Patent Application
20230058305
MYC is a proto-oncogene that is overexpressed in many human cancers. It plays an important role in many biological pathways related to neoplastic cell growth and proliferation. New and effective ways are required to target Myc for cancer therapeutics.
In some embodiments, disclosed herein are compositions comprising a synthetic nucleic acid sequence encoding a Plasmacytoma variant translocation 1_217 (PVT1_217) splice variant micropeptide, comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the PVT1_217 splice variant micropeptide is at least 12 amino acids in length. In some embodiments, the PVT1_217 splice variant micropeptide is at least 13 amino acids in length. In some embodiments, the PVT1_217 splice variant micropeptide is at least 14 amino acids in length. In some embodiments, disclosed herein are compositions comprising a synthetic nucleic acid sequence encoding a Plasmacytoma variant translocation 1_217 (PVT1_217) splice variant micropeptide, comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1, wherein the PVT1_217 splice variant micropeptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the PVT1_217 splice variant micropeptide comprises a maximum of 14 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is a messenger RNA. In some embodiments, the synthetic nucleic acid comprises one or more modified nucleotides. In some embodiments, the synthetic nucleic acid sequence encoding a PVT1_217 splice variant micropeptide is comprised in a vector.
In some embodiments, disclosed herein are vectors comprising: a nucleic acid sequence encoding a PVT1_217 splice variant micropeptide, wherein the PVT1_217 splice variant micropeptide is at least 12 amino acids in length, and comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the nucleic acid sequence encoding a PVT1_217 splice variant micropeptide has least 80% sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the vector comprises self-replicating RNA vector.
In some embodiments, disclosed herein are pharmaceutical compositions comprising: (i) a synthetic nucleic acid sequence encoding a Plasmacytoma variant translocation 1_217 (PVT1_217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1; and (ii) a pharmaceutically acceptable excipient. In some embodiments, the PVT1_217 splice variant micropeptide is at least 12 amino acids in length. In some embodiments, the PVT1_217 splice variant micropeptide is at least 13 amino acids in length. In some embodiments, the PVT1_217 splice variant micropeptide is at least 14 amino acids in length. In some embodiments, the PVT1_217 splice variant micropeptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the PVT1_217 splice variant micropeptide comprises a maximum of 14 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the synthetic nucleic acid sequence encoding the PVT1_217 splice variant micropeptide has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the synthetic nucleic acid is DNA. In some embodiments, the synthetic nucleic acid is a messenger RNA. In some embodiments, the synthetic nucleic acid comprises one or more modified nucleic acids. In some embodiments, the synthetic nucleic acid sequence encoding a PVT1_217 splice variant micropeptide is comprised in a vector. In some embodiments, the vector is a mammalian expression vector. In some embodiments, the vector is a lentiviral expression vector. In some embodiments, the vector comprises a promoter. In some embodiments, the promoter is inducible. In some embodiments, the pharmaceutical compositions disclosed herein further comprise a cancer cell targeting moiety.
In some embodiments, disclosed herein are methods for treating a subject having a MYC-driven cancer, the method comprising: administering to the subject a pharmaceutical composition comprising a nucleic acid sequence encoding a PVT1_217 splice variant micropeptide, comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid sequence encoding the PVT1_217 splice variant micropeptide comprises a sequence set forth in SEQ ID NO: 1. In some embodiments, the PVT1_217 splice variant micropeptide comprises 14 amino acids. In some embodiments, the PVT1_217 splice variant micropeptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the pharmaceutical composition results in reduction in cancer cell division.
Provided herein is an isolated peptide comprising an amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80% identity to SEQ ID NO: 1. In some embodiments, the isolated peptide may comprise the amino acid sequence is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.
In some embodiments, the amino acid sequence is at least 12 amino acids in length. In some embodiments, the amino acid sequence is at least 13 amino acids in length. In some embodiments, the amino acid sequence is at least 14 amino acids in length. In some embodiments, the peptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at a junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the peptide comprises a maximum of 14 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the amino acid sequence comprises at least 1 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence comprises at least 2 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence comprises at least 3 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence comprises at least 4 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence comprises at least 5 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the isolated peptide has an amino acid sequence of SEQ ID NO:1.
Provided herein is a pharmaceutical composition comprising: a Plasmacytoma variant translocation 1_217 (PVT1_217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1; and, a pharmaceutically acceptable excipient.
In some embodiments, the PVT1_217 splice variant micropeptide is at least 12 amino acids in length. In some embodiments, the PVT1_217 splice variant micropeptide is at least 13 amino acids in length. In some embodiments, the PVT1_217 splice variant micropeptide is at least 14 amino acids in length.
In some embodiments, the PVT1_217 splice variant micropeptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217. In some embodiments, the PVT1_217 splice variant micropeptide comprises a maximum of 14 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1_217.
In some embodiments, the micropeptide is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the micropeptide has a sequence of SEQ ID NO:1.
In some embodiments, the micropeptide comprises at least 1 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 2 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 3 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 4 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 5 conservative amino acid substitution within the sequence of SEQ ID NO: 1.
In some embodiments, the micropeptide is further conjugated with one or more biomolecules. In some embodiments, the biomolecule is a peptide. In some embodiments, the biomolecule is a lipid. In some embodiments, the micropeptide is further modified. In some embodiments, the modification is selected from the group consisting of: myristoylation, palmitoylation, isoprenylation, glypiation, lipolation, acylation, alkylation, amidation, phosphorylation, glycation, biotinylation, pegylation, sumoylation, ubiquitination, neddylation, or pupylation. In some embodiments, the pharmaceutical composition comprises a recombinant protein comprising the micropeptide described herein, having at least 80% sequence identity to SEQ ID NO:1.
In some embodiments, the peptide is associated with a carrier molecule. In some embodiments, the carrier molecule is a lipid.
In some embodiments, the pharmaceutical composition described herein is for use in preparing a medicament for the treatment of a cancer in a subject.
Provided herein is a method of treating cancer in a subject in need thereof, the method comprising: administering to the subject a pharmaceutical composition comprising a micropeptide having a sequence of SEQ ID NO: 1, or a sequence that is at least 80% identical to SEQ ID NO: 1.
In some embodiments, the cancer is a MYC-driven cancer.
Provided herein is the use of a composition comprising a Plasmacytoma variant translocation 1_217 (PVT1_217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1 in the treatment of a cancer in a subject.
Also provided herein is the use of a composition comprising a nucleic acid encoding Plasmacytoma variant translocation 1_217 (PVT1_217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1 in the treatment of a cancer in a subject.
The patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
Myc (avian myelocytomatosis viral oncogene homolog) is a transcription factor encoded by the oncogene MYC, and is associated with various cancers. It is estimated to contribute to at least 75% of all human cancers, including prostate, breast, colon and cervical cancers, myeloid leukemia, lymphomas, small-cell lung carcinomas, and neuroblastoma, among others. High expression of Myc can drive tumorigenesis in several tissue types. Myc is also associated with treatment resistant and lethal outcomes. The present disclosure is related to a novel finding that Myc driven cell proliferative function can be inhibited by the micropeptides disclosed and described herein, and that the micropeptides can be used for therapeutic intervention in Myc-associated cancers.
The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.
The terms “individual,” “patient,” or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly, or a hospice worker).
The term “gene,” as used herein, refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a “coding sequence” or “coding region”), optionally together with associated regulatory regions such as promoters, operators, terminators and the like, which may be located upstream or downstream of the coding sequence. As used herein, the term “polypeptide” encompasses amino acid molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to chemically modified amino acids such as amino acid analogs, naturally occurring non-protogenic amino acids such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid. In some embodiments, the at least one modified or unusual amino acid is selected from the group consisting of 2-aminoadipic acid; 3-aminoadipic acid; beta-alanine, beta-amino-propionic acid; 2-aminobutyric acid; 4-aminobutyric acid, piperidinic acid; 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2′-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylasparagine; hydroxylysine; allo-hydroxyline; 3-hydroxyproline; 4-hydroxyproline; isodemosine; allo-isoleucine; N-methylglycine, sarcosine; N-methylisoleucine; 6-N-methyllysine; N-methylvaline; norvaline; norleucine; and ornithine.
The terms “polypeptide,” “protein,” and “peptide” also encompass amino acid sequence variants of a protein or peptide. Amino acid sequence variants of the HNB polypeptides disclosed herein can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein that are not essential for function or immunogenic activity, as exemplified by variants of integral membrane proteins that lack a transmembrane sequence. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the polypeptide, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. As disclosed herein, sequences that have between about 70% and about 80%, or between about 81% and 90% or between about 91% and about 99% of amino acids that are identical or functionally equivalent to the amino acids of the HNB polypeptides disclosed herein are considered biologically functionally equivalent, provided the biological activity of the HNB polypeptide is maintained, and within the scope of the HNB polypeptides disclosed herein.
The terms “treat,” “treating,” and “treatment” is meant to include alleviating or abrogating a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself. Desirable effects of treatment can include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state and remission or improved prognosis.
The term “therapeutically effective amount” refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated. The term “therapeutically effective amount” also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or clinician.
The term “MYC-driven cancer,” refers to a cancer characterized by aberrant (typically augmented expression) expression of the c-MYC gene or the Myc protein. In some embodiments, an individual receiving therapy comprising the HNB polypeptides disclosed herein may be identified as having a susceptibility to a cancer therapy, including e.g., a MYC-dependent susceptibility to a cancer therapy or a KRas-dependent susceptibility to a cancer therapy. A MYC-driven neoplasm having a MYC-dependent susceptibility to a cancer therapy may be more susceptible to the cancer therapy than the corresponding neoplasm that lacks or displays reduced MYC expression (including e.g., where the MYC expression is conditionally controlled). A KRas-driven neoplasm having a KRas-dependent susceptibility to a cancer therapy may be more susceptible to the cancer therapy than the corresponding neoplasm that lacks or displays reduced KRas expression (including e.g., where the KRas expression is conditionally controlled). “Cancer therapy” as used herein, refers to any cancer therapy including but not limited to e.g., radiation therapy, chemotherapy, immunotherapy, and the like.
Myc tightly regulates a broad set of genes essential to growth and proliferation. In turn MYC is also tightly regulated at transcriptional, translational and post-translational levels. Myc levels are controlled by multiple mechanisms, including negative autoregulation, gene expression, mRNA, and protein stability and degradation, which all become deregulated in human cancers.
The MYC gene is found at locus 8q24.21 in a broader region on chromosome 8, which is frequently amplified in cancers. Its two paralogs, N-Myc and L-Myc, which are encoded by MYCN and MYCL genes, were respectively identified in neuroblastoma and lung cancer as tissue-specific factors. Human Myc contains several highly conserved regions that are functionally important and are organized in the same fashion among the three Myc paralogs, including: a largely unstructured N-terminal transactivation domain (TAD) and an intrinsically disordered C-terminal region comprising the basic, helix-loop-helix, leucine zipper (bHLHLZ) dimerization, and DNA-binding domains. Mechanisms that account for Myc deregulation include: amplifications or chromosomal translocations of the MYC locus that provoke its exacerbated expression, MYC mRNA destabilization through both direct and indirect regulatory events, and alteration in Myc protein turnover rate. The latter is due to either alterations in Myc protein stability normally dependent on Myc's phosphorylation status but caused by mutations in key phosphorylation sites or alterations of expression of proteins that are involved in Myc's post-translational modifications.
The human MYC gene is approximately 6 kilobases long. It contains three exons: a large non-coding exon 1, followed by coding exons 2 and 3. There are four distinct promoters, P0, P1, P2, and P3 that drive MYC transcription. There are two major translation start codons (CTG, and ATG), from which two universally expressed Myc proteins arise, and there are two polyadenylation signals and several DNAse 1-hypersensitive sites. P0 transcripts start at multiple initiation sites. P1 and P2 are the two major classical TATA-containing promoter start sites located at the 5′ end of exon 1, with greater than three-quarters of MYC transcripts originating from the P2 promoter. The MYC promoter region is regulated by a large number of signaling pathways, transcription factors, cis-regulatory elements, chromatin remodeling, and by its auto-suppression.
Plasmacytoma variant translocation 1 (PVT1) is a ‘long non-coding RNA’ transcribed from adjacent to the oncogene c-MYC, and has been shown to co-operate with c-Myc by stabilizing its protein product in 8q24 gain cancers. Long noncoding RNAs (lncRNAs) are a class of RNA transcripts which are longer than 200 nucleotides, evolutionarily conserved, and devoid of protein-coding potential. Recently, emerging studies have shown that lncRNAs are frequently deregulated in various tumors and exert multiple functions in a wide range of biological processes, such as proliferation, apoptosis, cell cycle arrest, cell migration and invasion. PVT1 was originally identified as a cluster of breakpoints for viral integration and translocation in T- and B-cell lymphomas. The PVT1 locus is syntenically conserved between the human and mouse. Although PVT1 is a mutational hotspot and frequently overexpressed in cancers, its role in tumorigenesis is poorly understood.
It has been shown that 98% of the 8q24 amplicons in a subset of cancers with 8q24 gain/amplification contain both MYC and PVT1. A tissue microarray analysis of 8 primary tumors (lung, colon, rectum, stomach, esophagus, liver, kidney, and breast) revealed a high correlation between PVT1 RNA and MYC protein expression in these primary tumors. These data provided strong evidence for PVT1/MYC co-operation in different human cancers.
The dependency of MYC-driven cancer cells on PVT1 was examined. The driver mutation in the colorectal cancer cell line HCT116 is a mutant β-catenin gene. A stable β-catenin protein recruits TCF4 to upregulate MYC transcription in these cells. Using the CRISPR/Cas9 system, PVT1 was deleted in these cells. PVT1-deficient HCT116 cells are impaired in their tumorigenic potential compared to their wild-type controls. Importantly, it was noticed ˜50% reduction in MYC protein levels in these PVT1-deficient cells. Thus, multiple lines of evidence suggest that PVT1 plays a crucial role in augmenting MYC protein in 8q24 gain cancers. Similarly, a recent study implicates another frequently amplified oncogenic lncRNA called FAL1 at 1q21 in the stabilization of BMI1 in ovarian cancers, suggesting a broader role of lncRNAs in the fine tuning of oncoproteins in cancer. A novel regulation of MYC via the lncRNA PVT1 was identified, at least in cancers where these loci are co-amplified.
PVT1 splice variants can regulate Myc. Targeting PVT1 in 8q24 gain cancers provides a means to target Myc, an otherwise notoriously undruggable candidate in cancers. Disclosed herein is the surprising finding that micro-peptides generated by PVT1, including micro-peptide fusion proteins, acts as a tumor suppressor and reduce Myc level in human cancers.
Micro-peptides (also referred to as micro-proteins) are polypeptides with a length of less than 100-150 amino acids that are encoded by short open reading frames (sORFs). In this respect, they differ from many other active small polypeptides, which are produced through the posttranslational cleavage of larger polypeptides. In terms of size, micro-peptides are considerably shorter than “canonical” proteins, which have an average length of 330 and 449 amino acids in prokaryotes and eukaryotes, respectively. Micro-peptides lack an N-terminal signaling sequences, suggesting that they are likely to be localized to the cytoplasm. However, some micro-peptides have been found in other cell compartments, as indicated by the existence of transmembrane micro-peptides. They are found in both prokaryotes and eukaryotes. The sORFs from which micro-peptides are translated can be encoded in 5′ UTRs, small genes, or polycistronic mRNAs.
Described herein are micropeptides that can inhibit Myc expression. In some embodiments, the micropeptides of the present disclosure are encoded by the PVT1 locus. In some embodiments, the micropeptides of the present disclosure are encoded by a splice variant of PVT1 locus. In some embodiments, the micropeptides of the present disclosure are encoded by a splice variant of PVT1 locus, designated as PVT1_217.
The micropeptides disclosed herein in some embodiments, are referred to as HONEYBADGER, HNB, HNB polypeptides, HNB peptides, HNB fragments, HNB polypeptide fragments, HNB micro-peptides, HNB mimetics, or HNB fusion proteins. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence MKTQLGAVKGFLHV (SEQ ID NO: 1). In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is 80% identical to SEQ ID NO: 1. In some aspects, the HNB polypeptide comprises a sequence that fits between the dimer interface of KRAS. In some aspects, the HNB polypeptide comprises a sequence that binds at the region of amino acid residues 147 to 156 of KRAS. In some aspects, the HNB polypeptide binds at the region of amino acid residues KTRQGVDDAF (SEQ ID NO:2) of KRAS. In some embodiments, the HNB polypeptide binds part of the region of amino acid residues 147 to 156 of KRAS, such as from 148 to 156, from 149 to 156, from 150 to 156, from 151 to 156, from 152 to 156, from 153 to 156, from 154 to 156, from 155 to 156, from 147 to 155, from 147 to 154, from 147 to 153, from 147 to 152, from 147 to 151, from 147 to 150, from 146 to 149, from 147 to 148, from 154 to 155, from 154 to 156, from 153 to 154, from 153 to 155, from 152 to 156, or from 151 to 156. In some embodiments, the binding of the HNB polypeptide disrupts formation of a salt bridge between residue D154 from a KRAS monomer and R161 from an opposing KRAS monomer. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is 96% identical to SEQ ID NO:1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is 97% identical to SEQ ID NO: 1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is 98% identical to SEQ ID NO: 1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is 99% identical to SEQ ID NO: 1. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least one conservative amino acid substitution. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least two conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least three conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least four conservative amino acid substitution. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least five conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least six conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least seven conservative amino acid substitution. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least eight conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least nine conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:1 with at least ten conservative amino acid substitutions. In some embodiments, disclosed herein are HNB polypeptides comprising SEQ ID NO:1 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions in SEQ ID NO: 1. In one aspect, the micropeptide of the present disclosure can be used for a therapeutic or pharmaceutical composition for treating cancer.
In some embodiments, the HNB polypeptides disclosed herein comprise HNB fusion proteins. In some embodiments, HNB fusion proteins comprise a HNB polypeptide and a heterologous polypeptide. In some embodiments, the heterologous polypeptide is selected from the group consisting of calmodulin, polyglutamine, E-tag, FLAG, HA, His, Myc, S-tag, SBP-tag, Softag 1, Softag3, Strep-tag, TC-tag, V5, VSV, Xpress, Isopeptag, SpyTag, SnoopTag, BCCP, GST, GFP, Halo-tag, MBP, Nus-tag, Thioredoxin, albumin, an antibody, Fc domain, and combinations thereof. In some embodiments, the heterologous polypeptide is an Fc domain. In some embodiments, the heterologous polypeptide targets the HNB fusion protein to a specific cell or tissue. In some embodiments, the heterologous polypeptide is fused to the N-terminus of the HNB polypeptide. In some embodiments, the heterologous polypeptide is at the C-terminus of the HNB polypeptide.
In some embodiments, the HNB fusion proteins comprise a HNB polypeptide and an exemplary sequence of a heterologous protein provided in Table 1 below:
In some embodiments, the HNB fusion polypeptides disclosed herein employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. In some embodiments, the HNB fusion polypeptides disclosed comprise immunologically active domains, such as an antibody epitope, to facilitate purification of the HNB fusion polypeptide. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals or transmembrane regions. Additional HNB fusion polypeptides as disclosed herein can comprise a cell-penetrating peptide linked to a polypeptide to promote uptake of the polypeptide by the cell.
In some embodiments, provided herein are polynucleotides encoding any one of the above HNB polypeptides. In some embodiments, provided herein are modified polypeptides comprising any one of the above HNB polypeptides. In some embodiments, the modification is selected from the group consisting of a glycosylation and a phosphorylation. In some embodiments, the modification is selected from the group consisting of: myristoylation, palmitoylation, isoprenylation, glypiation, lipolation, acylation, akylation, amidation, phosphorylation, glycation, biotinylation, pegylation, sumoylation, ubiquitination, neddylation, or pupylation. Modifications also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms. Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond. In some embodiments, there are provided compositions comprising any one of the above HNB fusion polypeptides, the HNB polynucleotides, or any one of the above modified polypeptides, and an excipient. In some embodiments, the excipient comprises at least one of the group consisting of maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and polyoxyethylene-sorbitan monooleate.
In some embodiments, the composition comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic. In some embodiments, there are provided any one of the above HNB fusion polypeptides, the above HNB polynucleotides, any one of the above modified polypeptides, or any one of the above compositions for use as a medicament. In some embodiments, there are provided any one of the above HNB fusion polypeptides, the above HNB polynucleotides, any one of the above modified polypeptides, or any one of the above compositions for preparation of a medicament for treatment of cancer. In some embodiments, there are provided any one of the above HNB fusion polypeptides, the above HNB polynucleotides, any one of the above modified polypeptides, or any one of the above compositions for use in treatment of cancer. In some embodiments, the cancer is a MYC driven cancer. In some embodiments, the cancer is a KRas driven cancer. In some embodiments, the cancer is selected from the group consisting of Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers (e.g., Kaposi Sarcoma, Lymphoma, etc.), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer (Extrahepatic), Bladder Cancer, Bone Cancer (e.g., Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma, etc.), Brain Stem Glioma, Brain Tumors (e.g., Astrocytomas, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniopharyngioma, Ependymoma, etc.), Breast Cancer (e.g., female breast cancer, male breast cancer, childhood breast cancer, etc.), Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor (e.g., Childhood, Gastrointestinal, etc.), Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Central Nervous System (e.g., Atypical Teratoid/Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumor, Lymphoma, etc.), Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Duct (e.g., Bile Duct, Extrahepatic, etc.), Ductal Carcinoma In Situ (DCIS), Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer (e.g., Intraocular Melanoma, Retinoblastoma, etc.), Fibrous Histiocytoma of Bone (e.g., Malignant, Osteosarcoma, ect.), Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor (e.g., Extracranial, Extragonadal, Ovarian, Testicular, etc.), Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis (e.g., Langerhans Cell, etc.), Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors (e.g., Pancreatic Neuroendocrine Tumors, etc.), Kaposi Sarcoma, Kidney Cancer (e.g., Renal Cell, Wilms Tumor, Childhood Kidney Tumors, etc.), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia (e.g., Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell, etc.), Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer (e.g., Non-Small Cell, Small Cell, etc.), Lymphoma (e.g., AIDS-Related, Burkitt, Cutaneous T-Cell, Hodgkin, Non-Hodgkin, Primary Central Nervous System (CNS), etc.), Macroglobulinemia (e.g., Waldenstrom, etc.), Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia (e.g., Chronic (CML), etc.), Myeloid Leukemia (e.g., Acute (AML), etc.), Myeloproliferative Neoplasms (e.g., Chronic, etc.), Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer (e.g., Lip, etc.), Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer (e.g., Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor, etc.), Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (e.g., Ewing, Kaposi, Osteosarcoma, Rhabdomyosarcoma, Soft Tissue, Uterine, etc.), Sezary Syndrome, Skin Cancer (e.g., Childhood, Melanoma, Merkel Cell Carcinoma, Nonmelanoma, etc.), Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer (e.g., with Occult Primary, Metastatic, etc.), Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Ureter and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer (e.g., Endometrial, etc.), Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, Wilms Tumor, and the like.
In some embodiments, provided herein are any one of the above HNB fusion polypeptides, the above HNB polynucleotides, any one of the above modified polypeptides, or any one of the above compositions for use in treatment of carcinoma. In some embodiments, the carcinoma is selected from the group consisting of acinar carcinoma, actinic cell carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, adrenocortical carcinoma, alveolar carcinoma, ameloblastic carcinoma, apocrine carcinoma, basal cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, cholangiocellular carcinoma, chorionic carcinoma, clear cell carcinoma, colloid carcinoma, cribriform carcinoma, ductal carcinoma in situ, embryonal carcinoma, carcinoma encuirasse, endometrioid carcinoma, epidermoid carcinoma, carcinoma ex mixed tumor, carcinoma ex pleomorphic adenoma, follicular carcinoma of thyroid gland, hepatocellular carcinoma, carcinoma in situ, intraductal carcinoma, Hurthle cell carcinoma, inflammatory carcinoma of the breast, large cell carcinoma, invasive lobular carcinoma, lobular carcinoma, lobular carcinoma in situ (LCIS), medullary carcinoma, meningeal carcinoma, Merkel cell carcinoma, mucinous carcinoma, mucoepidermoid carcinoma, nasopharyngeal carcinoma, non-small cell carcinoma, non-small cell lung carcinoma (NSCLC), oat cell carcinoma, papillary carcinoma, renal cell carcinoma, scirrhous carcinoma, sebaceous carcinoma, carcinoma simplex, signet-ring cell carcinoma, small cell carcinoma, small cell lung carcinoma, spindle cell carcinoma, squamous cell carcinoma, terminal duct carcinoma, transitional cell carcinoma, tubular carcinoma, verrucous carcinoma, and the like.
In some embodiments, the treatment reduces at least one symptom of a cancer. In some embodiments, the treatment (a) prevents the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting development of a disease and/or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and/or symptom(s). Those in need of treatment can include those already inflicted (e.g., those with cancer, e.g. those having tumors) as well as those in which prevention is desired (e.g., those with increased susceptibility to cancer; those with cancer; those suspected of having cancer; etc.).
In some embodiments, disclosed herein are cells expressing HNB polypeptides, including HNB fusion polypeptides as disclosed herein. In some embodiments, cell is a mammalian cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a bacterial cell. Examples of cells for expressing the HNB fusion polypeptides disclosed herein include, but are not limited to, a CHO cell, a ExpiCHO-S cell, a CHO DG44 cell, a CHO-K1 cell, a myeloma cell, a hybridoma cell, a NSO cell, a GS-NSO cell, aHEK293 cell, a HEK293T cell, aHTEK293E cell, a HEK293-6E cell, a HEK293F cell, and a per.C6 cell. In some embodiments, the cell is a CHO cell. In some embodiments, the cell is a myeloma cell. In some embodiments, the cell is selected from the group consisting of an E. coli cell, a P. mirabilis cell, a P. putidas cell, a B. brevis cell, a B. megaterium cell, a B. subtilis cell, a L. paracasei cell, a S. lividans cell, a Y. lipolytica cell, a K. lactis cell, a P. pastoris cell, a S. cerevisiae cell, a A. niger var. awamori cell, a A. oryzae cell, a L. tarentolae cell, a T. ni larvae cell, a S. frugiperda cell, a Drosophila S2 cell, a S. frugiperda SF9 cell, a T. ni cell, and a SfSWT-1 mimic cell
In some embodiments, provided herein are therapeutic compositions comprising a peptide or a polypeptide that comprises a micropeptide, having an sequence of amino acids that is at least 80% identical to the sequence MKTQLGAVKGFLHV (SEQ ID NO: 1). The therapeutic composition can be a vaccine, a prophylactic, or combined with other therapeutics for treating cancer.
Described herein are therapeutics comprising a peptide or a polypeptide that is less than 100 amino acids long, comprising an amino acid sequence of the micropeptide MKTQLGAVKGFLHV (SEQ ID NO: 1). In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 50 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 40 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 30 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 20 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 19 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 18 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 17 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 16 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 15 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 14 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1.
In some embodiments, the micropeptide comprises at least 1 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 2 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 3 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 4 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 5 conservative amino acid substitution within the sequence of SEQ ID NO: 1.
In some embodiments, the micropeptide is further conjugated with one or more biomolecules. In some embodiments, the biomolecule is a peptide. In some embodiments, the biomolecule is a lipid. In some embodiments, the micropeptide is further modified. In some embodiments, the modification is selected from the group consisting of: myristoylation, palmitoylation, isoprenylation, glypiation, lipolation, acylation, alkylation, amidation, phosphorylation, glycation, biotinylation, pegylation, sumoylation, ubiquitination, neddylation, or pupylation.
In some embodiments, the peptide is associated with a carrier molecule. In some embodiments, the carrier molecule is a lipid.
In some embodiments, the pharmaceutical composition described herein is for use in preparing a medicament for the treatment of a cancer in a subject.
The selection of peptides can be guided by the given tissue to avoid side effects. The selection may be dependent on the specific type of cancer, the status of the disease, earlier treatment regimens, the immune status of the patient, and, of course, the HLA-haplotype of the patient. Furthermore, the vaccine according to the disclosure can contain individualized components, according to personal needs of the particular patient. Examples include varying the amounts of peptides according to the Myc expression in the particular patient, unwanted side-effects due to personal allergies or other treatments, and adjustments for secondary treatments following a first round or scheme of treatment.
In some embodiments, the pharmaceutical composition comprises about 1-50,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-40,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-30,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-20,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-10,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-5,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-1,000 ug of the micro-peptide.
In some embodiments, the pharmaceutical compositions described herein comprise a nucleic acid that encodes a peptide or a polypeptide comprising a sequence that is at least 80% identical to SEQ ID NO: 1.
In some embodiments, the nucleic acid is DNA.
In some embodiments, the nucleic acid is RNA.
In some embodiments, the nucleic acid is messenger RNA (mRNA).
In some embodiments, the nucleic acid is an mRNA comprising a sequence encoding a sequence comprising MKTQLGAVKGFLHV (SEQ ID NO:1). In some embodiments, the mRNA comprises a sequence encoding a peptide that is at least 80% identical to SEQ ID NO: 1. In some embodiments the mRNA comprises a sequence: AUGAAGACCCAGCUGGGCGCCGUGAAGGGCUUCCUGCACGUG, or a sequence that is at least 95% identical to the same. In some embodiments the mRNA comprises more than one copy of the sequence encoding a peptide having the sequence denoted in SEQ ID NO: 1. In some embodiments, the mRNA comprises a concatemer of a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more sequences encoding a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more peptides each having the sequence denoted in SEQ ID NO: 1, or a peptide sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the concatemer comprises sequences for self-cleavable elements such as P2A, T2A or E2A in between two sequences each encoding the SEQ ID NO: 1 or a peptide sequence that is at least 80% identical to SEQ ID NO: 1.
Typically, in eukaryotic organisms, mRNA processing comprises the addition of a “cap” on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end. A typical cap is a 7-methylguanosine cap, which is a guanosine that is linked through a 5′-5′-triphosphate bond to the first transcribed nucleotide. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3′ end of the mRNA molecule. The presence of this “tail” serves to protect the mRNA from exonuclease degradation. mRNA is translated by the ribosomes into a series of amino acids that make up a protein.
mRNAs according to the present disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.
According to various embodiments, the present disclosure may be comprise a therapeutic or pharmaceutical composition comprising synthesized mRNA of a variety of lengths. In some embodiments, the present disclosure comprise in vitro synthesized mRNA of or greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length. In some embodiments, the present disclosure may comprise a therapeutic composition comprising a synthesized mRNA ranging from about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length. For example, typical mRNAs may be about 1 kb to about 5 kb in length. More typically, the mRNA will have a length of about 1 kb to about 3 kb. However, in some embodiments, the mRNA in the composition of the disclosure is much longer (greater than about 20 kb). In some embodiments, the present disclosure may comprise mRNA containing one or more modifications that typically enhance stability. In some embodiments, one or more modifications are selected from modified nucleotide, modified sugar phosphate backbones, 5′ and/or 3′ untranslated region.
Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the mRNA. A modified mRNA according to the disclosure can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, antibody encoding mRNAs (e.g., heavy chain and light chain encoding mRNAs) may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 3-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosure of which is included here in its full scope by reference.
Typically, mRNA synthesis includes the addition of a “cap” on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.
Thus, in some embodiments, mRNAs include a 5′ cap structure. A 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.
In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region. In some embodiments, a 5′ untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5′ untranslated region may be between about 50 and 500 nucleotides in length.
In some embodiments, a 3′ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3′ untranslated region comprises a poly A tail, that may be between 50 and 500 nucleotides in length or longer. In some embodiments, the poly A tail may be 50-250 nucleotides long.
There are various methods of delivery of a nucleic acid as a therapeutic into an organism, e.g., a human. Likewise, there are various methods of delivering a protein or a peptide into an organism. In some embodiments the therapeutic or pharmaceutical composition of the present disclosure is delivered to a subject in need thereof, wherein the therapeutic or pharmaceutical composition comprises a peptide, a polypeptide or a nucleic acid encoding the peptide or polypeptide, wherein the peptide or the polypeptide comprises a sequence as set forth in SEQ ID NO: 1, or a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 1.
For instance, the nucleic acid can be delivered directly, as “naked DNA”, or “naked mRNA”. The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253, which is herein incorporated by reference for purposes of describing ballistic delivery administration. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles. The nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for instance, in Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833; Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987) and others, which are hereby incorporated by reference for purposes of describing lipid-mediated gene delivery methods. In some embodiments, pharmaceutical formulations comprising the HNB polypeptides disclosed herein are made to be compatible with a particular local, regional or systemic administration or delivery route. Thus, pharmaceutical formulations include carriers, diluents, or excipients suitable for administration by particular routes. Specific non-limiting examples of routes of administration for compositions herein are parenteral, e.g., intravenous, intra-arterial, intradermal, intramuscular, subcutaneous, intra-pleural, transdermal (topical), transmucosal, intra-cranial, intra-spinal, intra-ocular, rectal, oral (alimentary), mucosal administration, and any other formulation suitable for the treatment method or administration protocol.
In some embodiments, pharmaceutical solutions or suspensions for parenteral application include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. In some embodiments, pH is adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
Pharmaceutical formulations for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), or suitable mixtures thereof. Fluidity is maintained, in some embodiments, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars; polyalcohols such as mannitol or sorbitol; or sodium chloride, in some embodiments, are included in the composition. In some cases, also included is an agent which delays absorption, in some embodiments, for example, aluminum monostearate or gelatin prolongs absorption of injectable compositions.
In some embodiments, sterile injectable formulations are prepared by incorporating the HNB polypeptides disclosed herein in the required amount in an appropriate solvent with one or more of the above ingredients. Generally, dispersions are prepared by incorporating the HNB polypeptides disclosed herein into a sterile vehicle containing a basic dispersion medium and any other ingredient. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include, for example, vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously prepared solution thereof.
For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. In some embodiments, transmucosal administration is accomplished through the use of nasal sprays, inhalation devices (e.g., aspirators) or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, creams or patches. In some embodiments, the pharmaceutical formulations are prepared with carriers that protect against rapid elimination from the body, such as a controlled release formulation or a time delay material such as glyceryl monostearate or glyceryl stearate. The formulations, in some embodiments, are also delivered using articles of manufacture such as implants and microencapsulated delivery systems to achieve local, regional or systemic delivery or controlled or sustained release.
In some embodiments, also disclosed herein are nanoparticle-polypeptide complexes comprising a HNB polypeptide as disclosed herein in association with a nanoparticle, wherein the HNB polypeptide is modified by the addition of a chemical moiety that facilitates cellular uptake of the complex. The nanoparticle may be a lipid-based nanoparticle, a superparamagnetic nanoparticle, a nanoshell, a semiconductor nanocrystal, a quantum dot, a polymer-based nanoparticle, a silicon-based nanoparticle, a silica-based nanoparticle, a metal-based nanoparticle, a fullerene or a nanotube. The nanoparticle may be a lipid-based nanoparticle. The lipid-based nanoparticle may be a liposome, a neutral liposome, a DOPC liposome or a DOTAP:cholesterol vesicle. The liposome may be a DOPC liposome. As used in cancer therapy, liposomes take advantage of the increased fenestrations in the cancer neo vasculature to enhance liposome concentration at tumor sites. In certain embodiments, the nanoparticle is a superparamagnetic nanoparticle. Superparamagnetic nanoparticles ranging in diameter from about 10 to 100 nm are small enough to avoid sequestering by the spleen, but large enough to avoid clearance by the liver. Particles this size can penetrate very small capillaries and can be effectively distributed in body tissues. In certain embodiments, the nanoparticle is a superparamagnetic nanoparticle, and the nanoparticle-polypeptide complex is within a liposome or a DOTAP:cholesterol vesicle. The liposome may be a DOPC liposome. The chemical moiety may be a fatty acid (e.g., a C4-C18 fatty acid, stearate or myristate). The chemical moiety may be a cell penetrating peptide. The cell penetrating peptide may be derived from HIV Tat, herpes virus VP22, or the Drosophila Antennapedia homeobox gene product. In some embodiments, the HNB polypeptides can be targeted to specific tissues and cells. For example, the nanoparticle-polypeptide complexes comprising a HNB polypeptide as disclosed herein can be conjugated to a cell targeting moiety. The targeting moiety can be, but is not limited to, a protein, peptide, lipid, steroid, sugar, carbohydrate or synthetic compound. Cell targeting moieties such as ligands recognize and bind to their cognate receptors on the surface of cells. Similarly, an antibody can act as cell targeting moieties by recognizing a cognate antigen on a cell surface. Targeted nanoparticle-polypeptide complexes can enhance the specificity of disease treatment and increase the amount of therapeutic agent entering a targeted cell.
Liposomes can be variously used to deliver nucleic acids or peptides. The peptides disclosed herein may also be administered via liposomes, which target the peptides to a particular cells tissue, such as lymphoid tissue. Liposomes are also useful in increasing the half-life of the peptides. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome. Thus, liposomes filled with a desired peptide of the disclosure can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions. Liposomes for use in the disclosure are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), USA; U.S. Pat. No. 4,235,871 and others, which are hereby incorporated by reference for purposes of describing methods for preparing liposomes. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated. Therapeutically effective amounts or dosages of the HNB polypeptides disclosed herein and pharmaceutical formulations comprising the HNB polypeptides disclosed herein are contemplated to include dosages of 0.01 mg to 20 mg, for example, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9 mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7 mg, 6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 10.1 mg, 10.2 mg, 10.3 mg, 10.4 mg, 10.5 mg, 10.6 mg, 10.7 mg, 10.8 mg, 10.9 mg, 11 mg, 11.1 mg, 11.2 mg, 11.3 mg, 11.4 mg, 11.5 mg, 11.6 mg, 11.7 mg, 11.8 mg, 11.9 mg, 12 mg, 12.1 mg, 12.2 mg, 12.3 mg, 12.4 mg, 12.5 mg, 12.6 mg, 12.7 mg, 12.8 mg, 12.9 mg, 13 mg, 13.1 mg, 13.2 mg, 13.3 mg, 13.4 mg, 13.5 mg, 13.6 mg, 13.7 mg, 13.8 mg, 13.9 mg, 14 mg, 14.1 mg, 14.2 mg, 14.3 mg, 14.4 mg, 14.5 mg, 14.6 mg, 14.7 mg, 14.8 mg, 14.9 mg, 15 mg, 15.1 mg, 15.2 mg, 15.3 mg, 15.4 mg, 15.5 mg, 15.6 mg, 15.7 mg, 15.8 mg, 15.9 mg, 16 mg, 16.1 mg, 16.2 mg, 16.3 mg, 16.4 mg, 16.5 mg, 16.6 mg, 16.7 mg, 16.8 mg, 16.9 mg, 17 mg, 17.1 mg, 17.2 mg, 17.3 mg, 17.4 mg, 17.5 mg, 17.6 mg, 17.7 mg, 17.8 mg, 17.9 mg, 18 mg, 18.1 mg, 18.2 mg, 18.3 mg, 18.4 mg, 18.5 mg, 18.6 mg, 18.7 mg, 18.8 mg, 18.9 mg, 19 mg, 19.1 mg, 19.2 mg, 19.3 mg, 19.4 mg, 19.5 mg, 19.6 mg, 19.7 mg, 19.8 mg, 19.9 mg, or 20 mg. Therapeutically effective amounts or dosages, in some cases, are contemplated to include dosages of 0.1 mg to 2.0 mg.
In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The concentration of peptides of the disclosure in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Pharmaceutical formulations include “pharmaceutically acceptable” and “physiologically acceptable” carriers, diluents or excipients. The terms “pharmaceutically acceptable” and “physiologically acceptable” include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration to a mammal, for example a human. In some embodiments, such formulations are contained in a liquid, e.g., emulsion, suspension, syrup or elixir; or solid form, i.e., tablet (e.g., coated or uncoated, immediate, delayed, continuous, or pulsatile release), capsule (e.g., hard or soft, immediate, delayed, continuous, or pulsatile release), powder, granule, crystal, or microbead. In some embodiments, supplementary compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) are also incorporated into the formulations.
Provided herein are methods for treating a subject in need thereof, the method comprising administering the subject a pharmaceutical composition comprising a peptide comprising an amino acid sequence set forth in SEQ ID NO: 1, or a sequence that is at least 80% identical to SEQ ID NO:1.
In some embodiments, provided herein are methods for treating a subject in need thereof, the method comprising administering the subject a pharmaceutical composition comprising a nucleic acid encoding a peptide comprising an amino acid sequence set forth in SEQ ID NO: 1, or a sequence that is at least 80% identical to SEQ ID NO:1.
Pharmaceutical compositions comprising the peptides disclosed herein may be administered to an individual already suffering from cancer. Pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. The compositions may be administered at the site of surgical excision to induce a local immune response to the tumor. The disclosure provides compositions for parenteral administration which comprise a solution of the peptides and vaccine compositions are dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. For solid compositions, conventional or nanoparticle nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and others. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the disclosure, and more preferably at a concentration of 25%-75%. For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included as desired, as with, e.g., lecithin for intranasal delivery.
The HNB polypeptides disclosed herein may also be combined with therapy designed to eliminate cancer cells present in a tumor, e.g. in a combination therapy with various other conventional methods of treating cancer cells, for example chemotherapy, radiation therapy, etc. Chemotherapeutic agents may include, for example, temozolomide, protein-bound paclitaxel, romidepsin. cyclophosphamide, vincristine, doxorubicin, methotrexate, ifosfamide, etoposide, and cytarabine (CODOX-M/IVAC) plus rituximab; rituximab plus etoposide, prednisone, vincristine (Oncovin), and doxorubicin (R-EPOCH) and rituximab plus cyclophosphamide, vincristine, doxorubicin (Adriamycin), and dexamethasone (R-Hyper CVAD). In some embodiments, the chemotherapeutic agent is selected from the group consisting of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar (Tositumomab and I 131 Iodine Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CeeNU (Lomustine), Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cometriq (Cabozantinib-S-Malate), COPP, COPP-ABV, Cosmegen (Dactinomycin), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine, Liposomal, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin Diftitox, Denosumab, Dinutuximab, DepoCyt (Liposomal Cytarabine), DepoFoam (Liposomal Cytarabine), Dexrazoxane Hydrochloride, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A (Recombinant Interferon Alfa-2b), Iodine 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Liposomal Cytarabine, Lomustine, Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megace (Megestrol Acetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized, Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilotinib, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, OEPA, Ofatumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), Rituximab, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Ruxolitinib Phosphate, Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synovir (Thalidomide), TAC, Tafinlar (Dabrafenib), Talc, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thiotepa, Toposar (Etoposide), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and I 131 Iodine Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), VAMP, Vandetanib, Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), XELIRI, Xeloda (Capecitabine), XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), and Zytiga (Abiraterone Acetate). An effective amount of such agents can readily be determined by routine experimentation, as can the most effective and convenient route of administration and the most appropriate formulation.
Further to the surprising and unexpected finding that Honeybadger has a positive role as a tumor suppressor, it was also noted in the observations depicted herein that the PVT1 locus plays a significant role in cancer, owing to the instability of the locus. It was also clear from our studies depicted herein that the Honeybadger is lost in patient's cancer cells. In one aspect, various methods of introducing HNB peptides or polypeptides, or nucleic acids encoding the same are provided herein in the sections above.
In some aspects, various methods of reducing the instability of the PVT1 locus are contemplated herein. In some embodiments, methods of preventing PVT1 breakpoint fusions are contemplated. In some embodiments, methods of preventing PVT1 chromosomal fusions are contemplated. In some embodiments, mechanisms of suppressing the chromosomal breakage and repair machinery of a cell in a spatio-temporal manner guided to the PVT1 locus is contemplated. In some embodiments, mechanisms for suppression of inter-chromosomal fusion involving the PVT1 locus is contemplated. In some embodiments, mechanisms for suppression of intra-chromosomal fusion involving the PVT1 locus is contemplated. In some embodiments, mechanisms for preventing extrachromosomal DNA formation fusion involving a nucleic acid sequence found within the PVT1 locus is contemplated.
An extrachromosomal DNA formed as a result of breakage at or within the PVT1 locus may be 10, 20, 30, 40, 50, 100, 1000, 10,000 nucleotides in length. An inhibitory RNA mediated decay of an extrachromosomal nucleic acid (ecDNA) comprising the 5′ region of PVT-1 may be contemplated herein. In some embodiments, a site-directed or sequence-targeted approach to prevent formation of extrachromosomal DNA comprising the PVT-1 locus may be contemplated.
A previously undescribed PVT1 splice variant that can regulate Myc protein was identified. Using the ENSEMBL database, PVT1 splice variants were analyzed. siRNAs were designed to target specific PVT1 splice variants and tested on MSTO-211H cell line for alteration in Myc expression levels. It was found that the siRNA dubbed Ex9 (si_Ex9) increased the expression of Myc protein in the MSTO-211H cell line compared to control siRNA (si_ctrl) or compared to si_C2 (an siRNA specific for CircPVT1, where CircPVT1 is a splice variant previously reported to upregulate Myc expression) (
A 3′RACE of PVT1_217 was performed to confirm its genomic co-ordinates (
Small inhibitory RNA was then generated against the PVT1ts splice variant. The si_E9, was shown to target a unique sequence in the exon 4 of PVT1ts (
A close inspection of PVT1ts revealed a short open reading frame (shORF) at the junction of Exon3 and 4. This shORF codes for a 14 amino acid micro-peptide (lclquery_280280,
To determine the function of the HNB, the Hi- and Lo-Myc cell lines were transfected with lentiviral vectors expressing GFP, HNB or HNB where the start site has been mutated (HNB(ATG>TGA)). All of the 8 cell lines where HNB is overexpressed (but not the ones which are transfected with GFP or HNB(ATG>TGA)) failed to proliferate, suggesting a role for HNB as a tumor suppressor (
In addition, an inducible lentiviral system was developed, in which the transgene could be expressed under a Doxycycline inducible promoter. The system was verified by inserting eGFP as the transgene and by flow analysis of GFP+ cells in absence and presence of doxycycline as shown in
In order to determine the mechanism by which HNB may act as a tumor suppressor, a computational analysis was carried out using the predicted structure of HNB (
In order to comprehensively determine the frequency of PVT1 gene fusions in human cancer, an analysis of gene fusion data from transcriptomic studies (RNA-seq) on the cancer cell line encyclopedia (CCLE) dataset was conducted. Strikingly, the analysis demonstrated that PVT1 is the most frequent participant in gene fusions in the entire CCLE dataset (
In this comprehensive analysis of PVT1 fusions, some remarkable observations were made. First, most PVT1 gene fusions were not frame-retaining, indicating that these fusions cannot form chimeric oncoproteins, unlike other well-characterized gene fusions such as the BCR-ABL or the KMT2A-MLLT3 fusions genes. It was observed that the fusion partners of PVT1 were both intrachromosomal and interchromosomal (
Second, 33% of these PVT1 fusions involved a partner gene on a different chromosome (interchromosomal fusions) (
Third, and most surprisingly, a vast majority of the intrachromosomal PVT1 gene fusions fuse PVT1 to a gene partner on 8q24 that is located upstream of PVT1, such as CASC11, CASC8 or MYC (
To test a hypothesis that genomic arrangements inside the PVT1 locus leads to unbalanced enrichment of the 5′- but not the 3′-region of PVT1, whole genome sequencing (WGS) of COLO-320DM, SK-PN-DW and D458 was carried out. All three Hi-MYC cell lines showed a remarkable enrichment of genomic sequencing reads from the 5′- compared to 3′-half of PVT1 locus (
It was next hypothesized that the breakpoints in the PVT1 locus could result in ecDNA formation. To test the hypothesis, dual color FISH in the Hi-MYC cell lines using separate probes spanning the 5′ and 3′ ends of the PVT1 locus were carried out (
This application claims the benefit of U.S. Provisional Application No. 62/902,890, filed on Sep. 19, 2019; which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/051626 | 9/18/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62902890 | Sep 2019 | US |
