeptide. This in turn is expected to result in higher proteasome mediated peptide processing and consequently higher TAP mediated peptide presentation to MHC and subsequent cell surface presentation.
The unique aspects of the codon-refined synthetic DNAs encoding TAP1 and TAP2 have been utilized to design PCR primer pairs that distinguish cDNAs derived from endogenous mRNA transcripts from cDNA derived from vector transcribed mRNA. The primer pairs were then used in PCR reactions using template DNA obtained from three sources: codon refined plasmid DNA for TAP1 and TAP2; cDNA template from non-transfected COS-7 and PANC-1 cells; and cDNA template from COS-7 cells transfected with TAP1 and TAP2 plasmid DNA.
COS-1 cells (70% confluent) were transfected in 60 mm plates using the Fugene HD reagent (Promega, Inc.). Approximately 40 hours post-transfection, total RNA was prepared. The total RNA was treated with DNase free RNase and cDNA was generated using M-MLV reverse transcriptase. HotStart Polymerase (Qiagen, Inc.) was used in the PCR reaction as follows: 95° hold, 95° 15′, [95° 30′′, 54/57° 30′′, 72° 1′30′′]40x 72° 5′, 12° hold; +/−Q solution.
The results demonstrated that the PCR primers designed to detect endogenous TAP1 and TAP2 do not recognize plasmid DNAs containing codon refined TAP1 or TAP2 (data not shown). In comparison, PCR primer pairs that are specific to the synthetic codon refined DNAs do prime. In addition, TAP1 primers do not cross-prime with TAP2 templates. Similarly, using total RNA prepared from non-transfected COS-7 and PANC-1 cells, PCR primer pairs recognizing endogenous TAP1 and TAP2 do not recognize cDNA derived from vector encoded mRNA but do recognize cDNA templates derived from endogenous TAP1 and TAP2 mRNA (data not shown). Finally, cDNA prepared from mRNA derived from COS-7 cells transfected with plasmid expression vectors containing codon preferred TAP1 and TAP2 sequences distinguished endogenous from transfected mRNAs (data not shown). These PCR conditions were used in order to follow/confirm TAP1 and TAP2 expression following presentation of therapeutic plasmid DNAs or following infection with recombinant vaccinia virus.
Further, since both TAP1 and TAP2 contain internalized non-cleaved signal sequences, leaving an exposed cytosolic NH2 portion of TAP1 and an exposed ER-lumen localized NH2 portion of TAP2, our synthetic constructs incorporate commonly used NH2-terminal epitope tags for the differential detection of vector encoded, as opposed to endogenous, TAP1 and/or TAP2. The NH2-terminal TAP1 tag is V5 and the NH2-terminally tag for TAP2 is AU5. Commercially available antibodies were used to detect expression and intracellular localization by immunoprecipitation, immunoblotting, and immunofluorescence.
Protein expression of both TAP1 and MTI-PAA was confirmed by immunoprecipitation (FIG. 2). Codon-optimized TAP2 protein expression was problematic and we are recreating the vectors using native human TAP2 sequence. Immunofluorescence data demonstrate the cytosolic expression of TAP1 using both anti-V5 and anti-TAP1 antibodies (FIG. 3). Control experiments characterizing full length expression of MTI-PAA was also confirmed by immunofluorescence staining of transfected COS cells using an anti-FGF2 Ab targeting the amino-terminus (data not shown) and an anti-VSV Ab directed against the C-terminal portion of MTI-PAA (FIG. 4). These data also demonstrate that MTI-PAA is predominantly localized to the nucleus. FIG. 2 also illustrates that MTI-PAA expression is enhanced in the presence of the proteosomal inhibitor MG132 (10 nM). IP data from transfected HEK cells also indicates increased levels of MTI-PAA in the presence of MG132 (FIG. 5) as well as detection of all four predicted translation products of the MTI-PAA (FIG. 5).
Example 3
Expression and Immune Recognition of Poly-Antigen Array Epitopes
FIG. 6 demonstrates that peptide-specific T cells (from peptide vaccinated mice) recognize THP-1 cells transfected (Lipofectamine LTX, Invitrogen) with PolyStartTM MTI-PAA, confirming PAA-driven expression, processing, and presentation of immunogenic peptides. Furthermore, as illustrated in FIG. 7, co-expression of TAP1 in HEK PAA-transfected cells influences antigen recognition by T cells.
Example 4
Murine Peptide Reactivity
Peptide-specific T cell responses from HLA-A2 transgenic mice intradermally vaccinated with Dryvax® were detected using IFNα ELISPOT. Positive responses are defined as ≧2 fold increase in spot count over background with p<0.05 and peptides exhibiting consistent reactivity in multiple mice are shown in Table 1. These same seven peptides were recognized by T cells from two different strains of HLA-A2 mice (chimeric A2/Kb on a BL/6×Balb/c F1 background and chimeric A2/Dd on a BL/6×CBA F1 background), increasing confidence in the immunologic relevance of these peptides following smallpox immunization







TABLE 1







Positive Peptides From HLA-A2 Mice











Peptide

Protein

SEQ ID


No.
Sequence
Source
Description
NO.





16
VLSLELPEV
D13L (127)
Virion Coat Protein
24


 


19
KIDYYIPYV
E2L (068)
Hypothetical Protein
25


 


22
SLSNLDFRL
F11L (058)
Unknown Function
26


 


25
ILMDNKGLGV
F1L (048)
Apoptosis Inhibitor
27


 


28
ILDDNLYKV
G5R (091)
Viral Morphogenesis
28


 


30
KLLLGELFFL
J3R (104)
Poly(A) Polymerase
29


 


33
GLLDRLYDL
O1L (079)
Unknown Function
30









Example 5
Peptide Immunogenicity Testing
HLA-A2 transgenic mice (n=3/group) were immunized twice with 10 μg of the individual peptides listed in Table 1. A2 peptides were emulsified in IFA along with 100 μg CpG1826 and 140 μg HBV core antigen (SEQ ID NO.: 31; TPPAYRPPNAPIL). Immune responses against peptide-pulsed target cells were readily detectable four weeks after the second vaccination (FIG. 8). Mice immunized with a combination of peptides had detectable responses 4-5 months (FIG. 9) after the second immunization. IFN-γ ELISPOT responses to individual peptides range from 2 to 19-fold increase over background with each peptide exhibiting a consistent magnitude of response (data not shown). These results demonstrate successfully elicited A2-restricted murine T cell responses to epitopes identified from infected human cell lines, thus the model system recapitulates the antigen processing and presentation of a natural human infection.
Example 6
Verification of Murine Model Epitopes in Human Vaccines
Immune responses against a peptide pool (all 7 peptides from Table 1) were tested in 83 HLA-A2 supertype positive, human subjects who had received the smallpox vaccine 1-4 years previously. 42.2% of the subjects (35/83) had positive IFNγ ELISPOT responses to the peptide pool (Spot count≧1.5 times that of background wells with t-test p<0.05). Responses ranged from 1.5 to 6.2-fold increase over background. Furthermore, 20.4% of an additional 54 HLA-A2 supertype positive subjects had detectable immune responses against one or more of the individual. Some peptides were not recognized by any of the subjects, while other peptides were recognized by multiple subjects (data not shown). These results support the use of the HLA transgenic mice to identify relevant peptides recognized by human vaccine recipients.
Example 7
Efficacy of Identified Peptides
Mice (n=5) were vaccinated subcutaneously on the right flank with four CTL peptides (#22, #25, #28, #30 from Table 1) 140 μg of the HBV T helper epitope, and 100 μg of CpG 1826 emulsified in IFA. Unvaccinated mice (n=5) served as controls. Three weeks after immunization, mice were challenged intranasally with 1×106 pfu Vaccinia Western Reserve. Survival, weight loss, and clinical symptoms of illness were monitored daily. Mice losing 25% of their body weight were euthanized.
Survival data are presented in FIG. 10. Peptide-vaccinated mice were completely protected from lethal challenge. In contrast, all of the unvaccinated mice developed severe clinical symptoms (ruffled fur, hunched posture, loss of mobility) and 80% of them succumbed to the infectious challenge.
Example 8
Epitope Identification in Additional Pathogens
Methods of this disclosure can be used to create a nucleic acid molecule vaccine that encodes antigenic peptide epitopes from seven HLA class I supertypes (A1, A2, A11, A24, B7, B27, B44) and three HLA-C alleles (Cw*0401, Cw*0602, Cw*0702), which would protect across a worldwide populations on average of >96% and is obtainable even without considering peptide binding promiscuity. Frequencies and resources for determine HLA class haplotypes are described in Robinson et al. Nucleic Acids Res. 41:D1222, 2012.
Example 9
HER2/Neu+ Cancer Vaccine Compositions
The following example illustrates a peptide-based and a nucleic acid-based immunization composition for treating cancers that overexpress a HER2/neu marker protein such as HER2/neu overexpressing breast cancer.
Compositions are prepared containing one or more HLA Class I and/or Class II antigenic T-cell peptide sequence generated by computer algorithms or identified by testing human patient samples. For example, computer-based predictions identified a panel of 84 Class II HLA-DR binding epitopes (Kalli et al., Cancer Res 68:4893, 2008; Karyampudi et al., Cancer Immunol Immunother 59:161, 2010). A pool containing four HLA-DR epitopes are immunogenic, naturally processed, and cover about 84% of Caucasians, African Americans and Asians (Hardy-Weinberg equilibrium analysis). The amino acid sequence of four individual peptide epitopes includes NLELTYLPTNASLSF (SEQ ID NO.: 32), HNQVRQVPLQRLRIV (SEQ ID NO.: 33), LSVFQNLQVIRGRIL (SEQ ID NO.: 34), and PIKWMALESILRRRF (SEQ ID NO.: 35). Compositions containing these four HLA-DR peptides are described in the Phase I Clinical Trial entitled “A Phase I Trial of a Multi-Epitope HER2/neu Peptide Vaccine for Previously Treated HER-2 Positive Breast Cancer”. In the trial, 500 μg of each peptide generate an immune response in up to about 90% of breast and ovarian cancer patients (Knutson et al., J. Clin. Invest. 107:477, 2001; Disis et al., J. Clin. Oncol. 20:2624, 2002).
The peptide or nucleic acid vaccine compositions may also contain one or more HLA Class I antigenic CD8+ T-cell antigenic epitope in combination with one or more HLA Class II epitopes. For example, the composition can include the epitopes as described in U.S. Provisional Application No. 61/600,480 entitled “Methods and materials for generating CD8+ T-cells having the ability to recognize cancer cells expressing a Her2/neu polypeptide.” An exemplary amino acid sequence is SLAFLPESFD (SEQ ID NO.:36)(amino acids 373-382).
An HLA Class I antigenic epitope may be modified by extending at its NH2 and/or COOH end by one or more naturally occurring amino acid. Such an extension may recapitulate an endogenously recognized naturally processed polypeptide sequence involved in proteolytic processing through a cells proteasome mediated degradation pathway, which may include sequences that promote ubiqutination. Accordingly, a plasmid-based nucleic acid immunization composition can incorporate the corresponding DNA coding sequences, which may include nucleic acid sequences reflecting natural NH2 and/or COOH terminal extensions, and which may incorporate linker regions positioned between the coding sequences of each peptide.
An exemplary vaccination strategy is to administer as a prime-and-boost, i.e., sequentially delivering one or more in a series of peptide vaccine composition(s), followed by one or more in a series of nucleic-acid (e.g., plasmid or viral) vaccine compositions, or vice versa. In one aspect, a peptide immunization composition is administered prior to administration of a nucleic acid immunization composition. In another aspect, the nucleic acid immunization composition is administered prior to administration of the peptide immunization composition.
In addition, some patients may have a better outcome if prior to administering a vaccine composition, the patient receives cyclophosphamide, or a similar composition, orally for 1 week followed by a 1 week rest, and then another week of cyclophosphamide treatment. About 7-10 days following cyclophosphamide treatment, patients are vaccinated intradermally, intramuscularly, or ex vivo using isolated cells. The peptides may be HLA-DR related and the nucleic acid may encode one or more HLA-Class I antigenic cytotoxic T-cell epitopes.
Compositions are prepared containing one or more HLA Class I and/or Class II antigenic T-cell peptide sequences from computer algorithms or testing human patient samples. The vaccine compositions may contain an adjuvant, such as GM-CSF (e.g., 125 μg/injection).
Example 10
Folate Receptor Alpha and Cancer Vaccine Compositions
This Example illustrates a combined or sequentially administered nucleic acid-based composition and peptide-based composition for treating a folate receptor alpha expressing cancer. Folate receptor alpha is overexpressed in a number of cancers such as ovarian, primary peritoneal, lung, uterine, testicular, colon, renal, HER2/neu+ breast, and triple negative breast cancer (Zhang et al., Arch. Pathol. Lab. Med. p 1-6, 2013; Weitman et al., Cancer Res. 52:3396, 1992; O'Shannesst et al., SpringerPlus 1:1, 2012; Kelemen, Int. J. Cancer 119:243, 2006; U.S. Pat. No. 8,486,412).
In one aspect, the vaccine composition contains one or more antigenic folate receptor alpha HLA Class II and/or HLA Class I antigenic peptide epitopes as described in a Phase I Clinical Trial entitled “A Phase I Trial of the Safety and Immunogenicity of a Multi-epitope Folate Receptor Alpha Peptide Vaccine in Combination with Cyclophosphamide in Subjects Previously Treated for Breast or Ovarian Cancer.” A vaccine composition comprising one or more HLA Class II folate receptor alpha antigenic T-cell peptides comprising for example peptides FR30 (amino acid sequence RTELLNVCMNAKHHKEK (SEQ ID NO.: 37)), peptide FR56 (amino acid sequence QCRPWRKNACCSTNT (SEQ ID NO.: 38)), peptide FR76 (amino acid sequence KDVSYLYRFNWNHCGEMA (SEQ ID NO.: 39)); peptide FR113 (amino acid sequence LGPWIQQVDQSWRKERV (SEQ ID NO.: 40)), and peptide FR238 (amino acid sequence PWAAWPPLLSLALMLLWL (SEQ ID NO.: 41)), and may optionally be formulated with one or more similarly identified HLA Class I folate receptor alpha antigenic T-cell peptides. It is of note that one or more Class I antigenic T-cell epitopes may be contained within the sequence of a HLA Class II epitope. For example, the Class II peptide FR56 and the peptide sequence of FR238 containing the HLA Class I epitope of folate receptor alpha peptides 245-253. Composition may also be formulated with an adjuvant such as GM-CSF and/or combined with prior treatment with an immune-regulatory substance such as cyclophosphamide or denileukin diftitox (Ontak).
The plasmid-based nucleic acid immunization composition incorporates the corresponding DNA coding sequences for a folate receptor alpha HLA Class II and/or HLA Class I antigenic T-cell peptide. The DNA may include nucleic acid sequences reflecting natural NH2 and/or COOH terminal extensions which are thought to recapitulate the naturally occurring protease processing site for proteasome mediated degradation. Further, the DNA may incorporate linker regions positioned between the coding sequences of each peptide.
A matched vaccination strategy is one in which the peptides and nucleic acid encoded sequences are derived from the same cancer related TAA. The matched vaccination strategy includes administering respective compositions as a prime-and-boost, i.e., sequentially delivering one or more of a series of folate receptor alpha antigenic T-cell peptide vaccine composition(s), followed by one or more in a series of folate receptor alpha peptide derived nucleic-acid vaccine compositions (e.g., plasmid or viral). In one aspect, the peptide immunization composition is administered prior to administration of a nucleic acid immunization composition. In another aspect, the nucleic acid immunization composition is administered prior to administration of the peptide immunization composition. The peptides may be HLA-DR related and the nucleic acid may encode one or more HLA-Class I antigenic cytotoxic T-cell epitopes.
Additional cancer TAAs may be included in the vaccine compositions, including antigenic T cell peptides from insulin-like growth factor binding protein 2 (Kalli et al., Cancer Res. 68:4893, 2008), carcinoembryonic antigen (Karyampudi et al., Cancer Immunol. Immunother. 59:161, 2010), and PD-1 antagonists (Krempski et al., J. Immunol. 186:6905, 2011).
Compositions are prepared containing one or more HLA Class I and/or Class II antigenic T-cell peptide sequences from computer algorithms or testing human patient samples. The vaccine compositions may contain an adjuvant such as GM-CSF (e.g., 125 μg/injection). In addition, some patients may have a better outcome if prior to administering a vaccine composition, the patient receives cyclophosphamide, or a similar composition, orally for 1 week followed by a 1 week rest, and then another week of cyclophosphamide treatment. About 7-10 days following cyclophosphamide treatment, patients are vaccinated intradermally, intramuscularly, or ex vivo using isolated cells.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including but not limited to U.S. Patent Application No. 61/954,588, filed Mar. 17, 2014, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

## Claims

1. A chimeric nucleic acid molecule, comprising a multiplex translation initiation (MTI) sequence comprising from two to about five translation initiation sites operatively linked in frame to a nucleic acid molecule encoding a fusion protein comprising from two to about ten human epidermal growth factor receptor 2 (HER2) antigenic peptides, wherein at least one of the MTI translation initiation sites is a non-AUG translation initiation site and the MTI allows the production of more than one mole of fusion protein per mole of mRNA.

2. The chimeric nucleic acid molecule of claim 1, wherein the MTI comprises one, two, three, or four non-AUG translation initiation sites.

3. The chimeric nucleic acid molecule of claim 2, wherein the non-AUG translation initiation sites are CUG translation initiation sites.

4. The chimeric nucleic acid molecule of claim 3, wherein the MTI comprises an AUG translation initiation site downstream of the CUG translation initiation sites.

5. The chimeric nucleic acid molecule of claim 1, wherein the MTI comprises (a) a nucleic acid molecule encoding one or two nuclear localization domains located downstream of two or three CUG translation initiation sites and upstream of an AUG translation initiation site, or (b) two or three CUG translation initiation sites upstream of a nucleic acid molecule encoding one or two nuclear localization domains and no AUG translation initiation site.

6. The chimeric nucleic acid molecule of claim 1, wherein the MTI comprises a 5′-portion of a human FGF2 gene, wherein the 5′-portion of the human FGF2 gene contains an AUG translation initiation site and about 123 nucleotides to about 385 nucleotides upstream of the AUG translation initiation site that is in frame with the nucleic acid molecule encoding the fusion protein.

7. The chimeric nucleic acid molecule of claim 6, wherein the MTI further comprises from about 15 nucleotides to about 45 nucleotides downstream of the AUG translation initiation site.

8. The chimeric nucleic acid molecule of claim 6, wherein the 5′-portion of the human FGF2 gene encodes a polypeptide having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.:8-12, or encodes a polypeptide as set forth in any one of SEQ ID NOS.:8-12.

9. The chimeric nucleic acid molecule of claim 1, wherein the MTI sequence has at least 90% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOS.:1-6, 95, or 96.

10. The chimeric nucleic acid molecule of claim 1, wherein the encoded fusion protein comprises from two to about five different or same antigenic HER2 peptides, or the encoded fusion protein comprises five different antigenic HER2 peptides.

11. The chimeric nucleic acid molecule of claim 1, wherein one or more of the antigenic peptides are an HLA Class I antigenic HER2 peptide, an HLA Class II antigenic HER2 peptide, an HLA Class II antigenic HER2 peptide comprising an embedded HLA Class I antigenic HER2 peptide, or any combination thereof.

12. The chimeric nucleic acid molecule of claim 1, wherein one or more of the encoded HER2 antigenic peptides of the fusion protein comprise on the N-terminus and/or C-terminus: (a) from one to about ten junction amino acids; (b) a spacer comprising from two to about 35 amino acids; (c) a spacer comprising a (Gly4Ser)n wherein n is an integer from 1 to 5; (d) a natural cleavage site comprising from one to about ten amino acids; (e) a self-cleaving amino acid sequence; (f) an intracellular trafficking sequence; or (g) any combination thereof.

13. The chimeric nucleic acid molecule of claim 1, wherein two or more of the HER2 antigenic peptides of the fusion protein are separated by a spacer comprising a (Gly4Ser)n, wherein n is an integer from 1 to 5.

14. The chimeric nucleic acid molecule of claim 1, wherein the encoded fusion protein comprises an amino acid cleavage sequence amino-terminal to one or more of the encoded polypeptide components of the fusion protein, wherein the amino acid cleavage sequence comprises a 2A peptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), or foot-and-mouth disease virus (F2A).

15. The chimeric nucleic acid molecule of claim 1, wherein the encoded fusion protein comprises a secretion signal amino acid sequence, a membrane localization amino acid sequence, an endosome targeting sequence, a dendritic cell targeting amino acid sequence, or any combination thereof.

16. The chimeric nucleic acid molecule of claim 1, wherein each encoded HER2 antigenic peptide of the fusion protein has:
(a) at least 90% amino acid sequence identity to any one of SEQ ID NOS.:121-135;
(b) an amino acid sequence of SEQ ID NOS.:121-135;
(c) at least 90% amino acid sequence identity to any one of SEQ ID NOS.:117-120;
(d) an amino acid sequence of SEQ ID NOS.:117-120;
(e) at least 90% amino acid sequence identity to any one of SEQ ID NOS.:117-135;
(f) an amino acid sequence of SEQ ID NOS.:117-135; or
(g) any one of (a)-(f) further comprising from one to about five spacer amino acids on the N-terminus, C-terminus or both of one or more of each HER2 antigenic peptide of the fusion protein.

17. The chimeric nucleic acid molecule of claim 16, wherein the encoded fusion protein encoding any one or more of SEQ ID NOS.:117-135 further comprises a secretion signal amino acid sequence, a membrane localization amino acid sequence, an endosome targeting sequence, a dendritic cell targeting amino acid sequence, or any combination thereof.

18. The chimeric nucleic acid molecule of claim 16, wherein the encoded fusion protein encoding any one or more of SEQ ID NOS.:117-120 comprises:
(a) a VSVG signal amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:43 that is operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the fusion protein, optionally comprising a nucleic acid molecule encoding an amino acid cleavage sequence operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the VSVG signal amino acid sequence;
(b) a VSVG trafficking amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:47 or 48 that is operably linked in frame to the MTI sequence;
(c) a VSVG membrane localization amino acid sequence as encoded by nucleotides 457 to 516 of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence;
(d) a VSVG trafficking amino acid sequence and membrane localization amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence; or
(e) any one of (a)-(d) further comprising a dendritic cell targeting amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:45;
(f) any one of (a) or (c)-(e) further comprising an intracellular trafficking sequence.

19. The chimeric nucleic acid molecule of claim 1, wherein the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:115 or 116; or wherein the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:138, 140, 142-144, 146-148 or 150.

20. The chimeric nucleic acid molecule of claim 1, wherein the chimeric nucleic acid molecule comprises:
(a) an mRNA molecule;
(b) a DNA molecule; or
(c) a DNA or RNA molecule contained in a vector and operably linked to an expression control sequence.

21. The chimeric nucleic acid molecule of claim 20, wherein the vector of subpart (c) comprises a plasmid vector or a viral vector.

22. The chimeric nucleic acid molecule of claim 21, wherein the vector comprises a viral vector selected from a rhabdoviral, adenoviral, herpesviral, poxviral, or retroviral vector.

23. A composition, comprising a chimeric nucleic acid molecule of claim 1 and a therapeutically acceptable carrier or excipient.

24. A method of eliciting an immune response, comprising administering to a human subject a therapeutically effective amount of a chimeric nucleic acid molecule, wherein the chimeric nucleic acid molecule comprises a multiplex translation initiation (MTI) sequence comprising from two to about five translation initiation sites operatively linked in frame to a nucleic acid molecule encoding a fusion protein comprising from two to about ten human epidermal growth factor receptor 2 (HER2) antigenic peptides, wherein at least one of the MTI translation initiation sites is a non-AUG translation initiation site and the MTI allows the production of more than one mole of fusion protein per mole of mRNA, thereby eliciting an immune response against one or more of the HER2 antigenic peptides.

25. The method of claim 24, wherein the elicited immune response comprises a cellular immune response that treats a HER2-associated cancer.

26. The method of claim 24, further comprising administering an effective amount of an antigenic peptide immunization composition comprising at least one HER2 antigenic peptide.

27. The method of claim 26, wherein:
(a) the chimeric nucleic acid molecule and the antigenic peptide immunization compositions are administered simultaneously;
(b) the chimeric nucleic acid molecule and the antigenic peptide immunization compositions are administered sequentially;
(c) the chimeric nucleic acid molecule is administered from 1 hour to 8 weeks after the antigenic peptide immunization composition;
(d) the antigenic peptide immunization composition is administered from 1 hour to 8 weeks after the chimeric nucleic acid molecule; or
(e) any one of subparts (a) to (d) wherein the chimeric nucleic acid molecule encodes one or more of the same HER2 antigenic peptides of the antigenic peptide composition.

28. The method of claim 27, further comprising one or more additional administrations of an effective amount of the antigenic peptide immunization composition and/or the chimeric nucleic acid molecule after the first administration of the antigenic peptide immunization composition and/or the chimeric nucleic acid molecule.

29. The method of claim 24, further comprising administering an adjunctive therapy selected from the group consisting of surgery, chemotherapy, radiation therapy, antibody therapy, immunosuppressive therapy, and any combination thereof.

30. The method of claim 24, further comprising administering an adjunctive therapy selected from the group consisting of cyclophosphamide, trastuzumab, anti-PD1, anti-PDL1, anti-CTLA4, and any combination thereof.

31. The method of claim 24, wherein the MTI comprises one, two, three, or four non-AUG translation initiation sites.

32. The method of claim 31, wherein the non-AUG translation initiation sites are CUG translation initiation sites.

33. The method of claim 32, wherein the MTI comprises an AUG translation initiation site downstream of the CUG translation initiation sites.

34. The method of claim 24, wherein the MTI comprises (a) a nucleic acid molecule encoding one or two nuclear localization domains located downstream of two or three CUG translation initiation sites and upstream of an AUG translation initiation site, or (b) two or three CUG translation initiation sites upstream of a nucleic acid molecule encoding one or two nuclear localization domains and no AUG translation initiation site.

35. The method of claim 24, wherein the MTI comprises a 5′-portion of a human FGF2 gene, wherein the 5′-portion of the human FGF2 gene contains an AUG translation initiation site and about 123 nucleotides to about 385 nucleotides upstream of the AUG translation initiation site that is in frame with the nucleic acid molecule encoding the fusion protein.

36. The method of claim 35, wherein the MTI further comprises from about 15 nucleotides to about 45 nucleotides downstream of the AUG translation initiation site.

37. The method of claim 35, wherein the 5′-portion of the human FGF2 gene encodes a polypeptide having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.:8-12, or encodes a polypeptide as set forth in any one of SEQ ID NOS.:8-12.

38. The method of claim 24, wherein the MTI sequence has at least 90% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOS.:1-6, 95, or 96.

39. The method of claim 24, wherein the encoded fusion protein comprises from two to about five different or same antigenic HER2 peptides, or the encoded fusion protein comprises five different antigenic HER2 peptides.

40. The method of claim 24, wherein one or more of the antigenic peptides are an HLA Class I antigenic HER2 peptide, an HLA Class II antigenic HER2 peptide, an HLA Class II antigenic HER2 peptide comprising an embedded HLA Class I antigenic HER2 peptide, or any combination thereof.

41. The method of claim 24, wherein one or more of the encoded HER2 antigenic peptides of the fusion protein comprise on the N-terminus and/or C-terminus: (a) from one to about ten junction amino acids; (b) a spacer comprising from two to about 35 amino acids; (c) a spacer comprising a (Gly4Ser)n wherein n is an integer from 1 to 5; (d) a natural cleavage site comprising from one to about ten amino acids; (e) a self-cleaving amino acid sequence; (f) an intracellular trafficking sequence; or (g) any combination thereof.

42. The method of claim 24, wherein two or more of the HER2 antigenic peptides of the fusion protein are separated by a spacer comprising a (Gly4Ser)n, wherein n is an integer from 1 to 5.

43. The method of claim 24, wherein the encoded fusion protein comprises an amino acid cleavage sequence amino-terminal to one or more of the encoded polypeptide components of the fusion protein, wherein the amino acid cleavage sequence comprises a 2A peptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), or foot-and-mouth disease virus (F2A).

44. The method of claim 24, wherein the encoded fusion protein comprises a secretion signal amino acid sequence, a membrane localization amino acid sequence, an endosome targeting sequence, a dendritic cell targeting amino acid sequence, or any combination thereof.

45. The method of claim 24, wherein each encoded HER2 antigenic peptide of the fusion protein has:
(a) at least 90% amino acid sequence identity to any one of SEQ ID NOS.:121-135;
(b) an amino acid sequence of SEQ ID NOS.:121-135;
(c) at least 90% amino acid sequence identity to any one of SEQ ID NOS.:117-120;
(d) an amino acid sequence of SEQ ID NOS.:117-120;
(e) at least 90% amino acid sequence identity to any one of SEQ ID NOS.:117-135;
(f) an amino acid sequence of SEQ ID NOS.:117-135; or
(g) any one of (a)-(f) further comprising from one to about five spacer amino acids on the N-terminus, C-terminus or both of one or more of each HER2 antigenic peptide of the fusion protein.

46. The method of claim 45, wherein the encoded fusion protein encoding any one or more of SEQ ID NOS.:117-135 further comprises a secretion signal amino acid sequence, a membrane localization amino acid sequence, a dendritic cell targeting amino acid sequence, or any combination thereof.

47. The method of claim 45, wherein the encoded fusion protein encoding any one or more of SEQ ID NOS.:117-120 comprises:
(a) a VSVG signal amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:43 that is operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the fusion protein, optionally comprising a nucleic acid molecule encoding an amino acid cleavage sequence operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the VSVG signal amino acid sequence;
(b) a VSVG trafficking amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:47 or 48 that is operably linked in frame to the MTI sequence;
(c) a VSVG membrane localization amino acid sequence as encoded by nucleotides 457 to 516 of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence;
(d) a VSVG trafficking amino acid sequence and membrane localization amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence; or
(e) any one of (a)-(d) further comprising a dendritic cell targeting amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:45;
(f) any one of (a) or (c)-(e) further comprising an intracellular trafficking sequence.

48. The method of claim 24, wherein the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:115 or 116; or wherein the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:138, 140, 142-144, 146-148 or 150.

49. The method of claim 24, wherein the chimeric nucleic acid molecule comprises:
(a) an mRNA molecule;
(b) a DNA molecule; or
(c) a DNA or RNA molecule contained in a vector and operably linked to an expression control sequence.

50. The method of claim 49, wherein the vector of subpart (c) comprises a plasmid vector or a viral vector.

51. The method of claim 50, wherein the vector comprises a viral vector selected from a rhabdoviral, adenoviral, herpesviral, poxviral, or retroviral vector.

52. The method of claim 24, wherein the chimeric nucleic acid molecule is formulated as a composition comprising a therapeutically acceptable carrier or excipient.

53. The method of claim 24, comprising contacting the chimeric nucleic acid molecule with an immune cell ex vivo before administration, and administering to the human subject a population of immune cells containing the chimeric nucleic acid molecule.