WO2024097894A1 - Compositions and methods of ribonucleic acid vaccines encoding nye-so-1 - Google Patents
Compositions and methods of ribonucleic acid vaccines encoding nye-so-1 Download PDFInfo
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- WO2024097894A1 WO2024097894A1 PCT/US2023/078543 US2023078543W WO2024097894A1 WO 2024097894 A1 WO2024097894 A1 WO 2024097894A1 US 2023078543 W US2023078543 W US 2023078543W WO 2024097894 A1 WO2024097894 A1 WO 2024097894A1
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- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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Definitions
- This disclosure relates to the field of RNA-based vaccines that generate an immune response to an epitope expressed by a cancer of interest.
- Cancer is a family of genetic disorders in which changes of genetic material drive a normal cell into a dysregulated state that manifests as malignant growth of tumor tissues. With aging of our society, cancer poses an increasing burden both in mortality and healthcare cost. According to data from the National Cancer Institute (NCI), in 2020, roughly 1.8 million people will be diagnosed with cancer and an estimated 606,520 people will die of cancer in the United States. Of all different types of cancers, lung cancer is responsible for the most deaths with 135,720 people expected to die from this disease. That is nearly three times the 53,200 deaths due to colorectal cancer, which is the second most common cause of cancer death. Pancreatic cancer is the third deadliest cancer, causing 47,050 deaths.
- NCI National Cancer Institute
- CTAs Cancer-testis antigens form a family of antigens that are encoded by 276 genes, comprising more than 70 gene families, whose expression is typically restricted to testicular germ cells and placenta trophoblasts with no or low expression in normal adult somatic cells (1, 2, 3).
- CTAs In addition to low expression in normal cells, several CTAs have been found to be reexpressed in a variety of epithelial cancers. For example, Rooney et al. identified 60 CTAs with absent expression in normal tissues and ectopic expression in numerous tumor types, including melanoma, head and neck, lung, liver, stomach, and ovarian cancer (4).
- CTA-rich tumors include melanoma, ovarian, lung, and bladder cancer.
- the group of CTA-moderate tumors comprises breast, bladder, and prostate cancer. Renal cell carcinoma, colorectal cancer, and lymphoma/leukemia are all categorized as CTA poor tumors (3, 5).
- CTA expression may be increased in bulk primary tumors, this may not necessarily be reflected on the single cell level. For instance, microdissection of ovarian cancer specimens demonstrated considerable intra-tumoral heterogeneity of NY-ESO-1 expression (6).
- Cancer-testis antigens are not only re-expressed in tumor tissues but they are also likely to be immunogenic proteins as many members of the family have been shown to elicit spontaneous cellular and humoral immune responses in cancer patients.
- MAGE-A1 The first CTA identified, MAGE-A1, was discovered through its ability to induce an autologous cytotoxic T lymphocyte response in a melanoma patient (7). Since then, several other CTAs have been identified as immunogenic tumor-associated antigens (TAAs) including SSX-2, NY-ESO-1 , and various members of the BAGE, GAGE, and MAGE families (2, 8).
- TAAs tumor-associated antigens
- NY-ESO-1 is a promising CTA for immune-based treatments as its tumor expression is clearly correlated with the induction of an immune response in a wide range of malignancies (11). Furthermore, NY-ESO-1 is an archetypical example of a CTA with restricted expression to germ cells and placental cells and re-expression in tumor cells.
- NY-ESO-1 expression has been reported in a wide range of tumor types, including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer (12, 13-27).
- tumor types including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer (12, 13-27).
- the expression frequency of NY-ESO-1 differs greatly with the most commonly expressing tumors being myxoid and round cell liposarcoma (89-100%), neuroblastoma (82%), synovial sarcoma (80%), melanoma (46%), and ovarian cancer (43
- NY-ESO- 1 provides one avenue for doing so.
- the present disclosure is directed towards an RNA useful as a vaccine that induces the in vivo production of NY-ESO-1.
- This production of NY-ESO-1 in turn elicits an immune response that can prevent or treat a cancer.
- DNA molecules encoding the RNA such as mRNA.
- vectors, host cells, and vaccines comprising the mRNAs and/or DNA molecules as disclosed herein, as well as compositions containing the vectors, host cells and vaccines, as well as in vitro and in vivo uses for the DNA, mRNAs, vectors, host cells, vaccines and compositions.
- Applicant increased the overall viability and expression level of mRNA vaccines by the use of chemical modifications such as an m7G cap and/or a polyA tail.
- this disclosure provides the mRNAs further comprising one or more of the m7G cap and/or polyA tail.
- Applicant provides in vitro transcription technology to increase mRNA vaccine yield. Thus, this disclosure provides this as well.
- the mRNA and vectors can be coupled to carrier technology such as for example, branched Histidine-Lysine (HK) polypeptides (HKP) to encapsulate mRNAs, as well as methods for coupling the mRNA to the carrier technology.
- carrier technology such as for example, branched Histidine-Lysine (HK) polypeptides (HKP) to encapsulate mRNAs, as well as methods for coupling the mRNA to the carrier technology.
- HK branched Histidine-Lysine
- HTP branched Histidine-Lysine
- Applicant’s encapsulated mRNA exhibit enhanced specificity and cellular penetration.
- a combination of mRNA vaccines encapsulated in H3K(+H)4b and MC3/DOTAP liposome carriers are more effective than the liposomes alone.
- an isolated deoxyribonucleic acid (DNA) or an isolated ribonucleic acid (RNA) comprising, or consisting essentially of, or yet further consisting of an open reading frame (ORF) encoding an NYE-SO-1 derived peptide.
- DNA deoxyribonucleic acid
- RNA isolated ribonucleic acid
- ORF open reading frame
- the isolated DNA encodes one or more of the following: 1) the RNA sequence as set forth in SEQ ID NO: 1 or the peptide as set forth in SEQ ID NO: 2; 2) the
- the isolated RNA comprises one or more of the following: 1) the RNA sequence as set forth in SEQ ID NO: 1; 2) the RNA sequence as set forth in SEQ ID NO: 3; 3) the RNA sequence as set forth in SEQ ID NO: 5; or 4) the RNA sequence as set forth in SEQ ID NO: 7.
- the RNA molecule further comprises a 3'-UTR, a 5'-UTR and optionally further comprises additional elements that (a) stabilize the molecule and (b) enhance expression of the polypeptide encoded by said RNA or open reading frame (ORF).
- the 5'-UTR comprises an m7G cap structure and/or a start codon.
- the 3'-UTR comprises a stop codon and/or a polyA tail.
- an immunogenic composition is provided that comprises an mRNA as disclosed herein and a carrier such as a pharmaceutically acceptable carrier.
- the RNA molecule in the composition further comprises a 3'-UTR, a 5'-UTR and optionally further comprises additional elements that (a) stabilize the molecule and (b) enhance expression of the polypeptide encoded by the mRNA or ORF.
- the 5'-UTR comprises an m7G cap structure and/or a start codon.
- the 3'-UTR comprises a stop codon and/or a poly A tail.
- the pharmaceutically acceptable carrier is a polymeric nanoparticle and/or a liposomal nanoparticle.
- the carrier comprises a branched Histidine-Lysine (HK) polypeptide (HKP) to encapsulate mRNAs, e.g., a H3K(+H)4b or MC3/DOTAP liposome carrier.
- HK Histidine-Lysine
- both carriers are used in a single composition.
- the mRNA of the immunogenic composition comprising a polymeric nanoparticle and/or a liposomal nanoparticle which comprises a branched Histidine-Lysine (HK) polypeptide (HKP) to encapsulate mRNAs, e.g., a H3K(+H)4b or MC3/DOTAP liposome carrier.
- HK Histidine-Lysine
- both carriers are used in a single composition and the mRNA further comprises a 3'-UTR, a 5'-UTR and optionally further comprises additional elements that (a) stabilize the molecule and (b) enhance expression of the polypeptide encoded by said mRNA or ORF.
- the 5'-UTR comprises an m7G cap structure and a start codon.
- the m7G cap comprises an m7GpppG structure or an m7GpppGm structure.
- the 3'-UTR comprises a stop codon and a polyA tail.
- the 3’- UTR follows the ORF and comprises the nucleotides AAUAAA.
- the mRNA of the composition may be expressed in a linear in vitro transcription (IVT) system or a plasmid DNA (pDNA) vector delivery system.
- IVT linear in vitro transcription
- pDNA plasmid DNA
- the polymeric nanoparticle carrier comprises a Histidine-Lysine copolymer (HKP).
- the HKP comprises H3K(+H)4b.
- the HKP can self assemble into nanoparticles upon admixture.
- the carrier comprises PLA or PLGA.
- the composition contains l,2-Dioleoyl-3-trimethylammonium propane (DOTAP).
- DOTAP l,2-Dioleoyl-3-trimethylammonium propane
- the carrier comprises aspermine-Lipid Cholesterol (SLiC).
- the SLiC is selected from the structures TM1-TM5 shown in FIG. 13.
- a method of treating a subject with a tumor expressing NYE-SO-1 by a method comprising, or consisting essentially of, or yet further consisting of administering to the subject an mRNA or composition as disclosed herein.
- An above composition can be provided in any acceptable manner as determined by the treating physician, researcher or veterinarian.
- Non-limiting modes of administration include: intratumorally, intravenously, intramuscularly, intradermally, or subcutaneously.
- the subject is a mammal such as for example a human.
- the method further comprises an assay to determine if the tumor expresses NYE-SO-1 to identify patients more likely to be treated by the compositions and methods as disclosed herein.
- FIGs. 1A and IB show the in vitro transcription of NYESO1 vaccine candidates.
- Four NYESO1 vaccine candidates were designed, synthesized and sequence verified.
- the plasmids encoding NYESO1 vaccine candidates were digested with Xhol at 37C for 4hr, followed by agarose gel verification (FIG. 1A) and in vitro transcription (FIG. IB).
- the integrity of mRNA was examined using agarose gel (FIG. IB).
- FIG. 2 shows the in vitro expression of NYESO1 vaccine candidates in 293T cells.
- Various amounts of NYESO1 vaccine candidate mRNA (0.5ug, lug and 2.5ug) were transfected into 293T cells. 48 hours post transfection, Western blot analysis was performed to detect the in vitro expression of each candidate. Wild-type NYESO1 showed the highest in vitro expression among all candidates.
- FIG. 3 shows an in vivo animal study plan to determine the vaccine dose.
- Three groups of mice (3 mice/group) were immunized with 1 pg, 5 pg, and 10 pg of NYESO1 vaccine at day 0 and day 21.
- Two weeks after the immunization blood was collected, and ELISA assay was performed to determine the immunogenicity of NYESO1 vaccine.
- mice were sacrificed, spleen was removed for isolation of splenocytes. Then ELISpot, intracellular staining and cytokine profiling assays were performed to measure T cell response induced by NYES01 vaccine.
- FIG. 4 shows the in vitro expression of NYESO1 vaccine (encoding SEQ ID NO: 2) in 293T cells.
- Newly formulated NYESO1 vaccine was transfected into 293T cells at 0.5 pg, 1 pg and 2.5 pg.
- Western Blot analysis was performed to detect in vitro expression of NYESO1 delivered by lipid nanoparticles (LNP).
- LNP lipid nanoparticles
- FIGs. 5A and 5B show the immunogenicity of NYESO1 (encoding SEQ ID NO: 2) measured by ELISA assay. Serum was collected two weeks post immunization for testing immunogenicity. Serum was serial diluted starting at 1 :200 dilution with 3-fold dilution up to 1 :437400. Dose-dependent immunogenicity ofNYESOl was observed 14 days (FIG. 5A) and 35 days (FIG. 5B) after immunization.
- FIGs. 6A and 6B show the IgG quantification of serum collected at day 35 for vaccine NEYSO1 (encoding SEQ ID NO: 2). IgG amount in sera collected from Day 35 post 1st immunization was measured using anti-CTAGIB antibody as standard. Standard curve (FIG. 6A) was plotted using Sigmoidal, 4PL, X function in GraphPad. The amount of IgG of each immunization dose (FIG. 6B) was calculated by interpolating the standard curve.
- FIGs. 7A and 7B show the IgG subtype determination using ELISA assay for Day 14 sera samples for vaccine NEYSO1 (encoding SEQ ID NO: 2). This figure shows IgG subtypes in sera collected two weeks post 1st immunization (FIG. 7A). Serum was diluted at 1 :200 dilution. The ratio of IgG2a/IgGl was calculated based on OD reading. The result shows that the 1st immunization induced biased Thl response (FIG. 7B).
- FIGs. 8A-8C show the IgG subtype determination using ELISA assay for Day 35 sera samples for vaccine NEYSO1 (encoding SEQ ID NO: 2). This figure shows IgG subtypes in sera collected two weeks post 2nd immunization. Serum was diluted starting at 1 :1800 dilution up to 1 :48600 (FIGs 8A-8B). The ratio of IgG2a/IgGl was calculated based OD reading (FIG. 8C). The result shows that the lug NYESO1 group has biased Thl response, while 5ug and lOug groups showed balanced Thl and Th2 response. [0037
- FIG. 9 shows the ELISpot assay for T cell response for vaccine NEYS01 (encoding SEQ ID NO: 2).
- NEYS01 encoding SEQ ID NO: 2
- spleens from immunized mice were removed, splenocytes were isolated, and stimulated using two peptide pools. One is full set NYESOl peptide mix, and the other is single NYES01 containing amino acids 157-165 of SEQ ID NO: 2.
- the number of IFNr spots in stimulated splenocytes showed dose-dependent increase with full set NYES01 peptide mix induce more IFNr secreting splenocytes.
- FIG- 10 shows the quantification of IFNr secreting cells in stimulated splenocytes for vaccine NEYSO1 (encoding SEQ ID NO: 2).
- FIGs. 11A-11D show intracellular staining to determine T cell response.
- spleens from immunized mice were removed, splenocytes were isolated, and stimulated using two peptide pools, one is full setNYESOl peptide mix, the other is single NYESO1 containing amino acids 157-165 of SEQ ID NO: 2.
- FACS analysis was performed to detect IFNr and IL4 secreting T cells. The results showed that the T cell response induced by NYESO1 vaccine is CD8+ cell-mediated response (FIGs. 11C and 11D) as opposed to a CD4+ cell-mediated response (FIGs. 11A and 11B).
- FIG. 12 provides a schematic representation of an optimized mRNA vaccine expression structure. This structure can be contained and/or transcribed in a linear in vitro transcription (IVT) expression system or plasmid DNA delivery vector.
- IVT linear in vitro transcription
- FIG. 13 shows the structures of the Spermine-Lipid Conjugates (SLiC) species.
- a cell includes a plurality of cells, including mixtures thereof.
- compositions and methods are intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others.
- Consisting essentially of when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination such as those that augment or diminish one or more of effectiveness, stability or toxicity for example.
- a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like.
- Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.
- comparative terms as used herein can refer to certain variation from the reference.
- such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 folds, or about 3 folds, or about 4 folds, or about 5 folds, or about 6 folds, or about 7 folds, or about 8 folds, or about 9 folds, or about 10 folds, or about 20 folds, or about 30 folds, or about 40 folds, or about 50 folds, or about 60 folds, or about 70 folds, or about 80 folds, or about 90 folds, or about 100 folds or more higher than the reference.
- such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.
- substantially or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
- first RNA and second RNA are used to distinguishing two RNAs.
- first line or “second line” or “third line” refers to the order of treatment received by a patient.
- First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively.
- the National Cancer Institute defines first line therapy as “the first treatment for a disease or condition.
- primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies.
- First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008.
- a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub- clinical response to the first line therapy or the first line therapy has stopped.
- a “3’ untranslated region” refers to a region of an mRNA that is directly downstream (i.e., 3’) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
- adenovirus is synonymous with the term “adenoviral vector” and refers to viruses of the genus adenoviridiae.
- adenoviridiae refers collectively to animal adenoviruses of the genus mastadenovirus including but not limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera.
- human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Adi 1 A and Ad 1 IP), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91.
- bovine adenoviruses includes but is not limited to bovine adenovirus types 1, 2, 3, 4, 7, and 10.
- canine adenoviruses includes but is not limited to canine types 1 (strains CLL, Glaxo, R1261, Utrect, Toronto 26-61) and 2.
- equine adenoviruses includes but is not limited to equine types 1 and 2.
- porcine adenoviruses includes but is not limited to porcine types 3 and 4.
- the adenovirus is derived from the human adenovirus serotypes 2 or 5.
- adenovirus vectors can be replication-competent or replication deficient in a target cell.
- the adenovirus vectors are conditionally or selectively replicating adenoviruses, wherein a gene(s] required for viral replication is/are operatively linked to a cell and/or context-specific promoter.
- a gene(s] required for viral replication is/are operatively linked to a cell and/or context-specific promoter.
- selectively replicating or conditionally replicating viral vectors are known in the art (see, for example, U.S. Pat. No. 7,691,370).
- RNA usually a dimer RNA comprising a cap at the 5’ end and a polyA tail at the 3’ end flanked by LTRs
- proteins such as a protease.
- U.S. Patent No. 6,924, 123 discloses that certain retroviral sequence facilitate integration into the target cell genome. This patent teaches that each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes.
- LTRs long terminal repeats
- the LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral genes.
- Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
- the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5.
- U3 is derived from the sequence unique to the 3' end of the RNA.
- R is derived from a sequence repeated at both ends of the RNA
- U5 is derived from the sequence unique to the 5'end of the RNA.
- the sizes of the three elements can vary considerably among different retroviruses.
- the site of poly (A) addition (termination) is at the boundary between R and U5 in the right hand side LTR.
- U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
- gag encodes the internal structural protein of the virus.
- Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
- the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
- RT reverse transcriptase
- I integrase
- the vector RNA genome is expressed from a DNA construct encoding it, in a host cell.
- the components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the "packaging system", which usually includes either or both of the gag/pol and env genes) expressed in the host cell.
- the set of sequences required for the production of the viral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways. The techniques involved are known to those skilled in the art.
- AAV adeno-associated virus
- AAV adeno-associated virus
- AAV refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered, AAV serotypes are known in the art.
- Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant or synthetic serotypes, e.g., AAV-DJ and AAV PHP.B.
- the AAV particle comprises, alternatively consists essentially of, or yet further consists of three major viral proteins: VP1, VP2 and VP3.
- the AAV refers to of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. These vectors are commercially available or have been described in the patent or technical literature.
- Plant viruses refers to a group of viruses that have been identified as being pathogenic to plants. These viruses rely on the plant host for replication, as they lack the molecular machinery to replicate without the plant host. Accordingly, a plant virus can be used as a vector for safely delivering a gene of interest to a non-plant animal subject.
- Plant viruses include but are not limited to tobacco mosaic virus, Maize chlorotic mottle virus; Maize rayado fino virus; Oat chlorotic stunt virus; Chayote mosaic tymovirus; Grapevine asteroid mosaic- associated virus; Grapevine fleck virus; Grapevine Red Globe virus; Grapevine rupestris vein feathering virus; Melon necrotic spot virus; Physalis mottle tymovirus; Prunus necrotic ringspot; Nigerian tobacco latent virus; Tobacco mild green mosaic virus; Tobacco necrosis virus; Eggplant mosaic virus; Kennedya yellow mosaic virus; Lycopersicon esculentum TVM viroid; Oat blue dwarf virus; Obuda pepper virus; Olive latent virus 1; Paprika mild mottle virus; PMMV; Tomato mosaic virus; Turnip vein-clearing virus; Carnation mottle virus; Cocksfoot mottle virus; Galinsoga mosaic virus; Johnsongrass chlorotic stripe mosaic virus;
- Odontoglossum ringspot virus Ononis yellow mosaic virus; Panicum mosaic virus; Poinsettia mosaic virus; Pothos latent virus; or Ribgrass mosaic virus.
- Gene delivery vehicles also include DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods disclosed herein.
- direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins disclosed herein are other non-limiting techniques.
- chemical modification and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population.
- the term refers to the ribonucleotide modifications in naturally occurring 5'-terminal mRNA cap moieties.
- the chemical modification is selected from pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy -pseudouridine, 4-thio-l- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine, 5-methoxyuridine, or 2'-O-methyl uridine.
- the extent of incorporation of chemically modified nucleotides has been optimized for improved immune responses to the vaccine formulation.
- the term excludes the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moi eties.
- Polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
- RNA polynucleotides such as mRNA polynucleotides
- polynucleotides in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties.
- the modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars.
- the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
- At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of residues of the RNA is chemically modified by one or more of modifications as disclosed herein.
- At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of uridine residues of the RNA is chemically modified by one or more of modifications as disclosed herein.
- complementary sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other.
- the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other.
- sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other.
- a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide.
- Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.
- NYE-SO-1 refers to an 18-kDa protein with 180 amino acids featuring a glycine-rich N-terminal region and a strongly hydrophobic C-terminal region with a Pcc-1 domain.
- SEQ ID NO: 2 The sequence of wild type NYE-SO-1 is provided as SEQ ID NO: 2.
- NYE-SO-1 is a member of a family of antigens known as cancer-testis antigens (CTAs).
- NY-ESO-1 expression has been reported in a wide range of tumor types, including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer.
- NYESO1 peptide mix is a pool of 43 peptides derived from a peptide scan (15mers with 11 amino acid overlap) through human NYE-SO-1. This can be used to restimulate the splenocytes to measure T-cell responses.
- a “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include carriers, such as pharmaceutically acceptable carriers.
- the carrier (such as the pharmaceutically acceptable carrier) comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier (for example, an HKP nanoparticle) or a lipid nanoparticle (LNP).
- Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e g., sugars, including monosaccharides, di-, tri, tetraoligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
- Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
- Representative amino acid components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
- Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
- monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
- disaccharides such as lactose, sucrose
- a composition as disclosed herein can be a pharmaceutical composition.
- a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
- the term “culturing” refers to the in vitro or ex vivo propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.
- the cell as disclosed herein is a eukaryotic cell or a prokaryotic cell.
- the cell is a human cell.
- the cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293 T cell, or an a549 cell.
- the cell is a host cell.
- Detectable label label
- detecttable marker or “marker” are used interchangeably, including, but not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, protein or composition described herein.
- DOTAP Dioleoyl-3 -trimethylammonium propane
- an “effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two or more agents, that, when administered for the treatment of a mammal or other subject, is sufficient to effect such treatment for the disease.
- the “effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
- encode refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed to produce the mRNA for the polypeptide or a fragment thereof, and optionally translated to produce the polypeptide or a fragment thereof.
- the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
- an amino acid sequence coding sequence refers to a nucleotide sequence encoding the amino acid sequence.
- the term “engineered” or “recombinant” refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain or the parental host strain of the referenced species.
- the term “engineered” or “recombinant” refers to being synthetized by human intervention.
- the term “recombinant protein” refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
- the term “enhancer”, as used herein, denotes sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed.
- An enhancer may enhance transcription from a single promoter or simultaneously from more than one promoter. As long as this functionality of improving transcription is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of wild-type activity, that is, activity of a full-length sequence), any truncated, mutated or otherwise modified variants of a wild-type enhancer sequence are also within the above definition.
- Eukaryotic cells comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus.
- the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, canine, bovine, porcine, murine, rat, avian, reptilian and human.
- the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of long-term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.
- the term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins.
- expression refers to the process by which polynucleotides are transcribed into mRNA or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
- F2A self-cleaving peptide or “T2A self-cleaving peptide” refers to a class of 18-22 aa-long peptides, which can induce ribosomal skipping during translation of a protein in a cell. The apparent cleavage is triggered by ribosomal skipping of the peptide bond between the Proline (P) and Glycine (G) in C-terminal of 2A peptide.
- a “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
- ORF open reading frame
- a “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
- the gene product may refer to an mRNA or other RNA, such as an interfering RNA, generated when a gene is transcribed.
- H3K(+H)4b refers to a specific type of HKP in which the dominant repeating sequence in its terminal branch is -HHHK- (SEQ ID NO: 82), there are four -HHHK- motifs (SEQ ID NO: 82) in each branch and one extra histidine is inserted in the second -HHHK- motif (SEQ ID NO: 82) of the terminal branch of H3K4b.
- Histidine-Lysine co-polymer refers to a type of polymeric nanoparticle that utilizes a polymer of histidine and lysine to deliver bio-relevant molecules to target cells.
- the HK polymer comprises four short peptide branches linked to a three-lysine amino acid core.
- the peptide branches consist of histidine and lysine amino acids, in different configurations.
- the general structure of these histidine-lysine peptide polymers (HK polymers) is shown in Formula I, where R represents the peptide branches and K is the amino acid L-lysine.
- R1-4 branches may be the same or different in the HK polymers of the invention.
- an R branch is “different”, the amino acid sequence of that branch differs from each of the other R branches in the polymer.
- Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non- homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. In some embodiments, the identity is calculated between two peptides or polynucleotides over their full-length, or over the shorter sequence of the two, or over the longer sequence of the two.
- a polynucleotide or polynucleotide region has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
- This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.
- default parameters are used for alignment.
- One alignment program is BLAST, using default parameters.
- the polynucleotide as disclosed herein is an RNA or an analog thereof. In some embodiments, the polynucleotide as disclosed herein is a DNA or an analog thereof. In some embodiments, the polynucleotide as disclosed herein is a hybrid of DNA and RNA or an analog thereof.
- an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide encodes the same sequence encoded by the reference. In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide hybridizes to the reference, a complement reference, a reverse reference, or a reverse-complement reference, optionally under conditions of high stringency.
- an equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 70% sequence identity, or at least 75% sequence identity, or at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence, or alternatively at least 99 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively an equivalent nucleic acid hybridizes under conditions of high stringency to a reference polynucleotide or its complementary.
- the equivalent must encode the same protein or a functional equivalent of the protein that optionally can be identified through one or more assays described herein.
- the equivalent of a polynucleotide would encode a protein or polypeptide of the same or similar function as the reference or parent polynucleotide.
- the host cell refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- the host cell can be a prokaryotic or a eukaryotic cell.
- the host cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293 T cell, or an a549 cell. Cultured cells lines are commercially available from the American Type Culture Collection, for example.
- Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
- the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
- the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
- a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
- Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg 2+ normally found in a cell.
- Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
- Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
- high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O.lx SSC, or deionized water.
- hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
- SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
- a double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
- “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
- the host cell can be a prokaryotic or a eukaryotic cell.
- the host cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293 T cell, or an a549 cell. Cultured cells lines are commercially available from the American Type Culture Collection, for example.
- immune cells includes, e.g., white blood cells (leukocytes, such as granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes (T cells, B cells, natural killer (NK) cells and NKT cells)) which may be derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells, and NKT cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
- HSC hematopoietic stem cells
- the immune cell is derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSc), hematopoietic stem cells (HSCs), or immortalized cells.
- the HSC are derived from umbilical cord blood of a subject, peripheral blood of a subject, or bone marrow of a subject.
- the subject from whom the immune cell is directly or indirectly obtained is the same subject to be treated.
- the subject from whom the immune cell is directly or indirectly obtained is different from the subject to be treated.
- the subject from whom the immune cell is directly or indirectly obtained is different from the subject to be treated and the subjects are from the same species, such as human.
- Immuno response broadly refers to the antigen-specific responses of lymphocytes to foreign substances.
- immunogen and “immunogenic” refer to molecules with the capacity to elicit an immune response. All immunogens are antigens, however, not all antigens are immunogenic.
- An immune response disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro.
- macromolecules including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to be immunogenic.
- nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen.
- immunogens are not limited to full-length molecules, but may include partial molecules.
- the term “immunoconjugate” comprises an antibody or an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
- a second agent such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
- immunogenic composition refers to a composition that induces clinically significant effector T cell activity against protein targets relevant to cancer cell activities.
- immune response broadly refers to the antigen-specific responses of lymphocytes to foreign substances.
- immunogen and “immunogenic” refer to molecules with the capacity to elicit an immune response. All immunogens are antigens, however, not all antigens are immunogenic.
- An immune response disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro.
- nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen.
- immunogens are not limited to full-length molecules, but may include partial molecules.
- the immune responses elicited can be measured by one of skill in the art, e.g., as illustrated in the Examples, antibodies can be generated in the immune responses; such antibodies can binds to the immunogen; and accordingly, an enzyme- linked immunosorbent assay (ELISA) can be used to detect and/or quantify the immunogen specific antibodies.
- ELISA enzyme- linked immunosorbent assay
- the term “immunoregulatory molecule” may refer to any molecule that may regulate or directly influence immune responses, including but not limited to chemokines such as CCL2, CCL5, CCL14, CCL19, CCL20, CXCL8, CXCL13, and LEC; lymphokines and cytokines such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), interferons a, P and y, factors stimulating cell growth (e.g., GM-CSF), and other factors (e.g., tumor necrosis factors, DC-SIGN, MIPla, MIPip, TGF-P or TNF); factors that provide co-stimulatory signals for T-cell activation such as B7 molecules and CD40; accessory molecules such as CD83; proteins involved in antigen processing and presentation such as TAP1/TAP2 transporter proteins, proteosome molecules such as LMP2 and L
- IVTT intra transcription system
- IVT refers to a protein production system that occurs outside a living cell that typically involves a composition that includes molecules necessary for the transcription and/or translation of target proteins.
- IVT involves template- directed synthesis of RNA molecules of almost any sequence.
- the size of the RNA molecules that can be synthesized using IVT methods range from short oligonucleotides to long nucleic acid polymers of several thousand bases.
- IVT methods permit synthesis of large quantities of RNA transcript (e.g., from microgram to milligram quantities) (Beckert et al., Methods Mol Biol. 703:29-41(2011); Rio et al. RNA: A Laboratory Manual.
- IVT utilizes a DNA template featuring a promoter sequence upstream of a sequence of interest.
- the promoter sequence is most commonly of bacteriophage origin (ex. the T7, T3 or SP6 promoter sequence) but many other promotor sequences can be tolerated including those designed de novo.
- Transcription of the DNA template is typically best achieved by using the RNA polymerase corresponding to the specific bacteriophage promoter sequence.
- Exemplary RNA polymerases include, but are not limited to T7 RNA polymerase, T3 RNA polymerase, or
- IVT is generally initiated at a dsDNA but can proceed on a single strand.
- isolated refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule.
- isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
- isolated is also used herein to refer to polypeptides, proteins and/or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
- the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, or protein, which are normally associated in nature.
- an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype.
- a non-naturally occurring polynucleotide, peptide, polypeptide, or protein does not require “isolation” to distinguish it from its naturally occurring counterpart.
- label or a detectable label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histidine tags (N-His), magnetically active isotopes, e.g., 115 Sn, 117 Sn and 119 Sn, a non-radioactive isotopes such as 13 C and 15 N, polynucleotide or protein such as an antibody so as to generate a “labeled” composition.
- N-terminal histidine tags N-His
- magnetically active isotopes e.g., 115 Sn, 117 Sn and 119 Sn
- a non-radioactive isotopes such as 13 C and 15 N
- polynucleotide or protein such as an antibody so as to generate a “labeled” composition.
- the term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequence
- the label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
- the labels can be suitable for small scale detection or more suitable for high- throughput screening.
- suitable labels include, but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
- the label may be simply detected, or it may be quantified.
- a response that is simply detected generally comprises a response whose existence merely is confirmed
- a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, or other property.
- the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
- luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence.
- Detectable luminescence response generally comprises a change in, or an occurrence of a luminescence signal.
- Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed).
- Examples of luminescent probes include, but are not limited to, aequorin and luciferases.
- fluorescent labels include, but are not limited to fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, and Texas Red.
- suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).
- the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
- Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
- the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
- a purification label or maker refers to a label that may be used in purifying the molecule or component that the label is conjugated to, such as an epitope tag (including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag), an affinity tag (including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)), or a fluorescent tag.
- an epitope tag including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag
- an affinity tag including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)
- fluorescent tag including but not limited to
- selection marker refers to a protein or a gene encoding the protein necessary for survival or growth of a cell grown in a selective culture regimen.
- Typical selection markers include sequences that encode proteins, which confer resistance to selective agents, such as antibiotics, herbicides, or other toxins.
- selection markers include genes for conferring resistance to antibiotics, such as spectinomycin, streptomycin, tetracycline, ampicillin, kanamycin, G 418, neomycin, bleomycin, hygromycin, methotrexate, dicamba, glufosinate, or glyphosate.
- liposomal nanoparticle refers to a nanoparticle typically comprise, or alternatively consist essentially of, or yet further consist of a lipid, in particular, an ionizable cationic lipid
- liposome refers to one or more lipids forming a complex, usually surrounded by an aqueous solution. Liposomes are generally spherical structures comprising lipids fatty acids, lipid bilayer type structures, unilamellar vesicles and amorphous lipid vesicles. Generally, liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. The liposomes may be unilamellar vesicles (possessing a single bilayer membrane), oligolamellar or multilamellar (an onion-like structure characterized by multiple membrane bilayers, each separated from the next by an aqueous layer).
- mRNA refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.
- an mRNA as disclosed herein comprises, or consists essentially of, or yet further consists of at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly- A tail.
- UTR 5' untranslated region
- micelle refers to a polymer assembly comprised of a hydrophilic shell (or corona) and a hydrophobic and/or ionic interior.
- the term micelle may refer to any poly ion complex assembly consisting of a multiblock copolymer possessing a net positive charge and a suitable negatively charged polynucleotide.
- a “normal cell corresponding to the cancer tissue type” refers to a normal cell from a same tissue type as the cancer tissue.
- a non-limiting example is a normal leukocyte from a patient, e.g., a patient with leukemia.
- nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine.
- nucleotide of a nucleic acid which can be DNA or an RNA
- adenosine cytidine
- guanosine thymidine
- thymidine a nucleotide having a uracil base
- nucleic acid sequence and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
- this term includes, but is not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
- oligonucleotide or “polynucleotide”, or “portion,” or “segment” thereof refer to a stretch of polynucleotide residues which is long enough to use in PCR or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules.
- the polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
- Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotri esters, phosphoamidates, carbamates, etc ), charged linkages (e g., phosphorothioates, phosphorodithioates, etc ), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
- uncharged linkages e.g., methyl phosphonates, phosphotri esters, phosphoamidates, carbamates, etc
- charged linkages e.g., phosphorothioates, phosphorodithioates, etc
- pendent moieties e.
- synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
- Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
- ORF open reading frame
- “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.
- an RNA as disclosed herein is “optimized”. Optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide.
- optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add
- PD-1 refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, or alternatively 90% sequence identity, or alternatively at least 95% sequence identity with the PD-1 sequence as shown herein and/or a suitable binding partner of PD-L1.
- Non-limiting example sequences of PD-1 are provided herein, such as but not limited to those under the following reference numbers - GCID:GC02M241849; HGNC: 8760; Entrez Gene: 5133; Ensembl: ENSG00000188389; OMIM: 600244; and UniProtKB: QI 5116 - and the sequence:
- Non-limiting examples of commercially available antibodies thereto include pembrolizumab (Merck), nivolumab (Bristol-Myers Squibb), pidilizumab (Cure Tech), AMP- 224 (GSK), AMP-514 (GSK), PDR001 (Novartis), and cemiplimab (Regeneron and Sanofi).
- PD-L1 refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, or alternatively 90% sequence identity, or alternatively at least 95% sequence identity with the PD-L1 sequence as shown herein and/or an suitable binding partner of PD-1.
- Non-limiting example sequences of PD-L1 are provided herein, such as but not limited to those under the following reference numbers - GCID: GC09P005450; HGNC: 17635; Entrez Gene: 29126; Ensembl: ENSG00000120217; OMIM: 605402; and UniProtKB: Q9NZQ7 - and the sequence:
- Non-limiting examples of commercially available antibodies thereto include atezolizumab (Roche Genentech), avelumab (Merck Soreno and Pfizer), durvalumab (AstraZeneca), BMS-936559 (Bristol-Myers Suibb), and CK-301 (Chekpoint Therapeutics).
- a pharmaceutically acceptable carrier refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein.
- a pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier (for example, an HKP nanoparticle) or a lipid nanoparticle (LNP).
- a polymeric nanoparticle carrier for example, an HKP nanoparticle
- LNP lipid nanoparticle
- pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
- buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen
- Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
- Plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Many plasmids are commercially available for such uses.
- the gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
- MCS multiple cloning site
- Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.
- Plasmid DNA vector delivery system refers to an extra- chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and doublestranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Many plasmids are commercially available for such uses.
- the gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
- MCS multiple cloning site
- Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass- producing a gene or the protein it then codes for.
- an mRNA further comprises a polyA tail.
- a “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3’), from the 3’ UTR that contains multiple, consecutive adenosine monophosphates.
- a polyA tail may contain 10 to 300 adenosine monophosphates (SEQ ID NO: 103). Additionally or alternatively, in a relevant biological setting (e.g., in cells, in vivo) the polyA tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation.
- a polyA tail as used herein comprises, or consists essentially of, or yet further consists of one or more of the following:
- poly(lactic-co-glycolic acid) or polylactic acid refers to a specific type of polymeric nanoparticle that consists of various ratios of glycolic and lactic acid. These molecules are known for biocompatibility and biodegradability.
- polymeric nanoparticle refers to a nanoparticle composed of polymer compound (e.g., compound composed of repeated linked units or monomers) including any organic polymers, such as a Histidine-Lysine (HK) polypeptide (HKP).a polymer-based particle that can transport a bio-relevant molecule either within the particle or in a surface-adsorbed manner.
- polymer compound e.g., compound composed of repeated linked units or monomers
- organic polymers such as a Histidine-Lysine (HK) polypeptide (HKP).
- Prokaryotic cells that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells’ genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about l-2pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coll bacterium, and Salmonella bacterium.
- protein refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
- the subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
- a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence.
- amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
- an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” or “aligned to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence.
- Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST.
- equivalent polynucleotides, proteins and corresponding sequences can be determined using BLAST (accessible at blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on August 1, 2021).
- an amino acid mutation is referred to herein as two letters separated by an integer, such as L19F.
- the first letter provides the one letter code of the original amino acid residue to be mutated; while the last letter provides the mutation, such as A indicating a deletion, or one letter code of the mutated amino acid residue.
- the integer is the numbering of the to-be-mutated amino acid residue in the amino acid sequence free of the mutation, optionally counting from the N terminus to the C terminus.
- the integer is the numbering of the mutated amino acid residue in the mutated amino acid sequence, optionally counting from the N terminus to the C terminus.
- an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity, or at least about 85 % homology or identity, or alternatively at least about 90 % homology or identity, or alternatively at least about 95 % homology or identity, or alternatively at least about 96 % homology or identity, or alternatively at least about 97 % homology or identity, or alternatively at least about 98 % homology or identity, or alternatively at least about 99 % homology or identity (in one aspect, as determined using the Clustal Omega alignment program) and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
- an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complementary sequence.
- An equivalent of a reference polypeptide comprises, consists essentially of, or alternatively consists of an polypeptide having at least 80%, or at least 85 %, or at least 90%, or at least 95%, or at least about 96%, or at least 97%, or at least 98%, or at least 99% amino acid identity to the reference polypeptide (as determined, in one aspect using the Clustal Omega alignment program), or a polypeptide that is encoded by a polynucleotide that hybridizes under conditions of high stringency to the complementary sequence of a polynucleotide encoding the reference polypeptide, optionally wherein conditions of high stringency comprises incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0. lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O.lx SSC, or deionized water.
- a first sequence (nucleic acid sequence or amino acid) is compared to a second sequence, and the identity percentage between the two sequences can be calculated.
- the first sequence can be referred to herein as an equivalent and the second sequence can be referred to herein as a reference sequence.
- the identity percentage is calculated based on the full-length sequence of the first sequence. In other embodiments, the identity percentage is calculated based on the full-length sequence of the second sequence.
- an equivalent of a reference polypeptide comprises, or consists essentially of, or yet further consists of the reference polypeptide with one or more amino acid residues replaced by a conservative substitution.
- the substitution can be “conservative” in the sense of being a substitution within the same family of amino acids.
- the naturally occurring amino acids can be divided into the following four families and conservative substitutions will take place within those families.
- Amino acids with basic side chains lysine, arginine, histidine.
- Amino acids with uncharged polar side chains asparagine, glutamine, serine, threonine, tyrosine.
- Amino acids with nonpolar side chains glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.
- polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
- Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
- polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
- a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
- modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
- the sequence of nucleotides can be interrupted by non-nucleotide components.
- a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
- the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
- a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
- A adenine
- C cytosine
- G guanine
- T thymine
- U uracil
- polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
- purification marker refers to at least one marker useful for purification or identification.
- a non-exhaustive list of this marker includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein.
- Suitable direct or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten
- a purified nucleic acid, peptide, protein, biological complexes or other active compound is one that is isolated in whole or in part from proteins or other contaminants.
- substantially purified peptides, proteins, biological complexes, or other active compounds for use within the disclosure comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide, protein, biological complex or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceutical formulation for therapeutic administration.
- the peptide, protein, biological complex or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients.
- the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.
- a regulatory sequence intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed or replicated, and facilitates the expression or replication of the target polynucleotide.
- a promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription.
- a promoter as used herein is corresponding to the RNA polymerase.
- a promoter as sued herein comprises, or consists essentially of, or yet further consists of a T7 promoter, or a SP6 promoter, or a T3 promoter.
- suitable promoters are provided in W02001009377A1.
- RNA refers to its generally accepted meaning in the art.
- RNA refers to a polynucleotide comprising at least one ribofuranoside moiety.
- the term can include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
- Such alterations can include addition of non-nucleotide material, for example at one or more nucleotides of the RNA.
- Nucleotides in the nucleic acid molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
- the RNA is a messenger RNA (mRNA).
- mRNA refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.
- an mRNA as disclosed herein comprises, or consists essentially of, or yet further consists of at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly -A tail.
- Vaccination is the most successful medical approach to disease prevention and control.
- the successful development and use of vaccines has saved thousands of lives and large amounts of money.
- a key advantage of RNA vaccines is that RNA can be produced in the laboratory from a DNA template using readily available materials, less expensively and faster than conventional vaccine production, which can require the use of chicken eggs or other mammalian cells.
- mRNA vaccines have the potential to streamline vaccine discovery and development, and facilitate a rapid response to emerging infectious diseases, see, for example, Maruggi et al., Mol Ther. 2019; 27(4):757-772.
- mRNA vaccines provide a safe and long- lasting immune response in animal models and humans.
- mRNA vaccines against infectious diseases may be developed as prophylactic or therapeutic treatments.
- mRNA vaccines expressing antigens of infectious pathogens have been shown to induce potent T cell and humoral immune responses. See, for example, Pardi et al., Nat Rev Drug Discov. 2018; 17:261-279.
- the production procedure to generate mRNA vaccines is cell-free, simple, and rapid, compared to production of whole microbe, live attenuated, and subunit vaccines. This fast and simple manufacturing process makes mRNA a promising bio-product that can potentially fill the gap between emerging infectious disease and the desperate need for effective vaccines.
- a “subject” of diagnosis or treatment is a cell or an animal such as a mammal, or a human.
- a subject is not limited to a specific species and includes non-human animals subject to diagnosis or treatment and are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals, and pets. Human patients are included within the term as well.
- Simultaneous use refers to the administration of the two compounds of the composition according to the invention in a single and identical pharmaceutical form or at the same time in two distinct pharmaceutical forms.
- solid tumor refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant, metastatic or non- metastatic. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.
- SRC Spermine-Lipid Cholesterol
- a “therapeutic agent” encompasses biological agents, such as an antibody, a peptide, a protein, an enzyme, and chemotherapeutic agents.
- the therapeutic agent also encompasses immuno-conjugates of cell-binding agents (CBAs) and chemical compounds, such as antibodydrug conjugates (ADCs).
- CBAs cell-binding agents
- ADCs antibodydrug conjugates
- the additional therapeutic is a chemotherapeutic agent.
- a “chemotherapeutic agent,” as used herein, refers to a substance which, when administered to a subject, treats or prevents the development of cancer in the subject's body.
- Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenesis agents, anti-estrogens, anti -androgens or immunomodulators.
- Cytoreductive therapy includes but is not limited to chemotherapy, cryotherapy, and radiation therapy. Agents that act to reduce cellular proliferation are known in the art and widely used. Chemotherapy drugs that kill cancer cells only when they are dividing are termed cell-cycle specific. These drugs include agents that act in S-phase, including topoisomerase inhibitors and anti-metabolites. [0168
- topoisomerase I inhibitors include the camptothecan analogs listed above, irinotecan and topotecan.
- topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.
- Antimetabolites are usually analogs of normal metabolic substrates, often interfering with processes involved in chromosomal replication. They attack cells at very specific phases in the cycle. Antimetabolites include folic acid antagonists, e.g., methotrexate; pyrimidine antagonist, e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine; purine antagonist, e.g., 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitor, e.g., cladribine, fludarabine, nelarabine and pentostatin; and the like.
- folic acid antagonists e.g., methotrexate
- pyrimidine antagonist e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine
- purine antagonist e.g., 6-mercaptopurine and 6-thi
- Plant alkaloids are derived from certain types of plants.
- the vinca alkaloids are made from the periwinkle plant Catharanthus rosed).
- the taxanes are made from the bark of the Pacific Yew tree (taxus).
- the vinca alkaloids and taxanes are also known as anti microtubule agents.
- the podophyllotoxins are derived from the May apple plant. Camptothecan analogs are derived from the Asian “Happy Tree” (Camptotheca acuminata). Podophyllotoxins and camptothecan analogs are also classified as topoisomerase inhibitors.
- the plant alkaloids are generally cell-cycle specific.
- vinca alkaloids e.g., vincristine, vinblastine and vinorelbine
- taxanes e.g., paclitaxel and docetaxel
- podophyllotoxins e.g., etoposide and tenisopide
- camptothecan analogs e.g., irinotecan and topotecan.
- Cryotherapy includes, but is not limited to, therapies involving decreasing the temperature, for example, hypothermic therapy.
- Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art.
- exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more than about 10 Gy and/or a dose of ultraviolet radiation at a range from at least about 5 J/m 2 to not more than about 50 J/m 2 , usually about 10 J/m 2 .
- a biological sample, or a sample can be obtained from a subject, cell line or cultured cell or tissue.
- exemplary samples include, but are not limited to, cell sample, tissue sample, tumor biopsy, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing
- ocular fluids
- transduce or “transduction” refers to the process whereby a foreign nucleotide sequence is introduced into a cell. In some embodiments, this transduction is done via a vector, viral or non-viral.
- treating or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
- treatment is an approach for obtaining beneficial or desired results, including clinical results.
- beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
- Treatments containing the disclosed compositions and methods can be first line, second line, third line, fourth line, fifth line therapy and are intended to be used as a sole therapy or in combination with other appropriate therapies. In one aspect, “treatment” excludes prevention.
- the disease is cancer
- the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor.
- Under transcriptional control which is also used herein as “directing expression of’ or any grammatical variation thereof, is a term well understood in the art and indicates that transcription and optionally translation of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription.
- RNA polymerase refers to an enzyme that produces a polyribonucleotide sequence, complementary to a pre-existing template polynucleotide (DNA or RNA).
- the RNA polymerase is a bacteriophage RNA polymerase, optionally a T7 RNA polymerase, or a SP6 RNA polymerase, or a T3 RNA polymerase.
- suitable polymerase are further detailed in US10526629B2.
- the term “vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and optionally integrate into the target cell’s genome.
- vectors include a plasmid, a nanoparticle, a liposome, a virus, a cosmid, a phage, a BAC, a YAC, etc.
- plasmid vectors may be prepared from commercially available vectors.
- viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art.
- the viral vector is a lentiviral vector. In one embodiment, the viral vector is a retroviral vector. In one embodiment, the vector is a plasmid. In one embodiment, the vector is a nanoparticle, optionally a polymeric nanoparticle or a lipid nanoparticle.
- Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
- a “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
- the DNA viruses constitute classes I and II.
- the RNA viruses and retroviruses make up the remaining classes.
- Class III viruses have a double-stranded RNA genome.
- Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA
- Class V viruses have a negative singlestranded RNA genome used as a template for mRNA synthesis.
- Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
- examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
- Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy.
- Multiplicity of infection refers to the number of viral particles that are added per cell during infection.
- compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage.
- unit dose or "dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen.
- the quantity to be administered depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
- solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
- the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.
- a combination as used herein intends that the individual active ingredients of the compositions are separately formulated for use in combination and can be separately packaged with or without specific dosages.
- the active ingredients of the combination can be administered concurrently or sequentially.
- the four-branched histidine-lysine (HK) peptide polymer H2K4b has been shown to be a good carrier of large molecular weight DNA plasmids (Leng et al. Nucleic Acids Res 2005;
- siRNA 33 :e40. but a poor carrier of relatively low molecular weight siRNA (Leng et al. J Gene Med 2005; 7:977-986.).
- H3K4b carrier of siRNA induced cytokines to a significantly greater degree in vitro and in vivo than H3K(+H)4b siRNA polyplexes (Leng et al. Mol Ther 2012; 20:2282-2290.).
- Suitable HK polypeptides are described in WO/2001/047496, WG/2003/090719, and WG/2006/060182, the contents of each of which are incorporated herein in their entireties. These polypeptides have a lysine backbone (three lysine residues) where the lysine side chain s-amino groups and the N- terminus are coupled to various HK sequences.
- HK polypeptide carriers can be synthesized by methods that are well-known in the art including, for example, solid-phase synthesis.
- HK polymers histidine-lysine peptide polymers
- HKP histidine-lysine peptide polymers
- HK polymers can be reproducibly made on a peptide synthesizer, their amino acid sequence can be easily varied, thereby allowing fine control of the binding and release of RNAs, as well as the stability of polypi exes containing the HK polymers and RNA (Chou et al. Biomaterials 2014; 35:846-855. Midoux et al. Bioconjug Chem (1999) 10:406-411; Henig et al. Journal of American Chemical Society (1999) 121:5123-5126).
- mRNA molecules are admixed with one or more HKP carriers the components self-assemble into nanoparticles.
- the HK polymer comprises four short peptide branches linked to a three-lysine amino acid core.
- the peptide branches consist of histidine and lysine amino acids, in different configurations.
- the general structure of these histidine-lysine peptide polymers (HK polymers) is shown in Formula I, where R represents the peptide branches and K is the amino acid L-lysine.
- R1-4 branches may be the same or different in the HK polymers of the invention.
- a R branch is “different”, the amino acid sequence of that branch differs from each of the other R branches in the polymer.
- Rc KHHHKHHHKHHHHKHHHK- ( SEQ ID NO : 16)
- RE HKHHHKHHHKHHHHKHHHK- ( SEQ ID NO : 18)
- RG KHHHHKHHHHKHHHHK- (SEQ ID NO: 20)
- RH KHHHKHHHKHHHHK- (SEQ ID NO: 21)
- Rj KHHHKHHHHKHHHKHHHHK- (SEQ ID NO: 23)
- HK polymers that may be used in the mRNA compositions include, but are not limited to, HK polymers where each of Ri, R2, R3 and R4 is the same and selected from RA - Rj (Table 1). These HK polymers are termed H2K4b, H3K4b, H3K(+H)4b, H3k(+H)4b, H- H3K(+H)4b, HH-H3K(+H)4b, H4K4b, H3K(1+H)4b, H3K(3+H)4b and H3K(1,3+H)4b, respectively.
- H3K4b the dominant repeating sequence in the branches is -HHHK- (SEQ ID NO: 82), thus “H3K” (SEQ ID NO: 82) is part of the name; the “4b” refers to the number of branches;
- H3K(+H)4b is an analogue of H3K4b in which one extra histidine is inserted in the second -HHHK- motif (SEQ ID NO: 82) (motif 2) of H3K4b; 4) for H3K(1+H)4b and H3K(3+H)4b peptides, there is an extra histidine in the first (motif 1) and third (motif 3) motifs, respectively;
- H2K4b RA KHKHHKHHKHHKHHKHHKHK- (SEQ ID NO: 14)
- H3K4b RB KHHHKHHHKHHHKHH- (SEQ ID NO : 15)
- H3K(+H)4b Rc KHHHKHHHKHHHHKHHHK- (SEQ ID NO : 16)
- H3k(+H)4b RD kHHHkHHHkHHHHkHHHk- (SEQ ID NO : 17)
- H-H3K(+H)4b RE HK HHHK HHHK HHHHK HHHK- (SEQ ID NO: 18)
- HH-H3K(+H)4b RF HHKHHHKHHHKHHHHKHHHK- (SEQ ID NO: 19)
- H4K4b RG KHHHHK HHHHI ⁇ HHHHI ⁇ HHHHI ⁇ - (SEQ ID NO: 20)
- H3K(1+H)4b KHHHKHHHKHHHHK- (SEQ ID NO: 21)
- H3K(3+H)4b Ri KHHHKHHHHKHHHKHHHK- (SEQ ID NO: 22)
- H3K(1,3+H)4b KHHHKHHHHKHHHKHHHHK- (SEQ ID NO: 23)
- mRNA encoding luciferase can be used to measure the efficiency of transfection. See, for example, He et al (J Gene Med. 2021 Feb;23(2):e3295) demonstrating the efficacy of delivering mRNA using a HKP and liposome formulation.
- H3K(+H)4b and DOTAP a cationic lipid
- the combination was about 3-fold and 8- fold more effective as carriers of mRNA than the polymer alone and the cationic lipid carrier, respectively.
- Not all HK peptides demonstrated the synergistic activity with DOTAP lipid.
- the combination of H3K4b and DOTAP was less effective than the DOTAP liposomes as carriers of luciferase mRNA.
- other cationic lipids that may be used with HK peptides include Lipofectin (ThermoFisher), Lipofectamine (ThermoFisher), and DOSPER.
- the D-isomer of H3k (+H)4b in which the L-lysines in the branches are replaced with D- lysines, was the most effective polymeric carrier (H3k(+H)4b vs. H3K(+H)4b, P ⁇ 0.05).
- the D- isomer/ liposome carrier of mRNA was nearly 4-fold and 10-fold more effective than the H3k(+H)4b alone and liposome carrier, respectively.
- the D-H3k(+H)4b/lipid combination was modestly more effective than the L-H3K(+H)4b/lipidmbination, this comparison was not statistically different.
- H3K4b and H3K(+H)4b can be used as carriers of nucleic acids in vitro See, for example, Leng et al. J Gene Med 2005; 7: 977-986; and Chou et al., Cancer Gene Ther 2011; 18: 707-716.
- H3K(+H)4b was markedly better as a carrier of mRNA compared to other similar analogues (Table 2).
- H3K4b has higher mRNA transfection efficiency than H3K4b in various weightweight (HK:mRNA) ratios.
- HK:mRNA weightweight
- luciferase expression was 10-fold higher with H3K(+H)4b than H3K4b in MDA-MB-231 cells without significant cytotoxicity.
- the buffering capacity does not seem to be an essential factor in their transfection differences since the percent of histidines (by weight) in H3K4b and H3K(+H)4b is 68.9 and 70.6 %, respectively
- H3K(+H)4b peptide binds more tightly to mRNA was demonstrated with a heparin-displacement assay.
- Various concentrations of heparin were added into the polyplexes formed with mRNA and HK and it was observed that, particularly at the lower concentrations of heparin, mRNA was released by the H3K4b polymer more readily than the H3K(+H)4b polymer.
- H3K(+H)4b polyplexes were imported into the cells more efficiently than H3K4b polyplexes. Similar to these results, fluorescent microscopy indicated that H3K(+H)4b polyplexes localized within the acidic endosomal vesicles significantly more than H3K4b polyplexes (H3K4b vs. H3K(+H)4b, P ⁇ 0.001). Interestingly, irregularly-shaped H3K4b polyplexes, which did not overlap endocytic vesicles, were likely extracellular and were not observed with H3K(+H)4b polyplexes.
- HK polymers and cationic lipids significantly and independently increase transfection with plasmids. See, for example, Chen et al. Gene Ther 2000; 7: 1698-1705. Consequently, whether these lipids together with HK polymers enhanced mRNA transfection was investigated. Notably, the H3K(+H)4b and H3k(+H)4b carriers were significantly better carriers of mRNA than the DOTAP liposomes. The combination of H3K(+H)4b and DOTAP lipid was synergistic in the ability to carry mRNA into MDA-MB-231 cells.
- the combination was about 3-fold and 8-fold more effective as carriers of mRNA than the polymer alone and the liposome carrier, respectively (H3K(+H)4b/lipid vs. liposomes or H3K(+H)4b). Notably, not all HK peptides demonstrated improved activity with DOTAP lipid.
- the combination of H3K4b and DOTAP carriers was less effective than the DOTAP liposomes as carriers of luciferase mRNA.
- the combination of DOTAP and H3K(+H)4b carriers were found to be synergistic in their ability to carry mRNA into cells. See, for example, He et al. (2020) J Gene Med., Nov 10:e3295.
- the carrier such as the HKP nanoparticle, further comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
- a cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.
- a cationic lipid is selected from 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA, or MC3), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
- DLin-KC2-DMA 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane
- DLin-MC3-DMA dilinoleyl-methyl-4- dimethylaminobutyrate
- L319 di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoy
- the carrier is a nanoparticle.
- nanoparticle refers to any particle having a diameter of less than 1000 nanometers (nm).
- the nanoparticles have dimensions small enough to allow their uptake by eukaryotic cells.
- the nanoparticles have a longest straight dimension (e.g., diameter) of 200 nm or less.
- the nanoparticles have a diameter of 100 nm or less. Smaller nanoparticles, e.g. having diameters of 50 nm or less, e.g., 5 nm-30 nm, are used in some embodiments.
- the carrier is a polymeric nanoparticle.
- polymeric nanoparticle refers to a nanoparticle composed of polymer compound (e.g., compound composed of repeated linked units or monomers) including any organic polymers, such as a Histidine-Lysine (HK) polypeptide (HKP).
- liposome refers to one or more lipids forming a complex, usually surrounded by an aqueous solution. Liposomes are generally spherical structures comprising lipids fatty acids, lipid bilayer type structures, unilamellar vesicles and amorphous lipid vesicles. Generally, liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. The liposomes may be unilamellar vesicles (possessing a single bilayer membrane), oligolamellar or multilamellar (an onion-like structure characterized by multiple membrane bilayers, each separated from the next by an aqueous layer).
- the carrier is a lipid nanoparticle (LNP, also referred to herein as a liposomal nanoparticle).
- LNP lipid nanoparticle
- the LNP has a mean diameter of about 50 nm to about 200 nm.
- Lipid nanoparticle carriers/formulations typically comprise, or alternatively consist essentially of, or yet further consist of a lipid, in particular, an ionizable cationic lipid, for example, SM-102 as disclosed herein, 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
- SM-102 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane
- DLin-MC3-DMA 2,2-dilinoleyl-4-methyl-4- dimethylaminobutyrate
- the LNP carriers/formulations further comprise a neutral lipid, a sterol (such as a cholesterol) and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid (also referred to herein as PEGylated lipid).
- a neutral lipid such as a cholesterol
- a sterol such as a cholesterol
- PEG-modified lipid also referred to herein as PEGylated lipid.
- Additional exemplary lipid nanoparticle compositions and methods of making same are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28: 172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51 :8529-8533; and Maier et al. (2013) Molecular Therapy 21 :1570-1578, the contents of each of which are incorporated herein by reference in their entirety.
- the term “disease” or “disorder” as used herein refers to a cancer, a status of being diagnosed with a cancer, a status of being suspect of having a cancer, or a status of at risk of having a cancer.
- the cancer or tumor cell expresses NYE-SO-1.
- a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and in some aspects, the term may be used interchangeably with the term “tumor.” In some embodiments, the cancer is a cell expressing NYE-SO-1.
- cancer or tumor antigen refers to an antigen known to be associated and expressed in a cancer cell or tumor cell (such as on the cell surface) or tissue
- cancer or tumor targeting antibody refers to an antibody that targets such an antigen.
- the neoantigen does not express in a non-cancer cell or tissue.
- the neoantigen expresses in a non-cancer cell or tissue at a level significantly lower compared to a cancer cell or tissue.
- “neoantigen” refers to a new protein that forms on cancer cells as a result of oncogenic DNA mutations.
- the cancer is selected from: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, and lipoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma
- SCLC small cell lung cancer
- the cancer is a colon cancer, colorectal cancer or rectal cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is an adenocarcinoma, an adenocarcinoma, an adenoma, a leukemia, a lymphoma, a carcinoma, a melanoma, an angiosarcoma, or a seminoma.
- the cancer is a solid tumor. In other embodiments, the cancer is not a solid tumor. In further embodiments, the cancer is a leukemia cancer. In some embodiments, the cancer is from a carcinoma, a sarcoma, a myeloma, a leukemia, or a lymphoma. In some embodiments, the cancer is a colon cancer, colorectal cancer or rectal cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a pancreatic cancer. [0210
- the cancer expresses NYE-SO-1 disclosed herein, such as a lung adenocarcinoma, a mucinous adenoma, a ductal carcinoma of the pancreas, a colorectal carcinoma; a rectal cancer, a follicular thyroid cancer, an autoimmune lymphoproliferative syndrome, a Noonan syndrome, a juvenile myelomonocytic leukemia; a bladder cancer, a follicular thyroid cancer, and an oral squamous cell carcinoma.
- the expression can be detected by sequencing a biopsy of the cancer, a Southern Blotting, a Northern Blotting, or by contacting with an antibody specifically binding to NYE-SO-1.
- the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
- the term “mammal” includes both human and non-human mammals such as non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, bat, rat, rabbit, guinea pig).
- non-human primates e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like
- domestic animals e.g., dogs and cats
- farm animals e.g., horses, cows, goats, sheep, pigs
- experimental animals e.g., mouse, bat, rat, rabbit, guin
- a mammal is a human.
- mammals include humans, non-human primates (e g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, bat, rabbit, guinea pig).
- a mammal is a human.
- a mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero).
- a mammal can be male or female.
- a subject is a human.
- the subject has or is diagnosed of having a disease.
- the subject is suspected of having a disease.
- the subject is at risk of having a disease.
- the subject is in fully (such as free of cancer) cancer remission.
- the subject is at risk of having a recurrence or relapse of a cancer.
- the subject is in partially cancer remission.
- the subject is at risk of cancer metastasis.
- treating or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
- treatment is an approach for obtaining beneficial or desired results, including clinical results.
- beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
- the disease is cancer
- the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor.
- treatment excludes prophylaxis.
- the terms “treating,” “treatment,” and the like, as used herein, mean ameliorating a disease, so as to reduce, ameliorate, or eliminate its cause, its progression, its severity, or one or more of its symptoms, or otherwise beneficially alter the disease in a subject.
- Reference to “treating,” or “treatment” of a patient is intended to include prophylaxis.
- Treatment may also be preemptive in nature, i.e., it may include prevention of disease in a subject exposed to or at risk for the disease. Prevention of a disease may involve complete protection from disease, for example as in the case of prevention of infection with a pathogen, or may involve prevention of disease progression.
- prevention of a disease may not mean complete foreclosure of any effect related to the diseases at any level, but instead may mean prevention of the symptoms of a disease to a clinically significant or detectable level. Prevention of diseases may also mean prevention of progression of a disease to a later stage of the disease.
- the following clinical endpoints are non-limiting examples of treatment: (1) elimination of a cancer in a subject or in a tissue/organ of the subject or in a cancer loci; (2) reduction in tumor burden (such as number of cancer cells, number of cancer foci, number of cancer cells in a foci, size of a solid cancer, concentrate of a liquid cancer in the body fluid, and/or amount of cancer in the body); (3) stabilizing or delay or slowing or inhibition of cancer growth and/or development, including but not limited to, cancer cell growth and/or division, size growth of a solid tumor or a cancer loci, cancer progression, and/or metastasis (such as time to form a new metastasis, number of total metastases, size of a metastasis, as well as variety of the tissues/organs to house metastatic cells); (4) less risk of having a cancer growth and/or development; (5) inducing an immune response of the patient to the cancer, such as higher number of tumor-infiltrating immune cell,
- the subject after treatment experiences one or more endpoints selected from tumor response, reduction in tumor size, reduction in tumor burden, increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of drug-related toxicity, and avoidance of sideeffects (e.g., decreased treatment emergent adverse events).
- endpoints selected from tumor response, reduction in tumor size, reduction in tumor burden, increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of drug-related toxicity, and avoidance of sideeffects (e.g., decreased treatment emergent adverse events).
- improvement of quality of life includes resolution or improvement of cancer-specific symptoms, such as but not limited to fatigue, pain, nausea/vomiting, lack of appetite, and constipation; improvement or maintenance of psychological well-being (e.g., degree of irritability, depression, memory loss, tension, and anxiety); improvement or maintenance of social well-being (e.g., decreased requirement for assistance with eating, dressing, or using the restroom; improvement or maintenance of ability to perform normal leisure activities, hobbies, or social activities; improvement or maintenance of relationships with family).
- improved patient quality of life that is measured qualitatively through patient narratives or quantitatively using validated quality of life tools known to those skilled in the art, or a combination thereof. Additional non-limiting examples of endpoints include reduced hospital admissions, reduced drug use to treat side effects, longer periods off-treatment, and earlier return to work or caring responsibilities. In one aspect, prevention or prophylaxis is excluded from treatment.
- a biological sample is obtained from a subject.
- exemplary samples include, but are not limited to, cell sample, tissue sample, biopsy, liquid samples such as blood and other liquid samples of biological origin, including, but not limited to, anterior nasal swab, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovi
- anterior nasal swab ocular
- the samples include fluid from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, or the like), umbilical cord blood, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), washings of female reproductive tract, urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof.
- a liquid biological sample is a blood plasma or serum sample.
- blood refers to a blood sample or preparation from a subject.
- the term encompasses whole blood, blood product or any fraction of blood, such as serum, plasma, huffy coat, or the like as conventionally defined.
- blood refers to peripheral blood.
- Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants.
- Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Fluid samples often are collected in accordance with standard protocols hospitals or clinics generally follow. For blood, an appropriate amount of peripheral blood (e.g., between 3-40 milliliters) often is collected and can be stored according to standard procedures prior to or after preparation.
- the term “adjuvant” refers to a substance or mixture that enhances the immune response to an antigen.
- the adjuvant can comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldi octadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (See e.g., US 8,241,610).
- the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant.
- the synthetic nanocarrier comprising and adjuvant can be formulated by the methods described in WO2011150240 and US20110293700, each of which is herein incorporated by reference in its entirety.
- contacting means direct or indirect binding or interaction between two or more.
- a particular example of direct interaction is binding.
- a particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity.
- Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.
- administering or “delivery” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval.
- the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer.
- one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more.
- Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
- Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application.
- the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.
- administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
- the disclosure is not limited by the route of administration, the formulation or dosing schedule.
- an RNA, polynucleotide, vector, cell or composition as disclosed herein is administered in an effective amount.
- An “effective amount” is an amount sufficient to effect beneficial or desired results.
- An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc.
- an RNA, polynucleotide, vector, cell or composition as disclosed herein is administered in a therapeutically or pharmaceutically effective amount.
- “Therapeutically effective amount” or “pharmaceutically effective amount” of an agent refers to an amount of the agent that is an amount sufficient to obtain a pharmacological response; or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient. The effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or pharmaceutically effective amount may be administered in one or more administrations.
- the treatment method as disclosed herein can be used as a first line treatment, or a second line treatment, or a third line treatment.
- first line or “second line” or “third line” refers to the order of treatment received by a patient.
- First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively.
- the National Cancer Institute defines first line therapy as “the first treatment for a disease or condition”.
- primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies.
- First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008.
- a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub-clinical response to the first line therapy or the first line therapy has stopped.
- an “anti-cancer therapy,” as used herein, includes but is not limited to surgical resection, chemotherapy, cryotherapy, radiation therapy, immunotherapy and targeted therapy. Agents that act to reduce cellular proliferation are known in the art and widely used. Chemotherapy drugs that kill cancer cells only when they are dividing are termed cell-cycle specific. These drugs include agents that act in S-phase, including topoisomerase inhibitors and anti-metabolites.
- Topoisomerase inhibitors are drugs that interfere with the action of topoisomerase enzymes (topoisomerase I and II). During the process of chemo treatments, topoisomerase enzymes control the manipulation of the structure of DNA necessary for replication and are thus cell cycle specific. Examples of topoisomerase I inhibitors include the camptothecan analogs listed above, irinotecan and topotecan. Examples of topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.
- Antimetabolites are usually analogs of normal metabolic substrates, often interfering with processes involved in chromosomal replication. They attack cells at very specific phases in the cycle. Antimetabolites include folic acid antagonists, e.g., methotrexate; pyrimidine antagonist, e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine; purine antagonist, e.g., 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitor, e.g., cladribine, fludarabine, nelarabine and pentostatin; and the like.
- folic acid antagonists e.g., methotrexate
- pyrimidine antagonist e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine
- purine antagonist e.g., 6-mercaptopurine and 6-thi
- Plant alkaloids are derived from certain types of plants.
- the vinca alkaloids are made from the periwinkle plant (Catharanthus rosea).
- the taxanes are made from the bark of the Pacific Yew tree (taxus).
- the vinca alkaloids and taxanes are also known as antimicrotubule agents.
- the podophyllotoxins are derived from the May apple plant. Camptothecan analogs are derived from the Asian “Happy Tree” (Camptotheca acuminata). Podophyllotoxins and camptothecan analogs are also classified as topoisomerase inhibitors.
- the plant alkaloids are generally cell-cycle specific.
- Examples of these agents include vinca alkaloids, e.g., vincristine, vinblastine and vinorelbine; taxanes, e.g., paclitaxel and docetaxel; podophyllotoxins, e.g., etoposide and tenisopide; and camptothecan analogs, e.g., irinotecan and topotecan.
- vinca alkaloids e.g., vincristine, vinblastine and vinorelbine
- taxanes e.g., paclitaxel and docetaxel
- podophyllotoxins e.g., etoposide and tenisopide
- camptothecan analogs e.g., irinotecan and topotecan.
- an anti-cancer therapy may comprises, or consists essentially of, or consists of a hematopoietic stem cell transplantation.
- a therapeutic agent such as a cell as disclosed herein
- a therapeutic agent may be combined in treating a cancer with another anti-cancer therapy or a therapy depleting an immune cell.
- lymphodepletion chemotherapy is performed followed by administration of a cell as disclosed herein, such as four weekly infusions.
- these steps may be repeated for once, twice, three or more times until a partial or complete effect is observed or a clinical end point is achieved.
- Cryotherapy includes, but is not limited to, therapies involving decreasing the temperature, for example, hypothermic therapy.
- Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art.
- exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more than about 10 Gy or a dose of ultraviolet radiation at a range from at least about 5 J/m 2 to not more than about 50 J/m 2 , usually about 10 J/m 2 .
- the immunotherapy regulates immune checkpoints.
- the immunotherapy comprises, or consists essentially of, or yet further consists of an immune checkpoint inhibitor, such as an Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) inhibitor, or a Programmed Cell Death 1 (PD-1) inhibitor, or a Programmed Death Ligand 1 (PD-L1) inhibitor.
- CTL4 Cytotoxic T-Lymphocyte Associated Protein 4
- PD-1 Programmed Cell Death 1
- PD-L1 inhibitor a Programmed Death Ligand 1
- the immune checkpoint inhibitor comprises, or consists essentially of, or yet further consists of an antibody or an equivalent thereof recognizing and binding to an immune checkpoint protein, such as an antibody or an equivalent thereof recognizing and binding to CTLA4 (for example, Yervoy (ipilimumab), CP- 675,206 (tremelimumab), AK104 (cadonilimab), or AGEN1884 (zalifrelimab)), or an antibody or an equivalent thereof recognizing and binding to PD-1 (for example, Keytruda (pembrolizumab), Opdivo (nivolumab), Libtayo (cemiplimab), Tyvyt (sintilimab), BGB-A317 (tislelizumab), JSOO1 (toripalimab), SHR1210 (camrelizumab), GB226 (geptanolimab), JSOO1 (toripalimab), AB122 (zimber), Yervoy
- a “targeted therapy” refers to a cancer therapy using drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, relapse, and spread of cancer, such as T cells or NK cells or other immune cells expressing a chimeric antigen receptor (CAR) which specifically targets and binds a neoantigen.
- the neoantigen targeted by this targeted therapy can be the same with one encoded by an RNA as disclosed herein.
- the neoantigen targeted by this targeted therapy is different from those encoded by an RNA as disclosed herein.
- a cleavable peptide which is also referred to as a cleavable linker, means a peptide that can be cleaved, for example, by an enzyme.
- One translated polypeptide comprising such cleavable peptide can produce two final products, therefore, allowing expressing more than one polypeptides from one open reading frame.
- cleavable peptides is a selfcleaving peptide, such as a 2A self-cleaving peptide.
- 2A self-cleaving peptides is a class of 18- 22 aa-long peptides, which can induce the cleaving of the recombinant protein in a cell.
- the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A and BmCPV2A. See, for example, Wang Y, et al. Sci Rep. 2015;5: 16273. Published 2015 Nov 5.
- T2A and 2A peptide are used interchangeably to refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or an artificial peptide comprising the requisite amino acids in a relatively short peptide sequence (on the order of 20 amino acids long depending on the virus of origin) containing the consensus polypeptide motif D-V/I-E-X-N-P-G-P, wherein X refers to any amino acid generally thought to be selfcleaving (SEQ ID NO: 99).
- the term “linker” refers to any amino acid sequence comprising from a total of 1 to 200 amino acid residues; or about 1 to 10 amino acid residues, or alternatively 8 amino acids, or alternatively 6 amino acids, or alternatively 5 amino acids that may be repeated from 1 to 10, or alternatively to about 8, or alternatively to about 6, or alternatively to about 5, or alternatively, to about 4, or alternatively to about 3, or alternatively to about 2 times.
- the linker may comprise up to 15 amino acid residues consisting of a pentapeptide repeated three times.
- the linker sequence is a (G4S)n, wherein n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 (SEQ ID NO: 100).
- an NYE-SO-1 derived peptide refers to a peptide engineered from a NYE-SO-1 gene or a NYE-SO-1 protein, such as a wild-type one.
- an NYE-SO-1 derived peptide is a NYE-SO-1 mutant, or a fragment thereof.
- a “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface and/or secreted outside of the cell.
- the signal peptide is at the N terminus of the protein and can be cleaved to produce the mature protein. In some embodiments, the signal peptide is about 15 to about 30 amino acid long.
- an open reading frame refers to a sequence of nucleotides that encodes a polypeptide or a portion thereof.
- the ORF is an RNA.
- Cancer treatment modalities traditionally include surgery, chemotherapy, and radiation therapy. More recently, with in-depth knowledge gained about the molecular pathology of cancer, targeted therapy and immunotherapy has been developed. Both have demonstrated promising results in cancer management. Cancer targeted therapy utilizes sequence information to inhibit the activities of protein products of cancer driver mutations. Because most somatic mutations extend beyond single anatomical sites or cancer types, targeted therapy can be applied to different tumors that share the same underlying mutations regardless their tissue locations. Since 2017, the U.S. Food and Drug Administration (FDA) has approved several treatments for specific genetic defect regardless tissue distribution.
- FDA U.S. Food and Drug Administration
- pembrolizumab that is approved for patients with unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors and entrectinib for patients with NTRK (neurotrophic tyrosine receptor kinase) gene fusion.
- MSI-H microsatellite instability-high
- dMMR mismatch repair deficient
- cancer immunotherapy also has potential to treat more than one type of cancer.
- Cancer immunotherapy utilizes a patient’s immune system to fight against tumor cells.
- Some cancer immunotherapies primarily focus on humoral components of immune systems, the antibodies, to kill cancer cells by inhibiting the function of proteins expressed by cancer cells.
- Other cancer immunotherapies exert their function through cytotoxic T cells that have the ability to destroy tumor cells directly.
- the human immune system as part of its normal functions, surveils and kills abnormal cells by recognizing mutated gene products that do not appear in normal cells and, thus, prevents or curbs the growth of cancers.
- the mutated version of proteins produced by cancer cells are often called tumor associated antigens, also known as neoantigens.
- cancer treatment vaccine Human tumor cell lysates or purified tumor neoantigens can be used to stimulate tumor specific immune response from cancer patients. Many different cell components of the immune system can be used to produce a cancer vaccine.
- a fusion protein that is consisted of a tumor neoantigen, prostatic acid phosphatase and an adjuvant, granulocyte-macrophage colony-stimulating factor, was loaded into patient’s own dendritic cells.
- Dendritic cells function as the primary antigen-presenting cells (APC) that are responsible for displaying the neoantigens to be recognized by cytotoxic cells. Other cells can also function as APCs.
- APC primary antigen-presenting cells
- Other cells can also function as APCs.
- RNA based vaccines are proposed as a possible solution to the challenges and have shown promise in preclinical and clinical studies.
- a key advantage of mRNA vaccines is that mRNA can be produced in the laboratory from a DNA template using readily available materials, less expensively and faster than conventional vaccine production, which can require the use of chicken eggs or other mammalian cells.
- mRNA vaccines have the potential to streamline vaccine discovery and development and facilitate a rapid response to emerging infectious diseases (see, for example, Maruggi et al., Mol Ther. 2019; 27(4): 757-772).
- RNA vaccines have been investigated extensively for infectious disease prevention, and for cancer prophylaxis and treatment. Preclinical and clinical trials have shown that mRNA vaccines provide a safe and long-lasting immune response in animal models and humans. mRNA vaccines expressing antigens of infectious pathogens induce potent T cell and humoral immune responses (Pardi et al. Nat Rev Drug Discov. 2018; 17: 261-279). As previously described, the production procedure to generate mRNA vaccines is entirely cell-free, simple, and rapid, if compared to production of whole microbe, live attenuated, and subunit vaccines. This fast and simple manufacturing process makes mRNA a promising bio-product that can potentially fill the gap between emerging infectious disease and the desperate need for effective vaccines.
- mRNA vaccines are safe, simple, and inexpensive and possess maximum flexibility. Particularly compared with peptide vaccines, they have self-adjuvanting properties, lack of MHC haplotype restriction, and do not need to enter the nucleus (Schlake et al., RNA Biol. 2012; 9(11): 1319-1330). mRNA does not integrate into the genome and therefore it avoids oncogenesis and mutagenesis (McNamara et al., J Immunol Res. 2015; 2015:794528). These vaccines are temporary information carriers due to early metabolic degradation within a few days. Last but not least is that any protein can be encoded for development of therapeutic and prophylactic vaccines, without affecting the properties of the mRNA.
- mRNA vaccines may benefit in that they have been demonstrated to induce balanced, long-lived and protective immunity to influenza A virus infections in even very young and very old mice.
- Vaccines based on mRNA or RNA replicons have also been shown to be immunogenic in a variety of animal models, including nonhuman primates (Maruggi et al., Vaccine. 2017; 35(2):361-368).
- an immunogenic composition that comprises, or consists essentially of, or further consists of a deoxyribonucleic acid or a messenger ribonucleic acid (mRNA) comprising, or consisting essentially of, or yet further consisting of an open reading frame (ORF) encoding one or multiple peptides of different NYE-SO-1 constructs, that can be formulated in a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a polymeric nanoparticle or a liposomal nanoparticle or both.
- composition can be administered to a subject in an amount effective to induce a specific immune response against NYE-SO-1 expression in such a subject.
- DNA deoxyribonucleic acid
- RNA isolated ribonucleic acid
- ORF open reading frame
- the isolated DNA encodes one or more of the following: 1) the RNA sequence as set forth in SEQ ID NO: 1 or the peptide as set forth in SEQ ID NO: 2; 2) the
- the isolated RNA comprises one or more of the following: 1) the RNA sequence as set forth in SEQ ID NO: 1; 2) the RNA sequence as set forth in SEQ ID NO: 3; 3) the RNA sequence as set forth in SEQ ID NO: 5; or 4) the RNA sequence as set forth in SEQ ID NO: 7.
- the RNA is formulated in a carrier, such as a pharmaceutically carrier.
- the RNA is encapsulated in a nanoparticle.
- the DNA or RNA further comprises a 3'-UTR and a 5'-UTR.
- the RNA further comprises one or more additional elements that stabilize the RNA and enhance expression of the peptides encoded by the ORF.
- the 5'-UTR comprises, or consists essentially of, or yet further comprises an m7G cap structure and a start codon.
- the 5’-UTR comprises, or consists essentially of, or yet further comprises UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCA ACCUCAAACAGACACCGCCACC (SEQ ID NO: 10) or an equivalent thereof.
- the 3'-UTR comprises, or consists essentially of, or yet further comprises a stop codon and a poly A tail.
- the 3’-UTR comprises, or consists essentially of, or yet further consists of AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC AACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUA AUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAA GCGAUCGCAAGCUUCUCGAGC (SEQ ID NO: 9) or an equivalent thereof.
- the poly A tail comprises, or consists essentially of, or yet further consists of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
- the RNA is prepared by transcribing a polynucleotide encoding the RNA in an in vitro transcription (IVT) system.
- the RNA is prepared by transcribing a plasmid DNA (pDNA) vector encoding the RNA.
- the vector is pUC57, or pSFVl, or pcDNA3, or pTK126.
- the vector comprises, or consists essentially of, or yet further consists of TCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTC AGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTG TACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAA TACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATC GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA GTGAATTCGAGCTCGGTACCTCGCGAATGCATCTAGATATCGGATCCCGGGCCCGTC GACTGCAGAGGCCTGCATGCAAGCCA GTGAATTCG
- the RNA is a messenger RNA (mRNA).
- the GC content of the full-length RNA is about 35 % to about 70 % (including any percentage or any subranges within the range) of the total RNA content, such as about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%.
- the RNA is chemically modified.
- the chemical modification comprises, or consists essentially or, or yet further consists of one or both of the incorporation of an Nl-methyl-pseudouridine residue or a pseudouridine residue.
- at least about 50% to about 100% of the uridine residues in the RNA are Nl- methyl pseudouridine or pseudouridine.
- At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at east about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of residues of the RNA is chemically modified by one or more of modifications as disclosed herein.
- At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of uridine residues of the RNA is chemically modified by one or more of modifications as disclosed herein.
- uridine residues are replaced by pseudouridines during in vitro transcription.
- This modification stabilizes the mRNA against enzymatic degradation in the cell, leading to enhanced translation efficiency of the mRNA.
- the pseudouridines used can be Nl-methyl-pseudouridine, or other modifications that are well known in the art such as N6m -ethyladenosine (m6A), inosine, pseudouridine, 5-methylcytidine (m5C), 5-hydroxymethylcytidine (hm5C), and N1 -methyladenosine (mlA).
- the modification optionally is made throughout the entire mRNA.
- the skilled artisan will recognize that other modified RNA residues can be used to stabilize the protein’s three-dimensional structure and increase protein translation.
- polynucleotide encoding a DNA or an RNA as disclosed herein, or a polynucleotide complementary thereto, or both.
- the polynucleotide is selected from the group of: a deoxyribonucleic acid (DNA), an RNA, a hybrid of DNA and RNA, or an analog of each thereof.
- the analog comprises, or consists essentially of, or yet further consists of a peptide nucleic acid or a locked nucleic acid or both.
- the polynucleotide further comprises a regulatory sequence directing the transcription thereof.
- the regulatory sequence is suitable for use in an in vitro transcription system.
- the regulatory sequence comprises, or consists essentially of, or yet further consists of a promotor.
- the promoter comprises, or consists essentially of, or yet further consists of: a bacteriophage RNA polymerase promoter, such as a T7 promoter, or a SP6 promoter, or a T3 promoter.
- the polynucleotide comprises a marker selected from a detectable marker, a purification marker, or a selection marker.
- a vector comprising, or consisting essentially of, or yet further consisting of a polynucleotide as disclosed herein.
- the vector further comprises a regulatory sequence operatively linked to the polynucleotide to direct the transcription thereof.
- the regulatory sequence is suitable for use in an in vitro transcription system.
- the regulatory sequence comprises, or consists essentially of, or yet further consists of a promotor.
- the promoter comprises, or consists essentially of, or yet further consists of: a bacteriophage RNA polymerase promoter, such as a T7 promoter, or a SP6 promoter, or a T3 promoter.
- the vector further comprises a marker selected from a detectable marker, a purification marker, or a selection marker.
- the vector further comprises a regulatory sequence operatively linked to the polynucleotide to direct the replication thereof.
- the regulatory sequence comprises, or alternatively consists essentially of, or yet further consists of one or more of the following: an origin of replication or a primer annealing site, a promoter, or an enhancer.
- the vector is a non-viral vector.
- the non- viral vector is a plasmid, or a liposome, or a micelle.
- the vector is pUC57, or pSFVl, or pcDNA3, or pTK126, or another plasmid available at addgene or Standard European Vector Architecture (SEVA).
- SEVA Standard European Vector Architecture
- the vector is a viral vector.
- the viral vector is selected from the group consisting of an adenoviral vector, or an adeno-associated viral vector, or a retroviral vector, or a lentiviral vector, or a plant viral vector.
- a cell comprising one or more of: a DNA or an RNA as disclosed herein, a polynucleotide as disclosed herein, or a vector as disclosed herein.
- the cell is suitable for replicating any one or more of: the DNA, the RNA, the polynucleotide, or the vector, thereby producing the one or more of: the RNA, the polynucleotide, or the vector.
- the cell is suitable for transcribing the polynucleotide or the vector to the RNA, thereby producing the RNA.
- the cell is a prokaryotic cell.
- the prokaryotic cell is an Escherichia coli cell.
- the cell is a eukaryotic cell.
- the eukaryotic cell is any one of a mammal cell, an insect cell, or a yeast cell.
- a cell as disclosed herein is suitable for producing (such as transcribing or expressing) an RNA as disclosed herein. Such production can be in vivo or in vitro.
- the cell can be used to produce the RNA in vitro.
- Such RNA is then administrated to a subject in need thereof optionally with a suitable pharmaceutical acceptable carrier.
- the cell can be used as a cell therapy and directly administrated to a subject in need thereof optionally with a suitable pharmaceutical acceptable carrier.
- the cell therapy can additionally deliver other prophylactic or therapeutic agent to the subject.
- the cell used as a cell therapy is an immune cell, such as a T cell, a B cell, an NK cell, an NKT cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage.
- an immune cell such as a T cell, a B cell, an NK cell, an NKT cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage.
- composition comprising, or consisting essentially of, or yet further consisting of a carrier, and one or more of: an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, or a cell as disclosed herein.
- the carrier is a pharmaceutically acceptable carrier.
- the composition further comprises an additional anti-cancer therapy. Additionally or alternatively, the composition further comprises an adjuvant.
- RNA RNA
- the method comprises, or consists essentially of, or yet further consists of culturing a cell as disclosed herein under conditions suitable for expressing the RNA (such as transcribing a DNA to the RNA).
- the cell comprises the DNA encoding the RNA of the disclosure.
- the method comprises, or consists essentially of, or yet further consists of contacting a polynucleotide as disclosed herein or a vector as disclosed herein with an RNA polymerase, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine-5'-triphosphate (GTP), and uridine triphosphate (UTP) or a chemically modified UTP under conditions suitable for expressing the RNA (such as transcribing a DNA to the RNA).
- the method further comprises isolating the RNA.
- the method further comprises storing the RNA.
- mRNA stability can be enhanced by partial chemical modification.
- short and double strand RNAs derived from aberrant RNA polymerase activities are removed.
- sequence optimization can be used, together with usage of modified nucleosides, such as pseudouridine ((p), 5-methylcytidine (5mC), Cap-1 structure and optimized codons, which in turn improve translation efficiency.
- modified nucleosides such as pseudouridine ((p), 5-methylcytidine (5mC), Cap-1 structure and optimized codons, which in turn improve translation efficiency.
- immature mRNA can be produced as a contaminant which inhibits translation through stimulating innate immune activation.
- FPLC and HPLC purification can be used to remove these contaminants.
- the template for in vitro transcription of mRNA contains five cis-acting structural elements, namely from 5’ to 3’ end: (i) an optimized cap structure, (ii) an optimized 5’ untranslated region (UTR), (iii) a codon optimized coding sequence, (iv) an optimized 3’ UTR and (v) a stretch of repeated adenine nucleotides (poly A tail) (FIG. 12).
- the 5'-UTR includes a start codon and some other elements, but does not encode polypeptide (i.e. it is non-coding).
- a 5'-UTR of the present disclosure comprises, or consists essentially of, or yet further consists of a cap structure with 7-methylguanosine (7mG) sequences.
- the 3'-UTR is directly downstream (3') from the stop codon (the codon of an mRNA transcript representing a termination signal) and does not encode a polypeptide (is non-coding).
- a polyA tail is a special region of mRNA that is downstream from 3 '-UTR and contains multiple consecutive adenosine monophosphates.
- a typical mRNA production cassette comprises, or consists essentially of, or yet further consists of a Cap structure at its 5 ’-UTR region, followed by an in-frame mRNA sequence coding for a corresponding protein or peptide.
- 3 ’-UTR with polyA tail is required for efficient mRNA production.
- an expression cassette is used not only for efficiency of mRNA production but also for the subsequent protein or peptide production (FIG. 12).
- mRNA is produced by in vitro transcription (IVT) from a linear DNA template containing a bacteriophage promoter, the optimized UTR’s and the codon optimized sequence by using an RNA polymerase (T7, T3 or SP6) and a mix of the different nucleosides.
- the linear DNA template can be cloned into a plasmid DNA (pDNA) as a delivery vector.
- pDNA plasmid DNA
- the plasmid vectors can be adapted for mRNA vaccine production. Commonly used plasmids include pSFVl, pcDNA3 and pTK126.
- One unique mRNA expression system is pEVL (see Grier et al. (2016) Mol Ther Nucleic Acids.
- the vaccine comprises, or consists essentially of, or yet further consists of an effective amount of an RNA, which comprises, or consists essentially of, or yet further consists of an open reading frame encoding one or more of NYE-SO-1, a variant of NYE- SO-1, and a pharmaceutically acceptable carrier.
- the effective amount is an amount effective to induce in the subject a neoantigen-specific, such as NYE-SO-l-specific, immune response.
- the carrier comprises, or consists essentially of, or yet further consists of a polymeric nanoparticle or a liposomal nanoparticle.
- the carrier is a Histidine-Lysine-copolymer or a Spermine-Liposome Conjugate.
- the carrier further comprises DOTAP or MC3 or both.
- the vaccine comprises, or consists essentially of, or yet further consists of an effective amount of an mRNA, which comprises, or consists essentially of, or yet further consists of an open reading frame encoding multiple neoantigens separated by selfcleaving 2A peptide sites, signal sequences to incorporate the neoantigen into the membrane and/or be secreted using different signal sequences, such as the albumin signal sequence.
- the vaccine comprises an effective amount of an mRNA, which comprises an open reading frame encoding one or more NYE-SO-1 -based neoantigens, and/or other neoantigens, together with a pharmaceutically acceptable carrier.
- the effective amount is an amount effective to induce in the subject a NYE- SOI -specific immune response.
- the carrier comprises a polymeric nanoparticle or a liposomal nanoparticle.
- the carrier is a Histidine-Lysine-copolymer or a Spermine-Liposome Conjugate.
- the carrier further includes DOTAP or MC3.
- Histidine-Lysine (HK) polypeptides as mRNA vaccine delivery systems
- a non-viral delivery system is more advantageous than the viral delivery system. See, for example, Brito et al. Adv Genet. 2015; 89: 179-233.
- One non-limiting examples is that non-viral methods are preferred over viral delivery systems for their safety and cost-effectiveness. See, for example, Juliano et al. Nucleic Acids Res. 2008; 36: 4158-4171 .
- Non-viral methods for delivery of vaccines include naked mRNA vaccines, gene gun, protamine condensation, adjuvant based vaccines, and encapsulated mRNA vaccines. Positive-sense RNA viruses, alpha viruses can be used for the viral delivery system.
- ex vivo transfected mRNA is an alternative to naked mRNA vaccination.
- mRNAs are transfected into monocytes, macrophages, T cells, dendritic cells (DCs) and mesenchymal stem cells (MSC), see, for example, Sahin et al., Nat Rev Drug Discov. 2014; 13: 759-780, before administration.
- a strong immune response can be induced by ex vivo transfected mRNA vaccination when compared to naked mRNA vaccination, which offers only optimal expression.
- HKP Histidine-Lysine polypeptides
- the HKPs used herein are a group of linear and branched peptides that consist of histidine and lysine residues and these peptides, in most cases, form spherical nanoparticles when mixed with nucleic acids.
- Such polypeptides are disclosed in US Patent No. 7,070,807 B2, issued July 4, 2006, and in US Patent No. 7,163,695 B2, issued January 16, 2007. The disclosures of each of these patents are incorporated herein by reference in their entireties.
- HKP carriers differ in their ability to carry various nucleic acids.
- the four-branched HK peptide (H2K4b) is a good carrier of plasmids (see, for example, Chen, et al., Nucleic Acids Res. 2001; 29: 1334-1340; and Zhang et al., Methods Mol Biol. 2004; 245: 33-52), but is a poor carrier for siRNA.
- H3K4b, H3K(+H)4b, and H3K8b are excellent carriers of siRNA (see, for example, Leng et al., J Gene Med.
- H3K(+H)4b shows effectiveness in carrying mRNA into the targeted cells.
- H3K(+H)4b is a more effective carrier of mRNA than DOTAP liposomes.
- a delivery carrier combination of H3K(+H)4b, MC3 and/or DOTAP can be used as described herein to enhance the efficacy of mRNA delivery. The results as described herein showed that the H3k(+H)4b, MC3 and/or DOTAP combination was the most effective carrier of mRNA. This combination was synergistic for its ability to carry mRNA into cells.
- composition comprising, or consisting essentially of, or yet further consisting of, for example an effective amount of, an RNA as disclosed herein formulated in a pharmaceutically acceptable carrier.
- the composition comprises, or consists essentially of, or yet further consists of the RNA and the pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle.
- the nanoparticle is a polymeric nanoparticle or a liposomal nanoparticle or both.
- the nanoparticle is a lipid nanoparticle (LNP).
- the pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a polymeric nanoparticle or a liposomal nanoparticle or both.
- the polymeric nanoparticle carrier comprises, or consists essentially of, or yet further consists of a Histidine-Lysine co-polymer (HKP).
- the HKP comprises, or consists essentially of, or yet further consists of H3K(+H)4b.
- the HKP comprises, or consists essentially of, or yet further consists of H3k(+H)4b.
- the HKP comprises a side chain selected from SEQ ID NOs: 14-23.
- the mass ratio of HKP and the RNA in the composition is about 10: 1 to about 1: 10, including any range or ratio there between, for example, about 5: 1 to 1 :5, about 5:1 to 1 : 1, about 10: 1, about 9.5:1, about 9: 1, about 8.5: 1, about 8: 1, about 7.5: 1, about 7: 1, about 6.5: 1, about 6: 1, about 5.5: 1, about 5: 1, about 4.5:1, about 4: 1, about 3.5: 1, about 3: 1, about 2:5 : 1, about 2: 1, about 1.5: 1, about 1 : 1, about 1 :1.5, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1:6, about 1:6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, or about 1 : 10.
- the polymeric nanoparticle carrier further comprises a lipid.
- the lipid is a cationic lipid.
- the cationic lipid is ionizable.
- the cationic lipid comprises, or consists essentially of, or yet further consists of Dlin-MC3-DMA (MC3) or dioleoyloxy-3-(trimethylammonio)propane (DOTAP) or both.
- MC3 Dlin-MC3-DMA
- DOTAP dioleoyloxy-3-(trimethylammonio)propane
- the lipid further comprises one or more of: a helper lipid, a cholesterol, or a PEGylated lipid. In some embodiments, the lipid further comprises PLA or PLGA.
- the HKP and the mRNA self-assemble into nanoparticles upon admixture.
- the liposomal nanoparticle carrier comprises, or consists essentially of, or yet further consists of a Spermine-Lipid Cholesterol (SLiC).
- the SLiC is selected from the group consisting of TM1-TM5, the structures of which are illustrated in FIG. 13.
- the pharmaceutical acceptable carrier is a lipid nanoparticle (LNP).
- the lipid is a cationic lipid.
- the cationic lipid is ionizable.
- the LNP comprises, or consists essentially of, or yet further consists of one or more of: 9-Heptadecanyl 8- ⁇ (2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino ⁇ octanoate (SM-102), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)
- the mass ratio of LNP and the RNA in the composition is about 10:1 to about 1: 10, including any range or ratio there between, for example, about 5:1 to 1:5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10.
- the mass ratio of LNP and the RNA in the composition is about 2.5:1. In another embodiment, the mass ratio of LNP and
- the helper lipid comprises, or consists essentially of, or yet further consists of one or more of: disteroylphosphatidyl choline (DSPC), Dipalmitoylphosphatidylcholine (DPPC), (2A)-3-(Hexadecanoyloxy)-2- ⁇ [(9Z)-octadec-9- enoyl] oxy ⁇ propyl 2-(trimethylazaniumyl)ethyl phosphate (POPC), or dioleoyl phosphatidylethanolamine (DOPE).
- DSPC disteroylphosphatidyl choline
- DPPC Dipalmitoylphosphatidylcholine
- the cholesterol comprises, or consists essentially of, or yet further consists of a plant cholesterol or an animal cholesterol or both.
- the PEGylated lipid comprises, or consists essentially of, or yet further consists of one or more of: PEG-c-DOMG (R-3-[(co-m ethoxy - poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine), PEG-DSG (1,2- Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) optionally PEG2000-DMG ((l,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N- [methoxy(polyethylene glycol)-2000)], or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
- PEG-c-DOMG R-3-[(co-m ethoxy - poly(ethyleneglycol
- the mass ratio of the cationic lipid and the helper lipid is about 10:1 to about 1: 10, including any range or ratio there between, for example, about 5: 1 to 1 :5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10. In one embodiment, the mass ratio of the cationic lipid and the helper lipid is about 1:1.
- the mass ratio of the cationic lipid and cholesterol is about 10:1 to about 1: 10, including any range or ratio there between, for example, about 5: 1 to 1 :5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10.
- the mass ratio of the cationic lipid and cholesterol is about 1:1.
- the mass ratio of the cationic lipid and PEGylated lipid is about 10:1 to about 1: 10, including any range or ratio there between, for example, about 5:1 to 1:5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about6.5:l, about6:l, about5.5:l, about5:l, about 4.5:1, about 4:1, about3.5:l, about3:l, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10.
- the mass ratio of the cationic lipid and PEGylated lipid is about 10:1 to about 1: 10, including any
- the mass ratio of the cationic lipid, helper lipid, cholesterol and PEGylated lipid can be calculated by one of skill in the art based on the ratios of the cationic lipid and the helper lipid, the cationic lipid and the cholesterol and the cationic lipid and the PEGylated lipid as disclosed herein.
- the LNP comprises, or consists essentially of, or yet further consists of SM-102, DSPC, cholesterol and PEG2000-DMG.
- the mass ratio of the SM-102, DSPC, cholesterol and PEG200-DMG is about 1 : 1 : 1 : 1.
- the molar ratio of the SM-102, DSPC, cholesterol and PEG2000-DMG is about 50:10:38.5:1.5.
- a mass ratio as provided here can be substituted with another parameter, such as a molar ratio, a weight percentage over the total weight, a component’s weight over the total volume, or a molar percentage over the total molar amount. Knowing the component and its molecular weight, one of skill in the art would have no difficulty in converting a mass ratio to a molar ratio or other equivalent parameters.
- a method of producing a composition as disclosed herein comprises, or consists essentially of, or yet further consists of contacting an RNA as disclosed herein with an HKP, thereby the RNA and the HKP are self-assembled into nanoparticles.
- the mass ratio of HKP and the RNA in the contacting step is about 10: 1 to about 1: 10, including any range or ratio there between, for example, about 5: 1 to 1 :5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10.
- the mass ratio of HKP and the RNA in the contacting step is about 2.5:1. In another embodiment,
- the method further comprises contacting the HKP and RNA with a cationic lipid.
- the cationic lipid comprises, or consists essentially of, or yet further consists of Dlin-MC3-DMA (MC3) or DOTAP (dioleoyloxy-3- (trimethylammonio)propane) or both.
- the mass ratio of the cationic lipid and the RNA in the contacting step is about 10:1 to about 1: 10, including any range or ratio therebetween, for example, about 5:1 to 1:5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1 :1.5, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1:4, about 1:4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1 :6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1:8.5, about 1 :9, about 1 :9.5, or about 1 : 10.
- the mass ratio of the RNA and the cationic lipid in the contacting step is about 1: 1. Accordingly, the mass ratio of the HKP, the RNA and the cationic lipid in the contacting step can be calculated based on the ratio between the HKP and the RNA and the ratio between the RNA and the cationic lipid. For example, if the ratio of the HKP to the RNA is about 4: 1 and the ratio of the RNA to the cationic lipid is about 1 : 1, the ratio of the HKP to the RNA to the cationic lipid is about 4: 1 : 1.
- a method of producing a composition as disclosed herein comprises, or consists essentially of, or yet further consists of contacting an RNA as disclosed herein with a lipid, thereby the RNA and the lipid are self-assembled into lipid nanoparticles (LNPs).
- LNPs lipid nanoparticles
- the LNPs comprise, or consist essentially of, or yet further consist of one or more of: 9-Heptadecanyl 8- ⁇ (2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino ⁇ octanoate (SM-102), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), or an equivalent of each thereof.
- the LNPs further comprise one or more of: a helper lipid, a cholesterol, or a PEGylated lipid.
- the helper lipid comprises, or consists essentially of, or yet further consists of one or more of: disteroylphosphatidyl choline (DSPC), Dipalmitoylphosphatidylcholine (DPPC), (27?)-3-(Hexadecanoyloxy)-2- ⁇ [(9Z)-octadec-9- enoyl] oxy ⁇ propyl 2-(trimethylazaniumyl)ethyl phosphate (POPC), or dioleoyl phosphatidylethanolamine (DOPE).
- DSPC disteroylphosphatidyl choline
- DPPC Dipalmitoylphosphatidylcholine
- DOPE dioleoyl phosphatidylethanolamine
- the cholesterol comprises, or consists essentially of, or yet further consists of a plant cholesterol or an animal cholesterol or both.
- the PEGylated lipid comprises, or consists essentially of, or yet further consists of one or more of: PEG-c-DOMG (R-3-[(co-m ethoxy - poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine), PEG-DSG (1,2- Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) optionally PEG2000-DMG ((l,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N- [methoxy(polyethylene glycol)-2000)], or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, me
- the LNPs comprise, or consist essentially of, or yet further consist of SM-102, DSPC, cholesterol and PEG2000-DMG.
- the mass ratio of the SM-102, DSPC, cholesterol and PEG200-DMG is about 1 : 1 : 1 : 1.
- the molar ratio of the SM-102, DSPC, cholesterol and PEG2000-DMG is about 50: 10:38.5: 1.5.
- the contacting step is performed in a microfluidic mixer.
- the microfluidic mixer is a slit interdigitial micromixer, or a staggered herringbone micromixer (SHM).
- composition produced by a method as disclosed herein.
- compositions comprising, or alternatively consisting essentially of, or yet further consisting of, a carrier and one or more of the products - e.g. an RNA comprising an ORF encoding one or more NYE-SO-1 derived peptides.
- the carrier may be a pharmaceutically acceptable carrier.
- a composition comprising, or alternatively consisting essentially of, or yet further consisting of the RNA comprising an ORF encoding one or more NYE-SO-1 derived peptides disclosed herein, and, optionally, a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle.
- the carrier comprises a Histidine-Lysine co-polymer (HKP).
- the HKP may comprise H3K(+H)4b.
- the polymeric nanoparticle carrier may comprise PLA or PLGA.
- the mRNA or the composition may comprise a plurality of mRNA molecules encoding the same or different polypeptide.
- the plurality of mRNA are encapsulated in the same or different nanoparticle or liposome.
- the mRNA in the composition is encapsulated in l,2-Dioleoyl-3- trimethylammonium propane (DOTAP).
- DOTAP l,2-Dioleoyl-3- trimethylammonium propane
- the liposomal nanoparticle carrier comprises a Spermine-Lipid Cholesterol (SLiC).
- the SLiC is selected from the group consisting of the structures TM1-TM5 shown in FIG. 13.
- compositions of the present disclosure include but are not limited to any one of the claimed compositions as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
- Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
- Compositions of the present disclosure may be formulated for oral, intravenous, topical, enteral, and/or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intravenous administration.
- Administration of the mRNA or compositions can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art.
- the cells and composition of the disclosure can be administered in combination with other treatments.
- the mRNA and compositions are administered to the host using methods known in the art.
- the carrier may be a polymeric nanoparticle or a liposomal nanoparticle.
- polymeric nanoparticles include H3K(+H)4b, PLA or PLGA.
- a non-limiting example of a liposomal nanoparticles is Spermine-Lipid Cholesterol (SLiC).
- a method of treating a subject having a cancer, or at risk of having a cancer, or suspect of having a cancer expresses NYE-SO-1.
- Methods to determine when the method is successful are known in the art and briefly described herein.
- the treatment method can be combined with an assay to determine if the cancer or a tumor cell expresses NYE-SO-1 .
- a method of inhibiting the growth of a tumor or cancer cell comprises, or consists essentially of, or yet further consists of contacting an immune cell with any one or more of an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, or a composition as disclosed herein, thereby activating the immune cell, and contacting the tumor or cancer cell with the activated immune cell.
- the cancer cell or tumor expresses NYE-SO-1.
- the method of treating a cancer subject with a tumor expressing NYE-SO-1 comprises, or alternatively consists essentially of, or yet further consists of contacting a cancer cell with an RNA described in this disclosure, optionally combined with a pharmaceutically acceptable carrier.
- the contacting can be in vitro or in vivo.
- compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated or prevented.
- the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
- they are administered directly by direct injection or systemically such as intravenous injection or infusion.
- the subject to be treated can be an animal, e.g., a mammal or a human patient.
- the cancer to be treated can be a liquid tumor or cancer or a solid tumor or cancer.
- the cancer expresses NYE-SO-1.
- the cancer can be a Stage I, II, III or IV.
- the treatment can be combined with other therapies as described herein.
- the treatment is adjuvant to tumor resection.
- the treatment can be first-line, second-line, third-line, fourth line, fifth line therapy.
- the method comprises, or consists essentially of, or yet further consists of detecting the expression of NYE-SO-1 as disclosed herein.
- expression of NYE-SO-1 can be detected using sequencing, southern blots, or northern blots.
- expression of NYE-SO-1 protein can be detected using flow cytometry or western blots.
- the method can be practiced in an animal to produce an animal model for treatment or to treat an animal, as determined by a treating veterinarian. Methods to determine when the method is successful are known in the art and briefly described herein.
- the cancer is an adenocarcinoma, an adenocarcinoma, an adenoma, a leukemia, a lymphoma, a carcinoma, a melanoma, an angiosarcoma, a pancreatic cancer, a colon cancer, a colorectal cancer, a rectal cancer, or a seminoma.
- the cancer can be primary or metastatic.
- the subject in need thereof may be suffering from an active cancer or be in remission, or at risk of developing a cancer, primary or secondary.
- the immune response comprises, or consists essentially of, or yet further consists of any one or more of: a Thl immune response, activation of CD8+ T cells, or production of a pro-inflammatory cytokine, such as interleukin-2 (IL-2), interferon-gamma (IFN-y), or tumor necrosis factor-beta (TNF-P).
- IL-2 interleukin-2
- IFN-y interferon-gamma
- TNF-P tumor necrosis factor-beta
- These methods comprise, or consist essentially of, or yet further consist of administering to the subject, for example an effective amount of (e.g., a pharmaceutically effective amount of), any one or more of a DNA or an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, or a composition as disclosed herein.
- an effective amount of e.g., a pharmaceutically effective amount of
- the DNA encodes one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.
- the RNA comprises one or more of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.
- the RNA further comprise a 5’UTR (for example comprising, or consisting essentially of, or yet further consisting of SEQ ID NO: 10) and a 3’UTR (for example comprising, or consisting essentially of, or yet further consisting of SEQ ID NO: 9).
- the vector comprises, or consists essentially of, or yet further consists of SEQ ID NO: 11.
- the composition comprises the RNA formulated in a carrier, such as an LNP or a HKP nanoparticle as disclosed herein.
- the administration is intratumoral, or intravenous, or intramuscular, or intradermal, or subcutaneous.
- the subject is a mammal, or a human.
- the method further comprises administering to the subject an additional anti-cancer therapy.
- the anti-cancer therapy is administrated prior to, or concurrently with, or after the administration of any one or more of the following: the RNA as disclosed herein, the polynucleotide as disclosed herein, the vector as disclosed herein, the cell as disclosed herein, or the composition as disclosed herein.
- the administration was repeated for at least one time, at least two times, at least three times, at least four times, or more.
- the interval between any two administrations can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer.
- the method further comprises detecting the expression of NYE- SO-1 as disclosed herein in a biological sample of the subject, such as a tumor biopsy or a circulating tumor DNA, prior to the administration.
- the method further comprises monitoring the expression of NYE- SO-1 as disclosed herein in a biological sample of the subject, such as a tumor biopsy or a circulating tumor DNA, after the administration.
- the method further comprises detecting antibodies recognizing and binding to NYE-SO-1 as disclosed herein in a biological sample of the subject, such as a blood sample, after the administration.
- an effective dose of an RNA, or polynucleotide, or vector, or cell or composition as disclosed herein is the dose required to produce a protective immune response in the subject to be administered.
- a protective immune response in the present context is one that treats a cancer in a subject.
- the RNA, or polynucleotide, or vector, or cell or composition as disclosed herein can be administered one or more times.
- An initial measurement of an immune response to the vaccine may be made by measuring production of antibodies in the subject receiving the RNA, or polynucleotide, or vector, or cell, or composition.
- Methods of measuring antibody production in this manner are also well known in the art, is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a cancer.
- the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the formulated composition.
- the RNA compositions can be administered at dosage levels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one or more times a day, per week, per month, etc. to obtain the desired therapeutic or prophylactic effect.
- the RNA composition is administered at a dosage of about 10 to about 500 pg/kg of body weight, or any dosage or subranges therein, such as about 28.5-285 pg/kg of body weight.
- the desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every 2 months, every three months, every 6 months, etc.
- the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein can be used.
- the RNA compositions can be administered at dosage levels sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg.
- the RNA compositions can be administered once or twice (or more) at dosage levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.
- the RNA compositions can be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.
- the RNA or composition is further combined with another therapy, such as a purified protein drug.
- a therapeutic mAb for example AvastinTM, HerceptinTM, YervoyTM, KeytrudaTM, OpdivoTM, TecentriqTM etc
- a therapeutic protein such as GM-CSF, an interleukin, an interferon, thymosin A, hexarelin or adiponectin.
- the combined treatment comprises administrating mRNA or composition as disclosed herein and a combined therapy concurrently, sequentially and/or separately to a subject in need thereof.
- the mRNA or composition and combined therapy are administrated to the subject via the same administration route.
- different administration route is used.
- any combination therapy as described herein achieves a synergistic effect in treating a disease, such as a cancer.
- RNA or composition as described herein to a subject in need thereof.
- the method comprising, or alternatively consisting of, or yet further consisting essentially of administering an effective amount of an RNA or composition as described herein.
- the method further comprises administering an effective amount of a combined treatment, whereby a synergistic effect is achieved in treating cancer.
- Anti-cancer and immune enhancing therapies are known in the art, several of which are described herein.
- RNA or composition as described herein is a method for delivering an RNA or composition as described herein to a subject in need thereof.
- the subject is suspected of having and/or is diagnosed with a cancer expressing NYE-SO-1.
- the cancer is a solid tumor (e.g., carcinoma or sarcoma) or a nonsolid cancer (for example, a blood cancer). Additionally or alternatively, the cancer or tumor is a primary or metastatic cancer or tumor.
- a solid tumor e.g., carcinoma or sarcoma
- a nonsolid cancer for example, a blood cancer.
- the cancer or tumor is a primary or metastatic cancer or tumor.
- the cancer is selected from a gastric cancer, a colorectal cancer, a prostate cancer, a breast cancer, a triple negative breast cancer, an ovarian carcinoma, a renal cell carcinoma, an Ewing sarcoma, a melanoma, a mesothelioma, a lung cancer, a non-small cell lung cancer, a stage IV lung cancer, a brain cancer, a glioblastoma, a lymphoma, a leukemia, or a multiple myeloma (MM).
- the cancer affects the blood and/or bone marrow.
- the methods further comprising identifying the subject for the therapy by assaying a cancer sample from the patient for a NYE-SO-1 expression.
- the therapy is then administered to the subject expressing NYE-SO-1.
- any of the methods as disclosed herein further comprise monitoring efficacy prior to, concurrently with, and/or after performing the method or any step(s) recited in the method.
- the efficacy is evaluated and/or quantified as a treatment of a disease (for instance a cancer) in a subject, an inhibition of a cancer cell growth, and/or a biomarker change.
- both efficacy outcomes and biomarker can be simultaneously determined in the cancer or tumor in a subject.
- a rodent bearing a human tumor xenograft is used.
- Human tumor cells preferably engineered to express a bioluminescent gene, such as Luciferase, are transplanted into a rodent (such as a mice or a rat), allowing visualization by injection of an activatable fluorescence optcial imaging agent in the rodent.
- the present disclosure provides methods for producing and administering an RNA comprising an ORF encoding NYE-SO-1 or a variant thereof.
- the present disclosure provides kits for performing these methods as well as instructions for carrying out the methods of the present disclosure such as administering to a subject an effective amount of mRNA described by the present disclosure.
- kits comprising, or alternatively consisting essentially of, or yet further consisting of, any one of the RNA molecules disclosed herein.
- kits comprising, or alternatively consisting essentially of, or yet further consisting of the composition as disclosed herein and optionally, instructions for use.
- Such a kit may also comprise, or alternatively consist essentially of, or yet further comprise media and other reagents appropriate for the administration of RNA molecules, such as those disclosed herein.
- the kits of this disclosure can also comprise, e.g., a buffering agent, a preservative or a protein-stabilizing agent.
- the kits can further comprise components necessary for detecting the detectable label, e.g., an enzyme or a substrate.
- kits can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample.
- Each component of a kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
- the kits of the present disclosure may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.
- these suggested kit components may be packaged in a manner customary for use by those of skill in the art.
- these suggested kit components may be provided in solution or as a liquid dispersion or the like.
- the kit comprises, or alternatively consists essentially of, or yet further consist of instructions for use and one or more of an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, or a composition as disclosed herein.
- the kit is suitable for use in a method of treatment as disclosed herein.
- the kit further comprises an anti-cancer therapy.
- the kit comprises, or alternatively consists essentially of, or yet further consist of instructions for use and one or more of: an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, a composition as disclosed herein, an HKP, or a lipid optionally a cationic lipid.
- the kit is suitable for use in a method producing an RNA or a composition as disclosed herein.
- the kit comprises, or alternatively consists essentially of, or yet further consist of instructions of use, a polynucleotide or a vector as disclosed herein, an RNA polymerase, ATP, CTP, GTP, and UTP or a chemically modified UTP.
- the kit is suitable for use in an in vitro method producing an RNA or a composition as disclosed herein.
- In vitro transcription was performed following RNAimmune’s In vitro transcription SOP.
- the NYESO-1 mRNA producing plasmid was linearized using Xhol (NEB) at 37°C for 4 hours and the linearized plasmid was purified using QIAquick PCR purification kit following manufacturer’s instruction (Qiagen).
- in vitro transcription reaction was performed in a 20 pl reaction with all the components added in the following order: 1 pg of linearized DNA; Ipl of ATP, CTP, GTP and Nl-methyl-pseudouri dine tri phosphate; 0.8pl CleanCap AG (3’0me); 2pl 10X IVT reaction buffer; 0.5 pl Murine RNase Inhibitor; 0.4pl E.coli Inorganic Pyrophosphatase; and 3.2pl T7 RNA polymerase. The IVT reaction was incubated at 37°C for 4 hours.
- 10X Transcription Buffer examples include: 400 mM Tris-HCL (pH 8), 100 mM DTT, 20 mMspermidine, 0.02% Triton X, 165 mM Magnesium Acetate, and DNase/RNase-Free Water. After in vitro transcription reaction, the mRNA was purified using RNeasy Mini Kit following manufacturer’s instruction (Qiagen).
- the IVT reaction was adjusted to a volume of lOOpl with RNase- free water. Then, mRNA was mixed well with 350pl buffer RLT.250pl of 100% ethanol was then added to the mixture, mix well by pipetting, and 700pl of the sample was transferred to a RNeasy Mini spin column, centrifuged for 15 seconds at >8000 x g (>10,000 rpm). After discarding the flow- through, the spin column was washed twice with 500pl Buffer RPE. Then the spin column was transferred to a 1.5 ml collection tube (kit supplied), mRNA was eluted with 30pl RNase-free water.
- RNA gel electrophoresis was employed to determine mRNA quality.
- the quality of purified mRNA was evaluated using 1% agarose gel in NorthernMaxTM-Gly Gel Prep with Sybr Gold (1 :20,000). The running buffer was prepared by diluting NorthernMaxTM-Gly Gel Prep from lOx stock solution in DEPC-treated ultrapure water.
- RNA sample was prepared by adding one volume of RNA (up to 30pg total RNA) to one volume NorthernMax®-Gly Sample Loading Buffer and incubated at 65C for 30 min to prevent formation of secondary structure.
- RNA sample and markers were run on 1% agarose gel at 5V/cm. Once the bromophenol blue dye front migrated approximately % the length of the gel, electrophoresis was stopped, and mRNA was visualized with blue light.
- each component was prepared as following: lOOmg of SM-102 in 5.63ml 100% ethanol; 39.5mg of DSPC in 10ml 100% ethanol; 74.4mg of Cholesterol in 100% ethanol; and 18.8mg of DMG-PEG2000 in 100% ethanol.
- the final concentration of 4x stock is 25mM SM-102, 5mM DSPC, 19.5mM Cholesterol and 0.75mM DMG-PEG2000.
- Each lipid component was then added to a 5ml tube at a ratio of 1 : 1 : 1 : 1 to make a lx working solution.
- the final concentration of working concentration is 6.25mM SM-102, 1.25mM DSPC, 4.815mM Cholesterol, and 0.1875mM DMG-PEG2000.
- the mRNA stock solution was diluted in 25 mM Sodium Acetate pH5.0 at a final concentration of 0.13 mg per ml.
- lipid nanoparticles were done using NanoAssmblr Ignite (PNI) following manufacturer's instructions with an mRNA:lipid ratio of 3 : 1.
- PNI NanoAssmblr Ignite
- the formulated LNPs were transferred into pre-soaked dialysis cassette and dialyzed in 20mM Tris- HC1, 8% sucrose dialysis buffer for at least 18 hours at 4°C.
- LNPs were removed from dialysis cassette using a 3-5ml syringe, filter sterilized using an acrodisc filter (0.2 pM pore size) and poured into the top compartment of an Amicon® Ultra- 15 centrifugal filtration tube.
- LNPs were transferred to a new tube and characterized by Dynamic Light Scattering to measure particle size, poly dispersity and zeta potential using Zetasizer Ultra (Malven Panalytical). The encapsulation efficiency was measured using Ribogreen assay (Invitrogen). The mRNA concentration was measured using Ribogreen assay and Nanodrop. The final LNPs were stored at -80C for future use.
- FIG. 293 T cell was transfected with various forms of NYESO1 mRNA using Lipofectamin MessengerMax transfection reagents. Forty-eight hours post transfection, cells were collected, cell lysates were prepared, and the protein concentration was determined using BCA assay. The expression level of each construct was compared using Western Blot analysis with anti-NYESOl antibody (anti-CTAGIB, Abeam).
- Balb/c female mice were immunized at week 0 and week 3 with 1, 5, and 10 pg of NYESO1 vaccine.
- Sera were collected 2 weeks post-immunization. The total IgG and the ratio of IgG2a/IgGl were measured. Briefly, 96-well plates were coated with 0.1 pg of NYESO1 protein (RayBiotech) per well and incubated overnight at 4°C. For measuring total IgG, individual sera were diluted starting 1 :200 in lx ELISA diluent buffer (Biolegnd) with 3- fold dilution up to 1 :437400 dilution and incubated for 2 h at room temperature.
- HRP horseradish peroxidase conjugated goat anti-mouse IgG
- FIGs. 5A-5B [0365
- HRP horseradish peroxidase
- Abeam horseradish peroxidase conjugated goat anti -mouse IgGl and IgG2a subtypes
- mice Eight weeks after the final boost immunization, the spleens of the mice were collected, dissociated into single cell suspension mechanically, passed through a 70-pm cell strainer (Miltenyi Biotec), and lysed by ACK lysis buffer (KD Medical). The cells were then resuspended in RPMI supplemented with 10% FBS, lx Pen/Strep and lx 2-Mercaptoethanol, counted and adjusted to 2 x 106 cells/ml. After this, 100 pl of splenocytes were added to a well of IFN-y coated 96-well ELISpot plate (Mabtech).
- Splenocytes were then incubated for 16 hours in cell culture incubator with (stimulated) or without (unstimulated) stimulation of either full set overlapping peptides pools of NYESO1 protein that was prepared according to manufacturer’s instruction (JPT peptide) or a single NYESO1 157-165 peptide (iba).
- JPT peptide JPT peptide
- iba NYESO1 157-165 peptide
- biotinylated anti-IFN-y R4-A2-biotin monoclonal antibody was used as detecting Ab; and streptavidin-ALP complex and BCIP/NBT-plus substrate (Mabtech) were used to reveal the presence of spots. Spots formed by fFN-y-secreting cells were counted using Cytation 7 (Biotek) and results are presented as spot-forming cells per 2x105 splenocytes. (FIGs. 9 and 10)
- Induction of antigen-specific T cells was determined using ICS. Splenocytes which were prepared as described above. IxlO 6 splenocytes were added in 12 x 75 mm plastic tubes in the presence of protein transport inhibitor, brefeldin A (BioLegend) with (stimulated) or without (unstimulated) stimulation of either full set overlapping peptides pools of NYESO1 protein that was prepared according to manufacturer’s instruction (JPT peptide) or single NYESO1 157-165 peptide (iba). After 16 hour incubation, FACS analysis was performed to determine cytokines splenocytes. Cell surface staining was performed using antibodies against CD3 (FITC), CD4 (BV421) and CD8 (BV650).
- FITC CD3
- CD4 BV421
- CD8 BV650
- Intracellular staining was performed using antibodies against IL-4 (APC), and IFN-y (PE).
- eFluor450 was used to distinguish live/dead cells (Invitrogen). Cells were acquired using BD Celesta Flow cytometer (BD Biosciences) and flow cytometry data were analyzed using Flow Jo software. (FIGs. 11A-11D)
- SEQ ID NO: 5 WT NYESO1 with COVID signal RNA
- SEQ ID NO: 7 (Codon Optimized NYESO1 with CO VID signal RNA)
- SEQ ID NO: 22 (HK peptide branch) KHHHKHHHHKHHHKHHHK
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Abstract
Compositions and methods are provided for potent NYE-SO-1 vaccines for treatment of cancer. The compositions include a pharmaceutical composition containing RNA molecules encoding NYE-SO-1 derived peptides together with a pharmaceutically acceptable carrier. Methods for stimulating system immune responses and treatment are provided, including intratumoral, intravenous, intramuscular, intradermal, and subcutaneous injection.
Description
Compositions and Methods of Ribonucleic Acid Vaccines Encoding NYE-SO-1
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/422,865, filed November 4, 2022, incorporated by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates to the field of RNA-based vaccines that generate an immune response to an epitope expressed by a cancer of interest.
BACKGROUND
[0003| Cancer is a family of genetic disorders in which changes of genetic material drive a normal cell into a dysregulated state that manifests as malignant growth of tumor tissues. With aging of our society, cancer poses an increasing burden both in mortality and healthcare cost. According to data from the National Cancer Institute (NCI), in 2020, roughly 1.8 million people will be diagnosed with cancer and an estimated 606,520 people will die of cancer in the United States. Of all different types of cancers, lung cancer is responsible for the most deaths with 135,720 people expected to die from this disease. That is nearly three times the 53,200 deaths due to colorectal cancer, which is the second most common cause of cancer death. Pancreatic cancer is the third deadliest cancer, causing 47,050 deaths.
[0004] Cancer-testis antigens (CTAs) form a family of antigens that are encoded by 276 genes, comprising more than 70 gene families, whose expression is typically restricted to testicular germ cells and placenta trophoblasts with no or low expression in normal adult somatic cells (1, 2, 3).
10005] In addition to low expression in normal cells, several CTAs have been found to be reexpressed in a variety of epithelial cancers. For example, Rooney et al. identified 60 CTAs with absent expression in normal tissues and ectopic expression in numerous tumor types, including melanoma, head and neck, lung, liver, stomach, and ovarian cancer (4).
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4859-4785-6012.1
[0006| Tumors can be classified as CTA-rich, intermediate, or poor based on the frequency of CTA expression. CTA-rich tumors include melanoma, ovarian, lung, and bladder cancer. The group of CTA-moderate tumors comprises breast, bladder, and prostate cancer. Renal cell carcinoma, colorectal cancer, and lymphoma/leukemia are all categorized as CTA poor tumors (3, 5). Additionally, while CTA expression may be increased in bulk primary tumors, this may not necessarily be reflected on the single cell level. For instance, microdissection of ovarian cancer specimens demonstrated considerable intra-tumoral heterogeneity of NY-ESO-1 expression (6).
[0007] Cancer-testis antigens are not only re-expressed in tumor tissues but they are also likely to be immunogenic proteins as many members of the family have been shown to elicit spontaneous cellular and humoral immune responses in cancer patients.
[0008] The first CTA identified, MAGE-A1, was discovered through its ability to induce an autologous cytotoxic T lymphocyte response in a melanoma patient (7). Since then, several other CTAs have been identified as immunogenic tumor-associated antigens (TAAs) including SSX-2, NY-ESO-1 , and various members of the BAGE, GAGE, and MAGE families (2, 8).
[0009] The function of CTAs has been studied, but is still unclear. Transgenic mouse models have revealed several CTA members to play a key role in male fertility (9). A handful of CTAs have been implicated in cell metabolism, cytoskeleton dynamics, double-strand break repair, maintenance of genomic integrity, and regulation of mRNA expression during spermatogenesis. Although an increasing number of studies are demonstrating CTA re-expression in cancer, their functional role in oncogenesis is largely unexplored. More recent data points to a role for CTAs in the regulation of epithelial-to-mesenchymal transition as well as tumor cell survival (10). Thus, a need exists in the art to better understand NY-ESO-1 expression in tumor progression and treatments based on its expression in the tumor environment. This disclosure satisfies this need and provides related advantages as well.
SUMMARY OF THE DISCLOSURE
[0010] NY-ESO-1 is a promising CTA for immune-based treatments as its tumor expression is clearly correlated with the induction of an immune response in a wide range of malignancies
(11). Furthermore, NY-ESO-1 is an archetypical example of a CTA with restricted expression to germ cells and placental cells and re-expression in tumor cells.
[0011 ] NY-ESO-1 expression has been reported in a wide range of tumor types, including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer (12, 13-27). Within these tumor types, the expression frequency of NY-ESO-1 differs greatly with the most commonly expressing tumors being myxoid and round cell liposarcoma (89-100%), neuroblastoma (82%), synovial sarcoma (80%), melanoma (46%), and ovarian cancer (43%) (18, 47-51). Other tumor types show protein expression of NY-ESO-1 in the range of 20-40%.
[0012] Important to note is that the majority of cancer types show a heterogeneous expression of NY-ESO-1, which could limit the treatment response to NY-ESO-1 targeted treatment. The most homogenous expression has been reported in myxoid and round cell liposarcomas (94%) and synovial sarcomas (70%) which might be related to the promising results that have been obtained in adoptive cellular immunotherapy trials (28, 30).
[0013] Based on the above cancer rates, a need to treat a cancer effectively exists, and NY-ESO- 1 provides one avenue for doing so.
[0014] In one aspect, the present disclosure is directed towards an RNA useful as a vaccine that induces the in vivo production of NY-ESO-1. This production of NY-ESO-1 in turn elicits an immune response that can prevent or treat a cancer. Also provided are DNA molecules encoding the RNA, such as mRNA.
[0015| Also provided are vectors, host cells, and vaccines comprising the mRNAs and/or DNA molecules as disclosed herein, as well as compositions containing the vectors, host cells and vaccines, as well as in vitro and in vivo uses for the DNA, mRNAs, vectors, host cells, vaccines and compositions.
[0016| As shown herein, Applicant increased the overall viability and expression level of mRNA vaccines by the use of chemical modifications such as an m7G cap and/or a polyA tail. Thus, in
one aspect, this disclosure provides the mRNAs further comprising one or more of the m7G cap and/or polyA tail. Additionally, Applicant provides in vitro transcription technology to increase mRNA vaccine yield. Thus, this disclosure provides this as well.
[0017] The mRNA and vectors can be coupled to carrier technology such as for example, branched Histidine-Lysine (HK) polypeptides (HKP) to encapsulate mRNAs, as well as methods for coupling the mRNA to the carrier technology. As disclosed herein, Applicant’s encapsulated mRNA exhibit enhanced specificity and cellular penetration. A combination of mRNA vaccines encapsulated in H3K(+H)4b and MC3/DOTAP liposome carriers are more effective than the liposomes alone.
[0018] In one aspect, provided is an isolated deoxyribonucleic acid (DNA) or an isolated ribonucleic acid (RNA) comprising, or consisting essentially of, or yet further consisting of an open reading frame (ORF) encoding an NYE-SO-1 derived peptide.
[0019] In some embodiments, the isolated DNA encodes one or more of the following: 1) the RNA sequence as set forth in SEQ ID NO: 1 or the peptide as set forth in SEQ ID NO: 2; 2) the
RNA sequence as set forth in SEQ ID NO: 3 or the peptide as set forth in SEQ ID NO: 4; 3) the
RNA sequence as set forth in SEQ ID NO: 5 or the peptide as set forth in SEQ ID NO: 6; or 4) the RNA sequence as set forth in SEQ ID NO: 7 or the peptide as set forth in SEQ ID NO: 8.
[0020] In other embodiments, the isolated RNA comprises one or more of the following: 1) the RNA sequence as set forth in SEQ ID NO: 1; 2) the RNA sequence as set forth in SEQ ID NO: 3; 3) the RNA sequence as set forth in SEQ ID NO: 5; or 4) the RNA sequence as set forth in SEQ ID NO: 7.
[00211 In one aspect, the RNA molecule further comprises a 3'-UTR, a 5'-UTR and optionally further comprises additional elements that (a) stabilize the molecule and (b) enhance expression of the polypeptide encoded by said RNA or open reading frame (ORF). In a further aspect, the 5'-UTR comprises an m7G cap structure and/or a start codon. In a further aspect, the 3'-UTR comprises a stop codon and/or a polyA tail.
[0022] In another aspect, an immunogenic composition is provided that comprises an mRNA as disclosed herein and a carrier such as a pharmaceutically acceptable carrier. In one aspect, the RNA molecule in the composition further comprises a 3'-UTR, a 5'-UTR and optionally further comprises additional elements that (a) stabilize the molecule and (b) enhance expression of the polypeptide encoded by the mRNA or ORF. In a further aspect, the 5'-UTR comprises an m7G cap structure and/or a start codon. In a further aspect, the 3'-UTR comprises a stop codon and/or a poly A tail.
[0023] In a further aspect, the pharmaceutically acceptable carrier is a polymeric nanoparticle and/or a liposomal nanoparticle. Yet further, the carrier comprises a branched Histidine-Lysine (HK) polypeptide (HKP) to encapsulate mRNAs, e.g., a H3K(+H)4b or MC3/DOTAP liposome carrier. In one aspect, both carriers are used in a single composition.
[0024] In another aspect, the mRNA of the immunogenic composition comprising a polymeric nanoparticle and/or a liposomal nanoparticle which comprises a branched Histidine-Lysine (HK) polypeptide (HKP) to encapsulate mRNAs, e.g., a H3K(+H)4b or MC3/DOTAP liposome carrier. In one aspect, both carriers are used in a single composition and the mRNA further comprises a 3'-UTR, a 5'-UTR and optionally further comprises additional elements that (a) stabilize the molecule and (b) enhance expression of the polypeptide encoded by said mRNA or ORF. In a further aspect, the 5'-UTR comprises an m7G cap structure and a start codon. In a further aspect, the m7G cap comprises an m7GpppG structure or an m7GpppGm structure. In another aspect, the 3'-UTR comprises a stop codon and a polyA tail. In a further aspect, the 3’- UTR follows the ORF and comprises the nucleotides AAUAAA.
[0025] In another aspect, the mRNA of the composition may be expressed in a linear in vitro transcription (IVT) system or a plasmid DNA (pDNA) vector delivery system. This linear system also is provided herein.
[0026] In another aspect, the polymeric nanoparticle carrier comprises a Histidine-Lysine copolymer (HKP). In a further aspect, the HKP comprises H3K(+H)4b. The HKP can self assemble into nanoparticles upon admixture. In another aspect, the carrier comprises PLA or PLGA. In a further aspect, the composition contains l,2-Dioleoyl-3-trimethylammonium propane (DOTAP).
[0027| In another aspect, the carrier comprises a Spermine-Lipid Cholesterol (SLiC). In a further aspect, the SLiC is selected from the structures TM1-TM5 shown in FIG. 13.
[0028] In another aspect, provided is a method of treating a subject with a tumor expressing NYE-SO-1 by a method comprising, or consisting essentially of, or yet further consisting of administering to the subject an mRNA or composition as disclosed herein. An above composition can be provided in any acceptable manner as determined by the treating physician, researcher or veterinarian. Non-limiting modes of administration include: intratumorally, intravenously, intramuscularly, intradermally, or subcutaneously. In a further aspect, the subject is a mammal such as for example a human. In a further aspect, the method further comprises an assay to determine if the tumor expresses NYE-SO-1 to identify patients more likely to be treated by the compositions and methods as disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIGs. 1A and IB show the in vitro transcription of NYESO1 vaccine candidates. Four NYESO1 vaccine candidates were designed, synthesized and sequence verified. The plasmids encoding NYESO1 vaccine candidates were digested with Xhol at 37C for 4hr, followed by agarose gel verification (FIG. 1A) and in vitro transcription (FIG. IB). The integrity of mRNA was examined using agarose gel (FIG. IB).
[0030] FIG. 2 shows the in vitro expression of NYESO1 vaccine candidates in 293T cells. Various amounts of NYESO1 vaccine candidate mRNA (0.5ug, lug and 2.5ug) were transfected into 293T cells. 48 hours post transfection, Western blot analysis was performed to detect the in vitro expression of each candidate. Wild-type NYESO1 showed the highest in vitro expression among all candidates.
[0031] FIG. 3 shows an in vivo animal study plan to determine the vaccine dose. Three groups of mice (3 mice/group) were immunized with 1 pg, 5 pg, and 10 pg of NYESO1 vaccine at day 0 and day 21. Two weeks after the immunization, blood was collected, and ELISA assay was performed to determine the immunogenicity of NYESO1 vaccine. At the end of study, mice were sacrificed, spleen was removed for isolation of splenocytes. Then ELISpot, intracellular staining
and cytokine profiling assays were performed to measure T cell response induced by NYES01 vaccine.
[0032] FIG. 4 shows the in vitro expression of NYESO1 vaccine (encoding SEQ ID NO: 2) in 293T cells. Newly formulated NYESO1 vaccine was transfected into 293T cells at 0.5 pg, 1 pg and 2.5 pg. Western Blot analysis was performed to detect in vitro expression of NYESO1 delivered by lipid nanoparticles (LNP).
[0033] FIGs. 5A and 5B show the immunogenicity of NYESO1 (encoding SEQ ID NO: 2) measured by ELISA assay. Serum was collected two weeks post immunization for testing immunogenicity. Serum was serial diluted starting at 1 :200 dilution with 3-fold dilution up to 1 :437400. Dose-dependent immunogenicity ofNYESOl was observed 14 days (FIG. 5A) and 35 days (FIG. 5B) after immunization.
[0034] FIGs. 6A and 6B show the IgG quantification of serum collected at day 35 for vaccine NEYSO1 (encoding SEQ ID NO: 2). IgG amount in sera collected from Day 35 post 1st immunization was measured using anti-CTAGIB antibody as standard. Standard curve (FIG. 6A) was plotted using Sigmoidal, 4PL, X function in GraphPad. The amount of IgG of each immunization dose (FIG. 6B) was calculated by interpolating the standard curve.
[0035] FIGs. 7A and 7B show the IgG subtype determination using ELISA assay for Day 14 sera samples for vaccine NEYSO1 (encoding SEQ ID NO: 2). This figure shows IgG subtypes in sera collected two weeks post 1st immunization (FIG. 7A). Serum was diluted at 1 :200 dilution. The ratio of IgG2a/IgGl was calculated based on OD reading. The result shows that the 1st immunization induced biased Thl response (FIG. 7B).
[0036| FIGs. 8A-8C show the IgG subtype determination using ELISA assay for Day 35 sera samples for vaccine NEYSO1 (encoding SEQ ID NO: 2). This figure shows IgG subtypes in sera collected two weeks post 2nd immunization. Serum was diluted starting at 1 :1800 dilution up to 1 :48600 (FIGs 8A-8B). The ratio of IgG2a/IgGl was calculated based OD reading (FIG. 8C). The result shows that the lug NYESO1 group has biased Thl response, while 5ug and lOug groups showed balanced Thl and Th2 response.
[0037| FIG. 9 shows the ELISpot assay for T cell response for vaccine NEYS01 (encoding SEQ ID NO: 2). At the end of study, spleens from immunized mice were removed, splenocytes were isolated, and stimulated using two peptide pools. One is full set NYESOl peptide mix, and the other is single NYES01 containing amino acids 157-165 of SEQ ID NO: 2. The number of IFNr spots in stimulated splenocytes showed dose-dependent increase with full set NYES01 peptide mix induce more IFNr secreting splenocytes.
[00381 FIG- 10 shows the quantification of IFNr secreting cells in stimulated splenocytes for vaccine NEYSO1 (encoding SEQ ID NO: 2).
[0039] FIGs. 11A-11D show intracellular staining to determine T cell response. At the end of study, spleens from immunized mice were removed, splenocytes were isolated, and stimulated using two peptide pools, one is full setNYESOl peptide mix, the other is single NYESO1 containing amino acids 157-165 of SEQ ID NO: 2. Sixteen hours post stimulation, FACS analysis was performed to detect IFNr and IL4 secreting T cells. The results showed that the T cell response induced by NYESO1 vaccine is CD8+ cell-mediated response (FIGs. 11C and 11D) as opposed to a CD4+ cell-mediated response (FIGs. 11A and 11B).
[0040] FIG. 12 provides a schematic representation of an optimized mRNA vaccine expression structure. This structure can be contained and/or transcribed in a linear in vitro transcription (IVT) expression system or plasmid DNA delivery vector.
(0041] FIG. 13 shows the structures of the Spermine-Lipid Conjugates (SLiC) species.
DETAILED DESCRIPTION OF THE DISCLOSURE
Definitions
[0042] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and/or separating the subject matter described.
[0043] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of this invention will be limited only by the appended claims.
[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.
[0045] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Techique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA
Interference: Technology and Application (CRC Press); and Plotkin et al., Plotkin; Human Vaccines, 7th edition (Elsevier).
[0046] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
100471 As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination such as those that augment or diminish one or more of effectiveness, stability or toxicity for example. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.
[0048] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.
[0049] The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1 % of the specified amount.
[0050] As used herein, comparative terms as used herein, such as high, low, increase, decrease, reduce, or any grammatical variation thereof, can refer to certain variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold,
or about 2 folds, or about 3 folds, or about 4 folds, or about 5 folds, or about 6 folds, or about 7 folds, or about 8 folds, or about 9 folds, or about 10 folds, or about 20 folds, or about 30 folds, or about 40 folds, or about 50 folds, or about 60 folds, or about 70 folds, or about 80 folds, or about 90 folds, or about 100 folds or more higher than the reference. In some embodiments, such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.
1'00511 As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.
[0052] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
[0053] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
[0054] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
[0055] The terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.
[0056] In some embodiments, the terms “first” “second” “third” “fourth” or similar in a component name are used to distinguish and identify more than one components sharing certain
identity in their names. For example, “first RNA” and “second RNA” are used to distinguishing two RNAs.
[0057] The phrase “first line” or “second line” or “third line” refers to the order of treatment received by a patient. First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively. The National Cancer Institute defines first line therapy as “the first treatment for a disease or condition. In patients with cancer, primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008. Typically, a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub- clinical response to the first line therapy or the first line therapy has stopped.
[0058] A “3’ untranslated region” (3’ UTR) refers to a region of an mRNA that is directly downstream (i.e., 3’) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
[0059] The term “adenovirus” is synonymous with the term “adenoviral vector” and refers to viruses of the genus adenoviridiae. The term adenoviridiae refers collectively to animal adenoviruses of the genus mastadenovirus including but not limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera. In particular, human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Adi 1 A and Ad 1 IP), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. The term bovine adenoviruses includes but is not limited to bovine adenovirus types 1, 2, 3, 4, 7, and 10. The term canine adenoviruses includes but is not limited to canine types 1 (strains CLL, Glaxo, R1261, Utrect, Toronto 26-61) and 2. The term equine adenoviruses includes but is not limited to equine types 1 and 2. The term porcine adenoviruses includes but is not limited to porcine types 3 and 4. In one
embodiment of the invention, the adenovirus is derived from the human adenovirus serotypes 2 or 5. For purposes of this invention, adenovirus vectors can be replication-competent or replication deficient in a target cell. In some embodiments, the adenovirus vectors are conditionally or selectively replicating adenoviruses, wherein a gene(s] required for viral replication is/are operatively linked to a cell and/or context-specific promoter. Examples of selectively replicating or conditionally replicating viral vectors are known in the art (see, for example, U.S. Pat. No. 7,691,370).
[0060] A retrovirus such as a gammaretrovirus and/or a lentivirus comprises (a) envelope comprising lipids and glycoprotein, (b) a vector genome, which is an RNA (usually a dimer RNA comprising a cap at the 5’ end and a polyA tail at the 3’ end flanked by LTRs) derived to the target cell, (c) a capsid, and (d) proteins, such as a protease. U.S. Patent No. 6,924, 123 discloses that certain retroviral sequence facilitate integration into the target cell genome. This patent teaches that each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome. The LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from the sequence unique to the 5'end of the RNA. The sizes of the three elements can vary considerably among different retroviruses. For the viral genome, the site of poly (A) addition (termination) is at the boundary between R and U5 in the right hand side LTR. U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
[0061] With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins
MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
[0062] For the production of viral vector particles, the vector RNA genome is expressed from a DNA construct encoding it, in a host cell. The components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the "packaging system", which usually includes either or both of the gag/pol and env genes) expressed in the host cell. The set of sequences required for the production of the viral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways. The techniques involved are known to those skilled in the art.
[9063] The term “adeno-associated virus” or “AAV” as used herein refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered, AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant or synthetic serotypes, e.g., AAV-DJ and AAV PHP.B. The AAV particle comprises, alternatively consists essentially of, or yet further consists of three major viral proteins: VP1, VP2 and VP3. In one embodiment, the AAV refers to of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. These vectors are commercially available or have been described in the patent or technical literature.
[0064] “Plant viruses” as used herein refers to a group of viruses that have been identified as being pathogenic to plants. These viruses rely on the plant host for replication, as they lack the molecular machinery to replicate without the plant host. Accordingly, a plant virus can be used as a vector for safely delivering a gene of interest to a non-plant animal subject. Plant viruses include but are not limited to tobacco mosaic virus, Maize chlorotic mottle virus; Maize rayado fino virus; Oat chlorotic stunt virus; Chayote mosaic tymovirus; Grapevine asteroid mosaic-
associated virus; Grapevine fleck virus; Grapevine Red Globe virus; Grapevine rupestris vein feathering virus; Melon necrotic spot virus; Physalis mottle tymovirus; Prunus necrotic ringspot; Nigerian tobacco latent virus; Tobacco mild green mosaic virus; Tobacco necrosis virus; Eggplant mosaic virus; Kennedya yellow mosaic virus; Lycopersicon esculentum TVM viroid; Oat blue dwarf virus; Obuda pepper virus; Olive latent virus 1; Paprika mild mottle virus; PMMV; Tomato mosaic virus; Turnip vein-clearing virus; Carnation mottle virus; Cocksfoot mottle virus; Galinsoga mosaic virus; Johnsongrass chlorotic stripe mosaic virus;
Odontoglossum ringspot virus; Ononis yellow mosaic virus; Panicum mosaic virus; Poinsettia mosaic virus; Pothos latent virus; or Ribgrass mosaic virus.
|'0065| Gene delivery vehicles also include DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods disclosed herein. In addition to the delivery of polynucleotides to a cell or cell population, direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins disclosed herein are other non-limiting techniques.
[0066] The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. In some embodiments, the term refers to the ribonucleotide modifications in naturally occurring 5'-terminal mRNA cap moieties. In further embodiments, the chemical modification is selected from pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy -pseudouridine, 4-thio-l- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine, 5-methoxyuridine, or 2'-O-methyl uridine. In some embodiments the extent of incorporation of chemically modified nucleotides has been optimized for improved immune
responses to the vaccine formulation. In other embodiments, the term excludes the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moi eties.
[0067] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
|0068| In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of residues of the RNA is chemically modified by one or more of modifications as disclosed herein. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of uridine residues of the RNA is chemically modified by one or more of modifications as disclosed herein.
[0069] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide
base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some embodiments, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.
[0070| As used herein, “NYE-SO-1” refers to an 18-kDa protein with 180 amino acids featuring a glycine-rich N-terminal region and a strongly hydrophobic C-terminal region with a Pcc-1 domain. The sequence of wild type NYE-SO-1 is provided as SEQ ID NO: 2. NYE-SO-1 is a member of a family of antigens known as cancer-testis antigens (CTAs). NY-ESO-1 expression has been reported in a wide range of tumor types, including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer.
[0071] The term “NYESO1 peptide mix” is a pool of 43 peptides derived from a peptide scan (15mers with 11 amino acid overlap) through human NYE-SO-1. This can be used to restimulate the splenocytes to measure T-cell responses.
[0072] A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or
the like and include carriers, such as pharmaceutically acceptable carriers. In some embodiments, the carrier (such as the pharmaceutically acceptable carrier) comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier (for example, an HKP nanoparticle) or a lipid nanoparticle (LNP).
[00731 Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e g., sugars, including monosaccharides, di-, tri, tetraoligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
[0074] A composition as disclosed herein can be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
[0075] The term “culturing” refers to the in vitro or ex vivo propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.
[0076| In some embodiments, the cell as disclosed herein is a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293 T cell, or an a549 cell. In some embodiments, the cell is a host cell.
|0077| “Detectable label”, “label”, “detectable marker” or “marker” are used interchangeably, including, but not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, protein or composition described herein.
[0078] As used herein, “Dioleoyl-3 -trimethylammonium propane” (DOTAP) refers to a cationic lipid surfactant that is commonly involved with liposomal transfection of DNA and RNA molecules.
[0079] An “effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two or more agents, that, when administered for the treatment of a mammal or other subject, is sufficient to effect such treatment for the disease. The “effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
[0080] The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed to produce the mRNA for the polypeptide or a fragment thereof, and optionally translated to produce the polypeptide or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. Further, as used herein an amino acid sequence coding sequence refers to a nucleotide sequence encoding the amino acid sequence.
[0081] In some embodiments, the term “engineered” or “recombinant” refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain or the parental host strain of the referenced species. In some embodiments, the term “engineered” or “recombinant” refers to being synthetized by human intervention. As used herein, the term “recombinant protein” refers to a polypeptide
which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
[0082] As used herein, the term “enhancer”, as used herein, denotes sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed.
An enhancer may enhance transcription from a single promoter or simultaneously from more than one promoter. As long as this functionality of improving transcription is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of wild-type activity, that is, activity of a full-length sequence), any truncated, mutated or otherwise modified variants of a wild-type enhancer sequence are also within the above definition.
[9083] Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, canine, bovine, porcine, murine, rat, avian, reptilian and human.
[0084] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of long-term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.
[0085] The term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins. As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[0086| As used herein, the term “F2A self-cleaving peptide” or “T2A self-cleaving peptide” refers to a class of 18-22 aa-long peptides, which can induce ribosomal skipping during translation of a protein in a cell. The apparent cleavage is triggered by ribosomal skipping of the peptide bond between the Proline (P) and Glycine (G) in C-terminal of 2A peptide.
10087] A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
[0088] A “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated. In some embodiments, the gene product may refer to an mRNA or other RNA, such as an interfering RNA, generated when a gene is transcribed.
[0089] As used herein, “H3K(+H)4b” refers to a specific type of HKP in which the dominant repeating sequence in its terminal branch is -HHHK- (SEQ ID NO: 82), there are four -HHHK- motifs (SEQ ID NO: 82) in each branch and one extra histidine is inserted in the second -HHHK- motif (SEQ ID NO: 82) of the terminal branch of H3K4b.
[0090] As used herein, “Histidine-Lysine co-polymer” (HKP) refers to a type of polymeric nanoparticle that utilizes a polymer of histidine and lysine to deliver bio-relevant molecules to target cells. The HK polymer comprises four short peptide branches linked to a three-lysine amino acid core. The peptide branches consist of histidine and lysine amino acids, in different configurations. The general structure of these histidine-lysine peptide polymers (HK polymers) is shown in Formula I, where R represents the peptide branches and K is the amino acid L-lysine.
[0(192] In Formula I where K is L-lysine and each of Ri, Rr, Rr and R4 is independently a histidine-lysine peptide. The R1-4 branches may be the same or different in the HK polymers of
the invention. When an R branch is “different”, the amino acid sequence of that branch differs from each of the other R branches in the polymer.
[0093] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non- homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. In some embodiments, the identity is calculated between two peptides or polynucleotides over their full-length, or over the shorter sequence of the two, or over the longer sequence of the two.
[0094] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; fdter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non- redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on August 1, 2021.
[0095] In some embodiments, the polynucleotide as disclosed herein is an RNA or an analog thereof. In some embodiments, the polynucleotide as disclosed herein is a DNA or an analog
thereof. In some embodiments, the polynucleotide as disclosed herein is a hybrid of DNA and RNA or an analog thereof.
[0096] In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide encodes the same sequence encoded by the reference. In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide hybridizes to the reference, a complement reference, a reverse reference, or a reverse-complement reference, optionally under conditions of high stringency.
[0097] Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 70% sequence identity, or at least 75% sequence identity, or at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence, or alternatively at least 99 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively an equivalent nucleic acid hybridizes under conditions of high stringency to a reference polynucleotide or its complementary. In one aspect, the equivalent must encode the same protein or a functional equivalent of the protein that optionally can be identified through one or more assays described herein. In addition or alternatively, the equivalent of a polynucleotide would encode a protein or polypeptide of the same or similar function as the reference or parent polynucleotide.
10098] “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The host cell can be a prokaryotic or a eukaryotic cell. In some embodiments, the host cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293 T cell, or an a549 cell. Cultured cells lines are commercially available from the American Type Culture Collection, for example.
[0099| “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
|0.1.00] Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg2+ normally found in a cell.
[0101 ] Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O.lx SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15
M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
[0102] When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called “annealing” and those polynucleotides are described as “complementary .” A double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. “Complementarity” or “homology” (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
[0103] “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The host cell can be a prokaryotic or a eukaryotic cell. In some embodiments, the host cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293 T cell, or an a549 cell. Cultured cells lines are commercially available from the American Type Culture Collection, for example.
[0104] As used herein, “immune cells” includes, e.g., white blood cells (leukocytes, such as granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes (T cells, B cells, natural killer (NK) cells and NKT cells)) which may be derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells, and NKT cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). In some embodiments, the immune cell is derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSc), hematopoietic stem cells (HSCs), or immortalized cells. In some embodiments, the HSC are derived from umbilical cord blood of a
subject, peripheral blood of a subject, or bone marrow of a subject. In some embodiments, the subject from whom the immune cell is directly or indirectly obtained is the same subject to be treated. In some embodiments, the subject from whom the immune cell is directly or indirectly obtained is different from the subject to be treated. In further embodiments, the subject from whom the immune cell is directly or indirectly obtained is different from the subject to be treated and the subjects are from the same species, such as human.
[0105] “Immune response” broadly refers to the antigen-specific responses of lymphocytes to foreign substances. The terms “immunogen” and “immunogenic” refer to molecules with the capacity to elicit an immune response. All immunogens are antigens, however, not all antigens are immunogenic. An immune response disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro. The skilled artisan will understand that a variety of macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to be immunogenic. The skilled artisan will further understand that nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen. The artisan will further understand that immunogens are not limited to full-length molecules, but may include partial molecules.
[0106] As used herein, the term “immunoconjugate” comprises an antibody or an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
[0107| As used herein, “immunogenic composition” refers to a composition that induces clinically significant effector T cell activity against protein targets relevant to cancer cell activities. In general, “immune response” broadly refers to the antigen-specific responses of lymphocytes to foreign substances. The terms “immunogen” and “immunogenic” refer to molecules with the capacity to elicit an immune response. All immunogens are antigens, however, not all antigens are immunogenic. An immune response disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro. The skilled artisan will understand that a variety of macromolecules,
including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to be immunogenic. The skilled artisan will further understand that nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen. The artisan will further understand that immunogens are not limited to full-length molecules, but may include partial molecules. The immune responses elicited can be measured by one of skill in the art, e.g., as illustrated in the Examples, antibodies can be generated in the immune responses; such antibodies can binds to the immunogen; and accordingly, an enzyme- linked immunosorbent assay (ELISA) can be used to detect and/or quantify the immunogen specific antibodies.
|'0108| As used herein, the term “immunoregulatory molecule” may refer to any molecule that may regulate or directly influence immune responses, including but not limited to chemokines such as CCL2, CCL5, CCL14, CCL19, CCL20, CXCL8, CXCL13, and LEC; lymphokines and cytokines such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), interferons a, P and y, factors stimulating cell growth (e.g., GM-CSF), and other factors (e.g., tumor necrosis factors, DC-SIGN, MIPla, MIPip, TGF-P or TNF); factors that provide co-stimulatory signals for T-cell activation such as B7 molecules and CD40; accessory molecules such as CD83; proteins involved in antigen processing and presentation such as TAP1/TAP2 transporter proteins, proteosome molecules such as LMP2 and LMP7, heat shock proteins such as gp96, HSP70 and HSP90, and MHC or HLA molecules; and biological equivalents thereof.
[0109] As used herein, “intro transcription system” (IVT) refers to a protein production system that occurs outside a living cell that typically involves a composition that includes molecules necessary for the transcription and/or translation of target proteins. IVT involves template- directed synthesis of RNA molecules of almost any sequence. The size of the RNA molecules that can be synthesized using IVT methods range from short oligonucleotides to long nucleic acid polymers of several thousand bases. IVT methods permit synthesis of large quantities of RNA transcript (e.g., from microgram to milligram quantities) (Beckert et al., Methods Mol Biol. 703:29-41(2011); Rio et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220; and Cooper, Geoffery M. The Cell: A Molecular
Approach. 4th ed. Washington D C.: ASM Press, 2007, 262-299). Generally, IVT utilizes a DNA template featuring a promoter sequence upstream of a sequence of interest. The promoter sequence is most commonly of bacteriophage origin (ex. the T7, T3 or SP6 promoter sequence) but many other promotor sequences can be tolerated including those designed de novo. Transcription of the DNA template is typically best achieved by using the RNA polymerase corresponding to the specific bacteriophage promoter sequence. Exemplary RNA polymerases include, but are not limited to T7 RNA polymerase, T3 RNA polymerase, or
SP6 RNA polymerase, among others. IVT is generally initiated at a dsDNA but can proceed on a single strand.
|0110| The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides, proteins and/or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, or protein, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, or protein, does not require “isolation” to distinguish it from its naturally occurring counterpart.
[0111] As used herein, the term “label” or a detectable label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histidine tags (N-His), magnetically active isotopes, e.g., 115Sn, 117Sn and 119Sn, a non-radioactive isotopes such as 13C and 15N, polynucleotide or protein such as an antibody so as to generate a “labeled” composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be
detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high- throughput screening. As such, suitable labels include, but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected, or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.
[0112] Examples of suitable fluorescent labels include, but are not limited to fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).
[0113] In some embodiments, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of
which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
[0114] As used herein, a purification label or maker refers to a label that may be used in purifying the molecule or component that the label is conjugated to, such as an epitope tag (including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag), an affinity tag (including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)), or a fluorescent tag.
101151 A “selection marker” refers to a protein or a gene encoding the protein necessary for survival or growth of a cell grown in a selective culture regimen. Typical selection markers include sequences that encode proteins, which confer resistance to selective agents, such as antibiotics, herbicides, or other toxins. Examples of selection markers include genes for conferring resistance to antibiotics, such as spectinomycin, streptomycin, tetracycline, ampicillin, kanamycin, G 418, neomycin, bleomycin, hygromycin, methotrexate, dicamba, glufosinate, or glyphosate.
[0116] As used herein, “liposomal nanoparticle” (LNP) refers to a nanoparticle typically comprise, or alternatively consist essentially of, or yet further consist of a lipid, in particular, an ionizable cationic lipid
[0117] As used herein, “liposome” refers to one or more lipids forming a complex, usually surrounded by an aqueous solution. Liposomes are generally spherical structures comprising lipids fatty acids, lipid bilayer type structures, unilamellar vesicles and amorphous lipid vesicles. Generally, liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. The liposomes may be unilamellar vesicles (possessing a single bilayer membrane), oligolamellar or multilamellar (an onion-like structure characterized by multiple membrane bilayers, each separated from the next by an aqueous layer).
[0118] As used herein, “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer
of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. In some embodiments, an mRNA as disclosed herein comprises, or consists essentially of, or yet further consists of at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly- A tail.
|0119| As used herein, the term “micelle” refers to a polymer assembly comprised of a hydrophilic shell (or corona) and a hydrophobic and/or ionic interior. In addition, the term micelle may refer to any poly ion complex assembly consisting of a multiblock copolymer possessing a net positive charge and a suitable negatively charged polynucleotide.
[ 0120] As used herein, a “normal cell corresponding to the cancer tissue type” refers to a normal cell from a same tissue type as the cancer tissue. A non-limiting example is a normal leukocyte from a patient, e.g., a patient with leukemia.
[0121] As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For purposes of clarity, when referring herein to a nucleotide of a nucleic acid, which can be DNA or an RNA, the terms “adenosine”, “cytidine”, “guanosine”, and “thymidine” are used. It is understood that if the nucleic acid is RNA, a nucleotide having a uracil base is uridine.
[0122] As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
[0123] The terms “oligonucleotide” or “polynucleotide”, or “portion,” or “segment” thereof refer to a stretch of polynucleotide residues which is long enough to use in PCR or various
hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules. The polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotri esters, phosphoamidates, carbamates, etc ), charged linkages (e g., phosphorothioates, phosphorodithioates, etc ), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
[0124] As used herein, “open reading frame” (ORF) refers to a sequence of nucleotides that encodes a polypeptide or a portion thereof. In some embodiments, the ORF is an mRNA.
[0125] “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.
[0126] In some embodiments, an RNA as disclosed herein is “optimized”. Optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide.
[0127] As used herein the term “PD-1” refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, or alternatively 90% sequence identity, or alternatively at least 95% sequence identity with the PD-1 sequence as shown herein and/or a suitable binding partner of PD-L1. Non-limiting example sequences of PD-1 are provided herein, such as but not limited to those under the following reference numbers - GCID:GC02M241849; HGNC: 8760; Entrez Gene: 5133; Ensembl: ENSG00000188389; OMIM: 600244; and UniProtKB: QI 5116 - and the sequence:
|0128| MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFT CSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVV RARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVV GVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQ WREKTPEPP VPC VPEQTEYATIVFP SGMGT S SP ARRGS ADGPRS AQPLRPEDGHC S WPL (SEQ ID NO: 101), and equivalents thereof.
[0129] Non-limiting examples of commercially available antibodies thereto include pembrolizumab (Merck), nivolumab (Bristol-Myers Squibb), pidilizumab (Cure Tech), AMP- 224 (GSK), AMP-514 (GSK), PDR001 (Novartis), and cemiplimab (Regeneron and Sanofi).
[0130] As used herein the term “PD-L1” refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, or alternatively 90% sequence identity, or alternatively at least 95% sequence identity with the PD-L1 sequence as shown herein and/or an suitable binding partner of PD-1. Non-limiting example sequences of PD-L1 are provided herein, such as but not limited to those under the following reference numbers - GCID: GC09P005450; HGNC: 17635; Entrez Gene: 29126; Ensembl: ENSG00000120217; OMIM: 605402; and UniProtKB: Q9NZQ7 - and the sequence:
[01311 MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQF VHGEEDLK VQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYR CMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDH
QVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHP PNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 102), and equivalents thereof. Non-limiting examples of commercially available antibodies thereto include atezolizumab (Roche Genentech), avelumab (Merck Soreno and Pfizer), durvalumab (AstraZeneca), BMS-936559 (Bristol-Myers Suibb), and CK-301 (Chekpoint Therapeutics).
[0132] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. In some embodiments, a pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier (for example, an HKP nanoparticle) or a lipid nanoparticle (LNP). Additionally or alternatively, pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
|0.t.33] A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act
as toxins under similar circumstances. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.
|'0134| As used herein, “plasmid DNA vector delivery system” (pDNA) refers to an extra- chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and doublestranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass- producing a gene or the protein it then codes for.
[0135] In some embodiments, an mRNA further comprises a polyA tail. A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3’), from the 3’ UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300
adenosine monophosphates (SEQ ID NO: 103). Additionally or alternatively, in a relevant biological setting (e.g., in cells, in vivo) the polyA tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation. In some embodiments, a polyA tail as used herein comprises, or consists essentially of, or yet further consists of one or more of the following:
[0136] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA (SEQ ID NO: 12); or
[0137] CGGCAAUAAAAAGACAGAAUAAAACGCACGGUGUUGGGUCGUUUGUUC (SEQ ID NO: 13).
[0138] As used herein, poly(lactic-co-glycolic acid) or polylactic acid (PLGA or PGA) refers to a specific type of polymeric nanoparticle that consists of various ratios of glycolic and lactic acid. These molecules are known for biocompatibility and biodegradability.
[0139] As used herein, “polymeric nanoparticle” refers to a nanoparticle composed of polymer compound (e.g., compound composed of repeated linked units or monomers) including any organic polymers, such as a Histidine-Lysine (HK) polypeptide (HKP).a polymer-based particle that can transport a bio-relevant molecule either within the particle or in a surface-adsorbed manner.
[0140] “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells’ genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about l-2pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coll bacterium, and Salmonella bacterium. Cultured cells lines are commercially available from the American Type Culture Collection, for example.
[01411 The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
|0142| As used herein, an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” or “aligned to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence. Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST. In one aspect, equivalent polynucleotides, proteins and corresponding sequences can be determined using BLAST (accessible at blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on August 1, 2021).
[0143] As used herein, an amino acid mutation is referred to herein as two letters separated by an integer, such as L19F. The first letter provides the one letter code of the original amino acid residue to be mutated; while the last letter provides the mutation, such as A indicating a deletion, or one letter code of the mutated amino acid residue. In some embodiments, the integer is the numbering of the to-be-mutated amino acid residue in the amino acid sequence free of the mutation, optionally counting from the N terminus to the C terminus. In some embodiments, the integer is the numbering of the mutated amino acid residue in the mutated amino acid sequence, optionally counting from the N terminus to the C terminus.
[0144] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof’ is intended to be synonymous with “equivalent thereof’
when referring to a reference protein, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity, or at least about 85 % homology or identity, or alternatively at least about 90 % homology or identity, or alternatively at least about 95 % homology or identity, or alternatively at least about 96 % homology or identity, or alternatively at least about 97 % homology or identity, or alternatively at least about 98 % homology or identity, or alternatively at least about 99 % homology or identity (in one aspect, as determined using the Clustal Omega alignment program) and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complementary sequence.
|0145| An equivalent of a reference polypeptide comprises, consists essentially of, or alternatively consists of an polypeptide having at least 80%, or at least 85 %, or at least 90%, or at least 95%, or at least about 96%, or at least 97%, or at least 98%, or at least 99% amino acid identity to the reference polypeptide (as determined, in one aspect using the Clustal Omega alignment program), or a polypeptide that is encoded by a polynucleotide that hybridizes under conditions of high stringency to the complementary sequence of a polynucleotide encoding the reference polypeptide, optionally wherein conditions of high stringency comprises incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0. lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O.lx SSC, or deionized water.
|0146| In some embodiments, a first sequence (nucleic acid sequence or amino acid) is compared to a second sequence, and the identity percentage between the two sequences can be calculated. In further embodiments, the first sequence can be referred to herein as an equivalent and the second sequence can be referred to herein as a reference sequence. In yet further embodiments, the identity percentage is calculated based on the full-length sequence of the first
sequence. In other embodiments, the identity percentage is calculated based on the full-length sequence of the second sequence.
[0147] In some embodiments, an equivalent of a reference polypeptide comprises, or consists essentially of, or yet further consists of the reference polypeptide with one or more amino acid residues replaced by a conservative substitution. The substitution can be “conservative” in the sense of being a substitution within the same family of amino acids. The naturally occurring amino acids can be divided into the following four families and conservative substitutions will take place within those families.
(1) Amino acids with basic side chains: lysine, arginine, histidine.
(2) Amino acids with acidic side chains: aspartic acid, glutamic acid
(3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine.
(4) Amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.
[0148] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and
single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
[0149] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
[0150] As used herein, the term “purification marker” refers to at least one marker useful for purification or identification. A non-exhaustive list of this marker includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein. Suitable direct or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten
[0151 [ As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid, peptide, protein, biological complexes or other active compound is one that is isolated in whole or in part from proteins or other contaminants. Generally, substantially purified peptides, proteins, biological complexes, or other active compounds for use within the disclosure comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide, protein, biological complex or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceutical formulation for therapeutic administration. More typically, the peptide, protein, biological complex or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation may be
essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.
[0152] The term “a regulatory sequence”, “an expression control element” or “promoter” as used herein, intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed or replicated, and facilitates the expression or replication of the target polynucleotide.
10153] A promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. In some embodiments, a promoter as used herein is corresponding to the RNA polymerase. In further embodiments, a promoter as sued herein comprises, or consists essentially of, or yet further consists of a T7 promoter, or a SP6 promoter, or a T3 promoter. Non-limiting examples of suitable promoters are provided in W02001009377A1.
[0154] The term “RNA” as used herein refers to its generally accepted meaning in the art. Generally, the term RNA refers to a polynucleotide comprising at least one ribofuranoside moiety. The term can include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, for example at one or more nucleotides of the RNA. Nucleotides in the nucleic acid molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. In some embodiments, the RNA is a messenger RNA (mRNA).
[0155] “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. In some embodiments, an mRNA as disclosed herein comprises, or consists essentially of,
or yet further consists of at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly -A tail.
[0156] Vaccination is the most successful medical approach to disease prevention and control. The successful development and use of vaccines has saved thousands of lives and large amounts of money. A key advantage of RNA vaccines is that RNA can be produced in the laboratory from a DNA template using readily available materials, less expensively and faster than conventional vaccine production, which can require the use of chicken eggs or other mammalian cells. In addition, mRNA vaccines have the potential to streamline vaccine discovery and development, and facilitate a rapid response to emerging infectious diseases, see, for example, Maruggi et al., Mol Ther. 2019; 27(4):757-772.
[0157] Preclinical and clinical trials have shown that mRNA vaccines provide a safe and long- lasting immune response in animal models and humans. mRNA vaccines against infectious diseases may be developed as prophylactic or therapeutic treatments. mRNA vaccines expressing antigens of infectious pathogens have been shown to induce potent T cell and humoral immune responses. See, for example, Pardi et al., Nat Rev Drug Discov. 2018; 17:261-279. The production procedure to generate mRNA vaccines is cell-free, simple, and rapid, compared to production of whole microbe, live attenuated, and subunit vaccines. This fast and simple manufacturing process makes mRNA a promising bio-product that can potentially fill the gap between emerging infectious disease and the desperate need for effective vaccines.
|0.l58] “Separate use” as used herein refers to the administration, at the same time, of the two compounds of the composition according to the invention in distinct pharmaceutical forms.
[0159] “Sequential use” as used herein refers to the successive administration of the two compounds of the composition according to the invention, each in a distinct pharmaceutical form.
[01.60] A “subject” of diagnosis or treatment is a cell or an animal such as a mammal, or a human. A subject is not limited to a specific species and includes non-human animals subject to diagnosis or treatment and are those subject to infections or animal models, for example,
simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals, and pets. Human patients are included within the term as well.
[0161 ] “ Simultaneous use” as used herein refers to the administration of the two compounds of the composition according to the invention in a single and identical pharmaceutical form or at the same time in two distinct pharmaceutical forms.
|01621 As used herein, “solid tumor” refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant, metastatic or non- metastatic. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.
[0163] As used herein, Spermine-Lipid Cholesterol (SLiC) refers to a specific type of a liposomal nanoparticle. Exemplary molecules can be found in FIG. 13.
[0164] A “therapeutic agent” encompasses biological agents, such as an antibody, a peptide, a protein, an enzyme, and chemotherapeutic agents. The therapeutic agent also encompasses immuno-conjugates of cell-binding agents (CBAs) and chemical compounds, such as antibodydrug conjugates (ADCs).
[0165] In one aspect, the additional therapeutic is a chemotherapeutic agent. A “chemotherapeutic agent,” as used herein, refers to a substance which, when administered to a subject, treats or prevents the development of cancer in the subject's body.
[01 6] Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenesis agents, anti-estrogens, anti -androgens or immunomodulators.
[0.167] “Cytoreductive therapy,” as used herein, includes but is not limited to chemotherapy, cryotherapy, and radiation therapy. Agents that act to reduce cellular proliferation are known in the art and widely used. Chemotherapy drugs that kill cancer cells only when they are dividing are termed cell-cycle specific. These drugs include agents that act in S-phase, including topoisomerase inhibitors and anti-metabolites.
[0168| Topoisomerase inhibitors are drugs that interfere with the action of topoisomerase enzymes (topoisomerase I and II). During the process of chemo treatments, topoisomerase enzymes control the manipulation of the structure of DNA necessary for replication and are thus cell cycle specific. Examples of topoisomerase I inhibitors include the camptothecan analogs listed above, irinotecan and topotecan. Examples of topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.
(0169) Antimetabolites are usually analogs of normal metabolic substrates, often interfering with processes involved in chromosomal replication. They attack cells at very specific phases in the cycle. Antimetabolites include folic acid antagonists, e.g., methotrexate; pyrimidine antagonist, e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine; purine antagonist, e.g., 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitor, e.g., cladribine, fludarabine, nelarabine and pentostatin; and the like.
[0170] Plant alkaloids are derived from certain types of plants. The vinca alkaloids are made from the periwinkle plant Catharanthus rosed). The taxanes are made from the bark of the Pacific Yew tree (taxus). The vinca alkaloids and taxanes are also known as anti microtubule agents. The podophyllotoxins are derived from the May apple plant. Camptothecan analogs are derived from the Asian “Happy Tree” (Camptotheca acuminata). Podophyllotoxins and camptothecan analogs are also classified as topoisomerase inhibitors. The plant alkaloids are generally cell-cycle specific.
[01711 Examples of these agents include vinca alkaloids, e.g., vincristine, vinblastine and vinorelbine; taxanes, e.g., paclitaxel and docetaxel; podophyllotoxins, e.g., etoposide and tenisopide; and camptothecan analogs, e.g., irinotecan and topotecan.
[0172] Cryotherapy includes, but is not limited to, therapies involving decreasing the temperature, for example, hypothermic therapy.
[0173] Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art. Exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more than about 10 Gy
and/or a dose of ultraviolet radiation at a range from at least about 5 J/m2 to not more than about 50 J/m2, usually about 10 J/m2.
[0174] As used herein, a biological sample, or a sample, can be obtained from a subject, cell line or cultured cell or tissue. Exemplary samples include, but are not limited to, cell sample, tissue sample, tumor biopsy, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In some instances, the sample is a tumor/cancer biopsy.
[0175] The term “transduce” or “transduction” refers to the process whereby a foreign nucleotide sequence is introduced into a cell. In some embodiments, this transduction is done via a vector, viral or non-viral.
[0176] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. Treatments containing the disclosed compositions and methods can be first line, second line, third line, fourth line, fifth line therapy
and are intended to be used as a sole therapy or in combination with other appropriate therapies. In one aspect, “treatment” excludes prevention. When the disease is cancer, the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor.
[0177] “Under transcriptional control”, which is also used herein as “directing expression of’ or any grammatical variation thereof, is a term well understood in the art and indicates that transcription and optionally translation of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription.
[0178] An "RNA polymerase" refers to an enzyme that produces a polyribonucleotide sequence, complementary to a pre-existing template polynucleotide (DNA or RNA). In some embodiments, the RNA polymerase is a bacteriophage RNA polymerase, optionally a T7 RNA polymerase, or a SP6 RNA polymerase, or a T3 RNA polymerase. Non-limiting examples of suitable polymerase are further detailed in US10526629B2.
[0179] In some embodiments, the term “vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and optionally integrate into the target cell’s genome. Non-limiting examples of vectors include a plasmid, a nanoparticle, a liposome, a virus, a cosmid, a phage, a BAC, a YAC, etc. In some embodiments, plasmid vectors may be prepared from commercially available vectors. In other embodiments, viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector. In one embodiment, the viral vector is a retroviral vector. In one embodiment, the vector is a plasmid. In one embodiment, the vector is a nanoparticle, optionally a polymeric nanoparticle or a lipid nanoparticle.
[0180] Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.)
and Promega Biotech (Madison, Wis.). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
[0181] A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. As is known to those of skill in the art, there are 6 classes of viruses. The DNA viruses constitute classes I and II. The RNA viruses and retroviruses make up the remaining classes. Class III viruses have a double-stranded RNA genome. Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA Class V viruses have a negative singlestranded RNA genome used as a template for mRNA synthesis. Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827. As used herein, Multiplicity of infection (MOI) refers to the number of viral particles that are added per cell during infection.
[0182] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in
association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.
[0183] A combination as used herein intends that the individual active ingredients of the compositions are separately formulated for use in combination and can be separately packaged with or without specific dosages. The active ingredients of the combination can be administered concurrently or sequentially.
[0184] The four-branched histidine-lysine (HK) peptide polymer H2K4b has been shown to be a good carrier of large molecular weight DNA plasmids (Leng et al. Nucleic Acids Res 2005;
33 :e40.), but a poor carrier of relatively low molecular weight siRNA (Leng et al. J Gene Med 2005; 7:977-986.). Two histidine-rich peptides analogs of H2K4b, namely H3K4b and H3K(+H)4b, were shown to be effective carriers of siRNA (Leng et al. J Gene Med 2005; 7: 977-986. Chou et al. Biomaterials 2014; 35:846-855.), although H3K(+H)4b appeared to be modestly more effective (Leng et al. Mol Ther 2012; 20:2282-2290.). Moreover, the H3K4b carrier of siRNA induced cytokines to a significantly greater degree in vitro and in vivo than H3K(+H)4b siRNA polyplexes (Leng et al. Mol Ther 2012; 20:2282-2290.). Suitable HK polypeptides are described in WO/2001/047496, WG/2003/090719, and WG/2006/060182, the contents of each of which are incorporated herein in their entireties. These polypeptides have a lysine backbone (three lysine residues) where the lysine side chain s-amino groups and the N- terminus are coupled to various HK sequences. HK polypeptide carriers can be synthesized by methods that are well-known in the art including, for example, solid-phase synthesis.
[0185] It was found that such histidine-lysine peptide polymers (“HK polymers” or “HKP”) were surprisingly effective as mRNA carriers, and that they can be used, alone or in combination with liposomes, to provide effective delivery of mRNA into target cells. Similar to PEI and other carriers, initial results suggested HK polymers differ in their ability to carry and release nucleic acids. However, because HK polymers can be reproducibly made on a peptide synthesizer, their amino acid sequence can be easily varied, thereby allowing fine control of the binding and release of RNAs, as well as the stability of polypi exes containing the HK polymers and RNA (Chou et al. Biomaterials 2014; 35:846-855. Midoux et al. Bioconjug Chem (1999) 10:406-411; Henig et al. Journal of American Chemical Society (1999) 121:5123-5126). When mRNA molecules are admixed with one or more HKP carriers the components self-assemble into nanoparticles.
[0186] As described herein, advantageously the HK polymer comprises four short peptide branches linked to a three-lysine amino acid core. The peptide branches consist of histidine and lysine amino acids, in different configurations. The general structure of these histidine-lysine peptide polymers (HK polymers) is shown in Formula I, where R represents the peptide branches and K is the amino acid L-lysine.
Formula I
[0187] In Formula I where K is L-lysine and each of Ri, Ri, R3 and R4 is independently a histidine-lysine peptide. The R1-4 branches may be the same or different in the HK polymers of the invention. When a R branch is “different”, the amino acid sequence of that branch differs from each of the other R branches in the polymer. Suitable R branches used in the HK polymers of the invention shown in Formula I include, but are not limited to, the following R branches RA - R-J:
RA = KHKHHKHHKHHKHHKHHKHK- ( SEQ ID NO : 14)
RB = KHHHKHHHKHHHKHHHK- (SEQ ID NO : 15)
Rc = KHHHKHHHKHHHHKHHHK- ( SEQ ID NO : 16)
RD = k H H H k H H H k H H H Hk H H H k- ( SEQ ID NO : 17)
RE = HKHHHKHHHKHHHHKHHHK- ( SEQ ID NO : 18)
RF = HHKHHHKHHHKHHH HKHHHK- (SEQ ID NO : 19)
RG = KHHHHKHHHHKHHHHKHHHHK- (SEQ ID NO: 20)
RH = KHHHKHHHKHHHKHHHHK- (SEQ ID NO: 21)
Ri = I<HHHI<HHHHI<H HHKHHHK- (SEQ ID NO : 22)
Rj = KHHHKHHHHKHHHKHHHHK- (SEQ ID NO: 23)
[0188| Specific HK polymers that may be used in the mRNA compositions include, but are not limited to, HK polymers where each of Ri, R2, R3 and R4 is the same and selected from RA - Rj (Table 1). These HK polymers are termed H2K4b, H3K4b, H3K(+H)4b, H3k(+H)4b, H- H3K(+H)4b, HH-H3K(+H)4b, H4K4b, H3K(1+H)4b, H3K(3+H)4b and H3K(1,3+H)4b, respectively. In each of these 10 examples, upper case “K” represents a L-lysine, and lower case “k” represents D-lysine. Extra histidine residues, in comparison to H3K4b, are underlined within the branch sequences. Nomenclature of the HK polymers is as follows:
1) for H3K4b, the dominant repeating sequence in the branches is -HHHK- (SEQ ID NO: 82), thus “H3K” (SEQ ID NO: 82) is part of the name; the “4b” refers to the number of branches;
2) there are four -HHHK- (SEQ ID NO: 82) motifs in each branch of H3K4b and analogues; the first -HHHK- motif (SEQ ID NO: 82) (“1”) is closest to the lysine core;
3) H3K(+H)4b is an analogue of H3K4b in which one extra histidine is inserted in the second -HHHK- motif (SEQ ID NO: 82) (motif 2) of H3K4b;
4) for H3K(1+H)4b and H3K(3+H)4b peptides, there is an extra histidine in the first (motif 1) and third (motif 3) motifs, respectively;
5) for H3K(1,3+H)4b, there are two extra histidines in both the first and the third motifs of the branches.
Table 1
Polymer Branch Sequence Sequence
Identifier
H2K4b RA = KHKHHKHHKHHKHHKHHKHK- (SEQ ID NO: 14)
H3K4b RB = KHHHKHHHKHHHKHHHK- (SEQ ID NO : 15)
H3K(+H)4b Rc = KHHHKHHHKHHHHKHHHK- (SEQ ID NO : 16)
H3k(+H)4b RD = kHHHkHHHkHHHHkHHHk- (SEQ ID NO : 17)
H-H3K(+H)4b RE = HK HHHK HHHK HHHHK HHHK- (SEQ ID NO: 18)
HH-H3K(+H)4b RF = HHKHHHKHHHKHHHHKHHHK- (SEQ ID NO: 19)
H4K4b RG = KHHHHK HHHHI<HHHHI<HHHHI< - (SEQ ID NO: 20)
H3K(1+H)4b RH = KHHHKHHHKHHHKHHHHK- (SEQ ID NO: 21)
H3K(3+H)4b Ri = KHHHKHHHHKHHHKHHHK- (SEQ ID NO: 22)
H3K(1,3+H)4b Rj = KHHHKHHHHKHHHKHHHHK- (SEQ ID NO: 23)
[0189] Methods well known in the art, including gel retardation assays, heparin displacement assays and flow cytometry can be performed to assess performance of different formulations containing HK polymer plus liposome in successfully delivering mRNA. Suitable methods are described in, for example, Gujrati et al, Mol. Pharmaceutics 11 :2734-2744 (2014), and Parnaste et al., Mol Ther Nucleic Acids. 7: 1-10 (2017).
[0190] Detection of mRNA uptake into cells can also be achieved using SmartFlare® technology (Millipore Sigma). These smart flares are beads that have a sequence attached that, when recognizing the RNA sequence in the cell, produce an increase in fluorescence that can be analyzed with a fluorescent microscope.
[01911 Other methods include measuring protein expressions from an mRNA, for example, an mRNA encoding luciferase can be used to measure the efficiency of transfection. See, for example, He et al (J Gene Med. 2021 Feb;23(2):e3295) demonstrating the efficacy of delivering mRNA using a HKP and liposome formulation.
[0192] In a study involving other RNA technology, the combination of H3K(+H)4b and DOTAP (a cationic lipid) surprisingly was synergistic in its ability to carry mRNA into MDA-MB-231 cells (H3K(+H)4b/liposomes vs liposomes, P<0.0001). The combination was about 3-fold and 8- fold more effective as carriers of mRNA than the polymer alone and the cationic lipid carrier, respectively. Not all HK peptides demonstrated the synergistic activity with DOTAP lipid. For example, the combination of H3K4b and DOTAP was less effective than the DOTAP liposomes as carriers of luciferase mRNA. Besides DOTAP, other cationic lipids that may be used with HK peptides include Lipofectin (ThermoFisher), Lipofectamine (ThermoFisher), and DOSPER.
[0193] The D-isomer of H3k (+H)4b, in which the L-lysines in the branches are replaced with D- lysines, was the most effective polymeric carrier (H3k(+H)4b vs. H3K(+H)4b, P<0.05). The D- isomer/ liposome carrier of mRNA was nearly 4-fold and 10-fold more effective than the H3k(+H)4b alone and liposome carrier, respectively. Although the D-H3k(+H)4b/lipid combination was modestly more effective than the L-H3K(+H)4b/lipidmbination, this comparison was not statistically different.
[01 4] Both H3K4b and H3K(+H)4b can be used as carriers of nucleic acids in vitro See, for example, Leng et al. J Gene Med 2005; 7: 977-986; and Chou et al., Cancer Gene Ther 2011; 18: 707-716. Despite these previous findings, H3K(+H)4b was markedly better as a carrier of mRNA compared to other similar analogues (Table 2).
Table 2
Polymer Ratio(wt:wt; mRNA:Polymer) RLU/ug-Protein
1:4 1532.9±122.9
H3K(+H)4b 1:8 1656.3±202.5
1:12 1033.4±197
1:4 1851.6±138.3
H3k(+H)4b 1:8 1787.2±195.2
1:12 1982.3±210.7
1:4 156.8±41.8
H3K4b 1:8 62.U13.2
1:12 18.H4.0
1:4 61.7±5.7
H3K(3+H)4b 1:8 68.7±3.1
1:12 59.0±7.5
1:4 24.3±4.5
H3K(1+H)4b 1:8 15.0±3.6
1:12 7.3±2.5
1:4 1107.5±140.4
H-H3K(+H)4b 1:8 874.6±65.2
1:12 676.4±25.7
1:4 1101.9±106.6
HH-H3K(+H)4b
1:8 832.2±75.3
1 : 12 739.8±105.4
1 :4 896.4±112.6
H4K4b 1 :8 821.8±115.6
1 : 12 522.4±69.2
1 :4 518.3±134.7
H3K(1,3+H)4b 1 :8 427.7±18.1
1 : 12 378±5.2
1 :4 546.7±70.1
H2K4b 1 :8 132.3±58.5
1 : 12 194.7±18.4
[0195| Especially it has higher mRNA transfection efficiency than H3K4b in various weightweight (HK:mRNA) ratios. At a 4:1 ratio, luciferase expression was 10-fold higher with H3K(+H)4b than H3K4b in MDA-MB-231 cells without significant cytotoxicity. Moreover, the buffering capacity does not seem to be an essential factor in their transfection differences since the percent of histidines (by weight) in H3K4b and H3K(+H)4b is 68.9 and 70.6 %, respectively
[0196| Gel retardation assays show that the electrophoretic mobility of mRNA was delayed by the HK polymers. The retardation effect increased with higher peptide to mRNA weight ratios. However, mRNA was completely retarded in 2:1 ratio of H3K(+H)4b, whereas it was not completely retarded by H3K4b. This suggested that H3K(+H)4b could form a more stable polyplex, which was advantageous for its ability to be a suitable carrier for mRNA delivery.
[0197| Further confirmation that the H3K(+H)4b peptide binds more tightly to mRNA was demonstrated with a heparin-displacement assay. Various concentrations of heparin were added into the polyplexes formed with mRNA and HK and it was observed that, particularly at the lower concentrations of heparin, mRNA was released by the H3K4b polymer more readily than
the H3K(+H)4b polymer. These data suggest H3K(+H)4b could bind to mRNA and form a more stable polyplex than H3K4b.
[01 8] With the mRNA labeled with cyanine-5, the uptake of H3K4b and H3K(+H)4b polyplexes into MDA-MB-231 cells was compared using flow cytometry. At different time points (1, 2, and 4 h), the H3K(+H)4b polyplexes were imported into the cells more efficiently than H3K4b polyplexes. Similar to these results, fluorescent microscopy indicated that H3K(+H)4b polyplexes localized within the acidic endosomal vesicles significantly more than H3K4b polyplexes (H3K4b vs. H3K(+H)4b, P<0.001). Interestingly, irregularly-shaped H3K4b polyplexes, which did not overlap endocytic vesicles, were likely extracellular and were not observed with H3K(+H)4b polyplexes.
[0199] It is known both that HK polymers and cationic lipids (i.e., DOTAP) significantly and independently increase transfection with plasmids. See, for example, Chen et al. Gene Ther 2000; 7: 1698-1705. Consequently, whether these lipids together with HK polymers enhanced mRNA transfection was investigated. Notably, the H3K(+H)4b and H3k(+H)4b carriers were significantly better carriers of mRNA than the DOTAP liposomes. The combination of H3K(+H)4b and DOTAP lipid was synergistic in the ability to carry mRNA into MDA-MB-231 cells. The combination was about 3-fold and 8-fold more effective as carriers of mRNA than the polymer alone and the liposome carrier, respectively (H3K(+H)4b/lipid vs. liposomes or H3K(+H)4b). Notably, not all HK peptides demonstrated improved activity with DOTAP lipid. The combination of H3K4b and DOTAP carriers was less effective than the DOTAP liposomes as carriers of luciferase mRNA. The combination of DOTAP and H3K(+H)4b carriers were found to be synergistic in their ability to carry mRNA into cells. See, for example, He et al. (2020) J Gene Med., Nov 10:e3295.
[0200] In some embodiments, the carrier, such as the HKP nanoparticle, further comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, a cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, a cationic lipid is selected from 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-
dimethylaminobutyrate (DLin-MC3-DMA, or MC3), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
[0201 ] In some embodiments, the carrier is a nanoparticle. As used herein, the term “nanoparticle” refers to any particle having a diameter of less than 1000 nanometers (nm). In some embodiments, the nanoparticles have dimensions small enough to allow their uptake by eukaryotic cells. Typically, the nanoparticles have a longest straight dimension (e.g., diameter) of 200 nm or less. In some embodiments, the nanoparticles have a diameter of 100 nm or less. Smaller nanoparticles, e.g. having diameters of 50 nm or less, e.g., 5 nm-30 nm, are used in some embodiments.
[02021 In some embodiments, the carrier is a polymeric nanoparticle. The term “polymeric nanoparticle” refers to a nanoparticle composed of polymer compound (e.g., compound composed of repeated linked units or monomers) including any organic polymers, such as a Histidine-Lysine (HK) polypeptide (HKP).
[0203] As used herein, “liposome” refers to one or more lipids forming a complex, usually surrounded by an aqueous solution. Liposomes are generally spherical structures comprising lipids fatty acids, lipid bilayer type structures, unilamellar vesicles and amorphous lipid vesicles. Generally, liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. The liposomes may be unilamellar vesicles (possessing a single bilayer membrane), oligolamellar or multilamellar (an onion-like structure characterized by multiple membrane bilayers, each separated from the next by an aqueous layer).
[0204] In some embodiments, the carrier is a lipid nanoparticle (LNP, also referred to herein as a liposomal nanoparticle). In some embodiments, the LNP has a mean diameter of about 50 nm to about 200 nm. In some embodiments, Lipid nanoparticle carriers/formulations typically comprise, or alternatively consist essentially of, or yet further consist of a lipid, in particular, an ionizable cationic lipid, for example, SM-102 as disclosed herein, 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the LNP
carriers/formulations further comprise a neutral lipid, a sterol (such as a cholesterol) and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid (also referred to herein as PEGylated lipid). Additional exemplary lipid nanoparticle compositions and methods of making same are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28: 172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51 :8529-8533; and Maier et al. (2013) Molecular Therapy 21 :1570-1578, the contents of each of which are incorporated herein by reference in their entirety.
[0205] In one embodiment, the term “disease” or “disorder” as used herein refers to a cancer, a status of being diagnosed with a cancer, a status of being suspect of having a cancer, or a status of at risk of having a cancer. In one aspect, the cancer or tumor cell expresses NYE-SO-1.
]0206| As used herein, a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and in some aspects, the term may be used interchangeably with the term “tumor.” In some embodiments, the cancer is a cell expressing NYE-SO-1.
[0207] The term “cancer or tumor antigen” or “neoantigen” refers to an antigen known to be associated and expressed in a cancer cell or tumor cell (such as on the cell surface) or tissue, and the term “cancer or tumor targeting antibody” refers to an antibody that targets such an antigen. In some embodiments, the neoantigen does not express in a non-cancer cell or tissue. In some embodiments, the neoantigen expresses in a non-cancer cell or tissue at a level significantly lower compared to a cancer cell or tissue. As used herein, “neoantigen” refers to a new protein that forms on cancer cells as a result of oncogenic DNA mutations.
[0208] In some embodiments, the cancer is selected from: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, and lipoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)
carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal system, for example, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), gastric, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); gastrointestinal stromal tumors and neuroendocrine tumors arising at any site; genitourinary tract, for example, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and/or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver, for example, hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma); bone, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system, for example, neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); reproductive system, for example, gynecological, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre- tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma),
vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), placenta, vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma) and other sites associated with female genital organs;, penis, prostate, testis, and other sites associated with male genital organs; hematologic system, for example, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cavity, for example, lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx; skin, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids; adrenal glands: neuroblastoma; and other tissues comprising connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa, breast, head or neck, anal region, thyroid, parathyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites. In some embodiments, the cancer is a colon cancer, colorectal cancer or rectal cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is an adenocarcinoma, an adenocarcinoma, an adenoma, a leukemia, a lymphoma, a carcinoma, a melanoma, an angiosarcoma, or a seminoma.
[0209] In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is not a solid tumor. In further embodiments, the cancer is a leukemia cancer. In some embodiments, the cancer is from a carcinoma, a sarcoma, a myeloma, a leukemia, or a lymphoma. In some embodiments, the cancer is a colon cancer, colorectal cancer or rectal cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a pancreatic cancer.
[0210| In some embodiments, the cancer is a primary cancer or a metastatic cancer. In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer reaches a remission, but can relapse. In some embodiments, the cancer is unresectable.
[0211] In some embodiments, the cancer expresses NYE-SO-1 disclosed herein, such as a lung adenocarcinoma, a mucinous adenoma, a ductal carcinoma of the pancreas, a colorectal carcinoma; a rectal cancer, a follicular thyroid cancer, an autoimmune lymphoproliferative syndrome, a Noonan syndrome, a juvenile myelomonocytic leukemia; a bladder cancer, a follicular thyroid cancer, and an oral squamous cell carcinoma. The expression can be detected by sequencing a biopsy of the cancer, a Southern Blotting, a Northern Blotting, or by contacting with an antibody specifically binding to NYE-SO-1.
[0212] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals such as non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, bat, rat, rabbit, guinea pig).
[0213] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, bat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, the subject has or is diagnosed of having a disease. In some embodiments, the subject is suspected of having a disease. In some embodiments, the subject is at risk of having a disease. In some embodiments, the subject is in fully (such as free of cancer) cancer remission. In further embodiments, the subject is at risk of having a recurrence or
relapse of a cancer. In some embodiments, the subject is in partially cancer remission. In some embodiments, the subject is at risk of cancer metastasis.
[0214] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. When the disease is cancer, the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor. In one aspect, treatment excludes prophylaxis.
[0215] In some embodiments, the terms “treating,” “treatment,” and the like, as used herein, mean ameliorating a disease, so as to reduce, ameliorate, or eliminate its cause, its progression, its severity, or one or more of its symptoms, or otherwise beneficially alter the disease in a subject. Reference to “treating,” or “treatment” of a patient is intended to include prophylaxis. Treatment may also be preemptive in nature, i.e., it may include prevention of disease in a subject exposed to or at risk for the disease. Prevention of a disease may involve complete protection from disease, for example as in the case of prevention of infection with a pathogen, or may involve prevention of disease progression. For example, prevention of a disease may not mean complete foreclosure of any effect related to the diseases at any level, but instead may mean prevention of the symptoms of a disease to a clinically significant or detectable level. Prevention of diseases may also mean prevention of progression of a disease to a later stage of the disease.
[0216| When the disease is cancer, the following clinical endpoints are non-limiting examples of treatment: (1) elimination of a cancer in a subject or in a tissue/organ of the subject or in a cancer loci; (2) reduction in tumor burden (such as number of cancer cells, number of cancer foci, number of cancer cells in a foci, size of a solid cancer, concentrate of a liquid cancer in the body fluid, and/or amount of cancer in the body); (3) stabilizing or delay or slowing or inhibition of cancer growth and/or development, including but not limited to, cancer cell growth and/or division, size growth of a solid tumor or a cancer loci, cancer progression, and/or metastasis (such as time to form a new metastasis, number of total metastases, size of a metastasis, as well as variety of the tissues/organs to house metastatic cells); (4) less risk of having a cancer growth and/or development; (5) inducing an immune response of the patient to the cancer, such as higher number of tumor-infiltrating immune cell, higher number of activated immune cells, or higher number cancer cell expressing an immunotherapy target, or higher level of expression of an immunotherapy target in a cancer cell; (6) higher probability of survival and/or increased duration of survival, such as increased overall survival (OS, which may be shown as 1-year, 2- year, 5-year, 10-year, or 20-year survival rate), increased progression free survival (PFS), increased disease free survival (DFS), increased time to tumor recurrence (TTR) and increased time to tumor progression (TTP). In some embodiments, the subject after treatment experiences one or more endpoints selected from tumor response, reduction in tumor size, reduction in tumor burden, increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of drug-related toxicity, and avoidance of sideeffects (e.g., decreased treatment emergent adverse events). In some embodiments, improvement of quality of life includes resolution or improvement of cancer-specific symptoms, such as but not limited to fatigue, pain, nausea/vomiting, lack of appetite, and constipation; improvement or maintenance of psychological well-being (e.g., degree of irritability, depression, memory loss, tension, and anxiety); improvement or maintenance of social well-being (e.g., decreased requirement for assistance with eating, dressing, or using the restroom; improvement or maintenance of ability to perform normal leisure activities, hobbies, or social activities; improvement or maintenance of relationships with family). In some embodiments, improved patient quality of life that is measured qualitatively through patient narratives or quantitatively
using validated quality of life tools known to those skilled in the art, or a combination thereof. Additional non-limiting examples of endpoints include reduced hospital admissions, reduced drug use to treat side effects, longer periods off-treatment, and earlier return to work or caring responsibilities. In one aspect, prevention or prophylaxis is excluded from treatment.
|0217| As used herein, a biological sample, or a sample, is obtained from a subject. Exemplary samples include, but are not limited to, cell sample, tissue sample, biopsy, liquid samples such as blood and other liquid samples of biological origin, including, but not limited to, anterior nasal swab, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In some embodiments, the biological sample is a tumor biopsy.
[0218] In some embodiments, the samples include fluid from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, or the like), umbilical cord blood, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), washings of female reproductive tract, urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof. In some embodiments, a liquid biological sample is a blood plasma or serum sample. The term "blood" as used herein refers to a blood sample or preparation from a subject. The term encompasses whole blood, blood product or any fraction of blood, such as serum, plasma, huffy coat, or the like as conventionally defined. In some embodiments, the term “blood” refers to peripheral blood. Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants. Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Fluid samples often are collected in accordance with standard protocols hospitals or clinics generally
follow. For blood, an appropriate amount of peripheral blood (e.g., between 3-40 milliliters) often is collected and can be stored according to standard procedures prior to or after preparation.
[0219] The term “adjuvant” refers to a substance or mixture that enhances the immune response to an antigen. As non-limiting example, the adjuvant can comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldi octadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (See e.g., US 8,241,610). In another embodiment, the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant. As a non-limiting example, the synthetic nanocarrier comprising and adjuvant can be formulated by the methods described in WO2011150240 and US20110293700, each of which is herein incorporated by reference in its entirety.
[9220] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.
[0221] “Administration” or “delivery” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice,
three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.
|0222] The term administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The disclosure is not limited by the route of administration, the formulation or dosing schedule.
(0223] In some embodiments, an RNA, polynucleotide, vector, cell or composition as disclosed herein is administered in an effective amount. An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a
variety of factors including the activity of the specific agent employed, bioavailability of the agent, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the agent that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.
[0224] In some embodiments, an RNA, polynucleotide, vector, cell or composition as disclosed herein is administered in a therapeutically or pharmaceutically effective amount.
“Therapeutically effective amount” or “pharmaceutically effective amount” of an agent refers to an amount of the agent that is an amount sufficient to obtain a pharmacological response; or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient. The effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or pharmaceutically effective amount may be administered in one or more administrations.
[0225] In some embodiments, the treatment method as disclosed herein can be used as a first line treatment, or a second line treatment, or a third line treatment. The phrase “first line” or “second line” or “third line” refers to the order of treatment received by a patient. First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively. The National Cancer Institute defines first line therapy as “the first treatment for a disease or condition”. In patients with cancer, primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008. Typically, a patient is given a subsequent chemotherapy regimen because the
patient did not show a positive clinical or sub-clinical response to the first line therapy or the first line therapy has stopped.
[0226] An “anti-cancer therapy,” as used herein, includes but is not limited to surgical resection, chemotherapy, cryotherapy, radiation therapy, immunotherapy and targeted therapy. Agents that act to reduce cellular proliferation are known in the art and widely used. Chemotherapy drugs that kill cancer cells only when they are dividing are termed cell-cycle specific. These drugs include agents that act in S-phase, including topoisomerase inhibitors and anti-metabolites.
[0227] Topoisomerase inhibitors are drugs that interfere with the action of topoisomerase enzymes (topoisomerase I and II). During the process of chemo treatments, topoisomerase enzymes control the manipulation of the structure of DNA necessary for replication and are thus cell cycle specific. Examples of topoisomerase I inhibitors include the camptothecan analogs listed above, irinotecan and topotecan. Examples of topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.
[0228] Antimetabolites are usually analogs of normal metabolic substrates, often interfering with processes involved in chromosomal replication. They attack cells at very specific phases in the cycle. Antimetabolites include folic acid antagonists, e.g., methotrexate; pyrimidine antagonist, e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine; purine antagonist, e.g., 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitor, e.g., cladribine, fludarabine, nelarabine and pentostatin; and the like.
[0229] Plant alkaloids are derived from certain types of plants. The vinca alkaloids are made from the periwinkle plant (Catharanthus rosea). The taxanes are made from the bark of the Pacific Yew tree (taxus). The vinca alkaloids and taxanes are also known as antimicrotubule agents. The podophyllotoxins are derived from the May apple plant. Camptothecan analogs are derived from the Asian “Happy Tree” (Camptotheca acuminata). Podophyllotoxins and camptothecan analogs are also classified as topoisomerase inhibitors. The plant alkaloids are generally cell-cycle specific.
[0230] Examples of these agents include vinca alkaloids, e.g., vincristine, vinblastine and vinorelbine; taxanes, e.g., paclitaxel and docetaxel; podophyllotoxins, e.g., etoposide and tenisopide; and camptothecan analogs, e.g., irinotecan and topotecan.
[0231] In some embodiments where the cancer is an immune cell cancer, an anti-cancer therapy may comprises, or consists essentially of, or consists of a hematopoietic stem cell transplantation.
[0232] In some embodiments, a therapeutic agent, such as a cell as disclosed herein, may be combined in treating a cancer with another anti-cancer therapy or a therapy depleting an immune cell. For example, lymphodepletion chemotherapy is performed followed by administration of a cell as disclosed herein, such as four weekly infusions. In further embodiments, these steps may be repeated for once, twice, three or more times until a partial or complete effect is observed or a clinical end point is achieved.
[0233] Cryotherapy includes, but is not limited to, therapies involving decreasing the temperature, for example, hypothermic therapy.
[0234] Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art. Exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more than about 10 Gy or a dose of ultraviolet radiation at a range from at least about 5 J/m2 to not more than about 50 J/m2, usually about 10 J/m2.
[0235] In some embodiments, the immunotherapy regulates immune checkpoints. In further embodiments, the immunotherapy comprises, or consists essentially of, or yet further consists of an immune checkpoint inhibitor, such as an Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) inhibitor, or a Programmed Cell Death 1 (PD-1) inhibitor, or a Programmed Death Ligand 1 (PD-L1) inhibitor. In yet further embodiments, the immune checkpoint inhibitor comprises, or consists essentially of, or yet further consists of an antibody or an equivalent thereof recognizing and binding to an immune checkpoint protein, such as an antibody or an equivalent thereof recognizing and binding to CTLA4 (for example, Yervoy (ipilimumab), CP- 675,206 (tremelimumab), AK104 (cadonilimab), or AGEN1884 (zalifrelimab)), or an antibody
or an equivalent thereof recognizing and binding to PD-1 (for example, Keytruda (pembrolizumab), Opdivo (nivolumab), Libtayo (cemiplimab), Tyvyt (sintilimab), BGB-A317 (tislelizumab), JSOO1 (toripalimab), SHR1210 (camrelizumab), GB226 (geptanolimab), JSOO1 (toripalimab), AB122 (zimberelimab), AK105 (penpulimab), HLX10 (serplulimab), BCD-100 (prolgolimab), AGEN2034 (balstilimab), MGA012 (retifanlimab), AK104 (cadonilimab), HX008 (pucotenlimab), PF-06801591 (sasanlimab), JNJ-63723283 (cetrelimab), MGD013 (tebotelimab), CT-011 (pidilizumab), or Jemperli (dostarlimab)), or an antibody or an equivalent thereof recognizing and binding to PD-L1 (for example, Tecentriq (atezolizumab), Imfinzi (durvalumab), Bavencio (avelumab), CS1001 (sugemalimab), or KN035 (envafolimab)).
|'O2361 As used herein, a “targeted therapy” refers to a cancer therapy using drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, relapse, and spread of cancer, such as T cells or NK cells or other immune cells expressing a chimeric antigen receptor (CAR) which specifically targets and binds a neoantigen. In some embodiments, the neoantigen targeted by this targeted therapy can be the same with one encoded by an RNA as disclosed herein. In other embodiments, the neoantigen targeted by this targeted therapy is different from those encoded by an RNA as disclosed herein.
[0237] As used herein, a cleavable peptide, which is also referred to as a cleavable linker, means a peptide that can be cleaved, for example, by an enzyme. One translated polypeptide comprising such cleavable peptide can produce two final products, therefore, allowing expressing more than one polypeptides from one open reading frame. One example of cleavable peptides is a selfcleaving peptide, such as a 2A self-cleaving peptide. 2A self-cleaving peptides, is a class of 18- 22 aa-long peptides, which can induce the cleaving of the recombinant protein in a cell. In some embodiments, the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A and BmCPV2A. See, for example, Wang Y, et al. Sci Rep. 2015;5: 16273. Published 2015 Nov 5.
[0238] As used herein, the terms “T2A” and “2A peptide” are used interchangeably to refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or an artificial peptide comprising the requisite amino acids in a relatively short peptide sequence (on the order
of 20 amino acids long depending on the virus of origin) containing the consensus polypeptide motif D-V/I-E-X-N-P-G-P, wherein X refers to any amino acid generally thought to be selfcleaving (SEQ ID NO: 99).
[0239] In some embodiments, the term “linker” refers to any amino acid sequence comprising from a total of 1 to 200 amino acid residues; or about 1 to 10 amino acid residues, or alternatively 8 amino acids, or alternatively 6 amino acids, or alternatively 5 amino acids that may be repeated from 1 to 10, or alternatively to about 8, or alternatively to about 6, or alternatively to about 5, or alternatively, to about 4, or alternatively to about 3, or alternatively to about 2 times. For example, the linker may comprise up to 15 amino acid residues consisting of a pentapeptide repeated three times. In one embodiment, the linker sequence is a (G4S)n, wherein n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 (SEQ ID NO: 100).
[0240] As used herein, the phrase “derived” means isolated, purified, mutated, or engineered, or any combination thereof. For example, an NYE-SO-1 derived peptide refers to a peptide engineered from a NYE-SO-1 gene or a NYE-SO-1 protein, such as a wild-type one. In some embodiments, an NYE-SO-1 derived peptide is a NYE-SO-1 mutant, or a fragment thereof.
[0241 [ In some embodiments, a “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface and/or secreted outside of the cell. In some embodiments, the signal peptide is at the N terminus of the protein and can be cleaved to produce the mature protein. In some embodiments, the signal peptide is about 15 to about 30 amino acid long.
[0242] As used herein, an open reading frame (ORF) refers to a sequence of nucleotides that encodes a polypeptide or a portion thereof. In some embodiments, the ORF is an RNA.
Modes For Carrying Out the Disclosure
[0243] Cancer treatment modalities traditionally include surgery, chemotherapy, and radiation therapy. More recently, with in-depth knowledge gained about the molecular pathology of
cancer, targeted therapy and immunotherapy has been developed. Both have demonstrated promising results in cancer management. Cancer targeted therapy utilizes sequence information to inhibit the activities of protein products of cancer driver mutations. Because most somatic mutations extend beyond single anatomical sites or cancer types, targeted therapy can be applied to different tumors that share the same underlying mutations regardless their tissue locations. Since 2017, the U.S. Food and Drug Administration (FDA) has approved several treatments for specific genetic defect regardless tissue distribution. Examples include pembrolizumab that is approved for patients with unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors and entrectinib for patients with NTRK (neurotrophic tyrosine receptor kinase) gene fusion.
[0244] Like targeted therapy, cancer immunotherapy also has potential to treat more than one type of cancer. Cancer immunotherapy utilizes a patient’s immune system to fight against tumor cells. Some cancer immunotherapies primarily focus on humoral components of immune systems, the antibodies, to kill cancer cells by inhibiting the function of proteins expressed by cancer cells. Other cancer immunotherapies exert their function through cytotoxic T cells that have the ability to destroy tumor cells directly. The human immune system, as part of its normal functions, surveils and kills abnormal cells by recognizing mutated gene products that do not appear in normal cells and, thus, prevents or curbs the growth of cancers. The mutated version of proteins produced by cancer cells are often called tumor associated antigens, also known as neoantigens. By exposing the immune system to cancer neoantigens, it is possible to enhance the human immune system’s ability to target and kill tumor cells. This modality is called cancer treatment vaccine. Human tumor cell lysates or purified tumor neoantigens can be used to stimulate tumor specific immune response from cancer patients. Many different cell components of the immune system can be used to produce a cancer vaccine. As the first FDA approved cancer treatment vaccine, a fusion protein that is consisted of a tumor neoantigen, prostatic acid phosphatase and an adjuvant, granulocyte-macrophage colony-stimulating factor, was loaded into patient’s own dendritic cells. Dendritic cells function as the primary antigen-presenting cells (APC) that are responsible for displaying the neoantigens to be recognized by cytotoxic cells. Other cells can also function as APCs.
[0245| Despite the great promise of cancer treatment vaccines, there are a great number of technical challenges from immune-epitope discovery to vaccine manufacturing. RNA based vaccines are proposed as a possible solution to the challenges and have shown promise in preclinical and clinical studies. A key advantage of mRNA vaccines is that mRNA can be produced in the laboratory from a DNA template using readily available materials, less expensively and faster than conventional vaccine production, which can require the use of chicken eggs or other mammalian cells. In addition, mRNA vaccines have the potential to streamline vaccine discovery and development and facilitate a rapid response to emerging infectious diseases (see, for example, Maruggi et al., Mol Ther. 2019; 27(4): 757-772).
|'0246| During the last two decades, there has been broad interest in RNA-based technologies for the development of prophylactic and therapeutic vaccines. In this field, mRNA vaccines have been investigated extensively for infectious disease prevention, and for cancer prophylaxis and treatment. Preclinical and clinical trials have shown that mRNA vaccines provide a safe and long-lasting immune response in animal models and humans. mRNA vaccines expressing antigens of infectious pathogens induce potent T cell and humoral immune responses (Pardi et al. Nat Rev Drug Discov. 2018; 17: 261-279). As previously described, the production procedure to generate mRNA vaccines is entirely cell-free, simple, and rapid, if compared to production of whole microbe, live attenuated, and subunit vaccines. This fast and simple manufacturing process makes mRNA a promising bio-product that can potentially fill the gap between emerging infectious disease and the desperate need for effective vaccines.
[0247] Compared with traditional plasmid and viral-based approaches, this approach allows design of patient-personalized mRNAs that also benefit from eliminating needing to pass through the nuclear membrane (unlike DNA) and thus carries little to no risk of genomic integration. Furthermore, mRNA vaccines are safe, simple, and inexpensive and possess maximum flexibility. Particularly compared with peptide vaccines, they have self-adjuvanting properties, lack of MHC haplotype restriction, and do not need to enter the nucleus (Schlake et al., RNA Biol. 2012; 9(11): 1319-1330). mRNA does not integrate into the genome and therefore it avoids oncogenesis and mutagenesis (McNamara et al., J Immunol Res. 2015; 2015:794528). These
vaccines are temporary information carriers due to early metabolic degradation within a few days. Last but not least is that any protein can be encoded for development of therapeutic and prophylactic vaccines, without affecting the properties of the mRNA.
[0248] Recently, self-amplifying mRNA vaccines have been proved to be safe and effective against human viral pathogens (e g., influenza). Influenza mRNA vaccines hold great promises, being an egg-free platform and leading to production of antigen with high fidelity in mammalian cells. Recent published results demonstrated that the loss of a glycosylation site by a mutation in the hemagglutinin (HA) of the egg-adapted H3N2 vaccine strain resulted in poor neutralization of circulating H3N2 viruses in vaccinated humans and ferrets (Zost et al.,. Proc Natl Acad Sci USA. 2017; 114: 12578-12583). By contrast, the process of mRNA vaccine production is egg- free, and mRNA-encoded proteins are properly folded and glycosylated in host cells after vaccine administration, thus avoiding the risk of producing incorrect antigens.
[0249] Generation of a robust immune response in infants and the elderly has always been an issue for influenza vaccines. However, mRNA vaccines may benefit in that they have been demonstrated to induce balanced, long-lived and protective immunity to influenza A virus infections in even very young and very old mice. Vaccines based on mRNA or RNA replicons have also been shown to be immunogenic in a variety of animal models, including nonhuman primates (Maruggi et al., Vaccine. 2017; 35(2):361-368).
[0250] As described herein, Applicant has developed several NYE-SO-1 vaccines using an immunogenic composition that comprises, or consists essentially of, or further consists of a deoxyribonucleic acid or a messenger ribonucleic acid (mRNA) comprising, or consisting essentially of, or yet further consisting of an open reading frame (ORF) encoding one or multiple peptides of different NYE-SO-1 constructs, that can be formulated in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a polymeric nanoparticle or a liposomal nanoparticle or both. The composition can be administered to a subject in an amount effective to induce a specific immune response against NYE-SO-1 expression in such a subject.
[02511 Accordingly, in one aspect, provided is an isolated deoxyribonucleic acid (DNA) or an isolated ribonucleic acid (RNA), e.g., mRNA comprising, or consisting essentially of, or yet further consisting of an open reading frame (ORF) encoding an NYE-SO-1 derived peptide.
[0252] In some embodiments, the isolated DNA encodes one or more of the following: 1) the RNA sequence as set forth in SEQ ID NO: 1 or the peptide as set forth in SEQ ID NO: 2; 2) the
RNA sequence as set forth in SEQ ID NO: 3 or the peptide as set forth in SEQ ID NO: 4; 3) the
RNA sequence as set forth in SEQ ID NO: 5 or the peptide as set forth in SEQ ID NO: 6; or 4) the RNA sequence as set forth in SEQ ID NO: 7 or the peptide as set forth in SEQ ID NO: 8.
[0253] In other embodiments, the isolated RNA comprises one or more of the following: 1) the RNA sequence as set forth in SEQ ID NO: 1; 2) the RNA sequence as set forth in SEQ ID NO: 3; 3) the RNA sequence as set forth in SEQ ID NO: 5; or 4) the RNA sequence as set forth in SEQ ID NO: 7. In some embodiments, the RNA is formulated in a carrier, such as a pharmaceutically carrier. In further embodiments, the RNA is encapsulated in a nanoparticle.
[0254] In some embodiments, the DNA or RNA further comprises a 3'-UTR and a 5'-UTR. In some embodiments, the RNA further comprises one or more additional elements that stabilize the RNA and enhance expression of the peptides encoded by the ORF.
[0255] In some embodiments, the 5'-UTR comprises, or consists essentially of, or yet further comprises an m7G cap structure and a start codon. In some embodiments, the 5’-UTR comprises, or consists essentially of, or yet further comprises UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCA ACCUCAAACAGACACCGCCACC (SEQ ID NO: 10) or an equivalent thereof.
[0256| In some embodiments, the 3'-UTR comprises, or consists essentially of, or yet further comprises a stop codon and a poly A tail. In some embodiments, the 3’-UTR comprises, or consists essentially of, or yet further consists of AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC AACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUA AUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAA GCGAUCGCAAGCUUCUCGAGC (SEQ ID NO: 9) or an equivalent thereof. In some
embodiments, the poly A tail comprises, or consists essentially of, or yet further consists of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAA (SEQ ID NO: 12); or CGGCAAUAAAAAGACAGAAUAAAACGCACGGUGUUGGGUCGUUUGUUC (SEQ ID NO: 13).
[0257] In some embodiments, the RNA is prepared by transcribing a polynucleotide encoding the RNA in an in vitro transcription (IVT) system. In some embodiments, the RNA is prepared by transcribing a plasmid DNA (pDNA) vector encoding the RNA. In some embodiments, the vector is pUC57, or pSFVl, or pcDNA3, or pTK126. In some embodiments, the vector comprises, or consists essentially of, or yet further consists of TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTC AGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTG TACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAA TACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATC GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA GTGAATTCGAGCTCGGTACCTCGCGAATGCATCTAGATATCGGATCCCGGGCCCGTC GACTGCAGAGGCCTGCATGCAAGCTTTAATACGACTCACTATAAGGACATTTGCTTC TGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTTGTTTTT CTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTATGACTGAATATAAACTTGTGGTAG TTGGAGCTGATGACGTAGGCAAGAGTGCCTTTACGATACAGCTAATTCAGAATCATT TTGTGGACGAATATGATCCAACAATAGAGGATTCCTACAGGAAGCAAGTAGTAATT GATGGAGAAACCTGTCTCTTGGATATTCTCGACACAACAGATCACGAGGAGTACAG TGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCAT AAATAATACTAAATCATTTGAAGATATTCACCATTATAGAGAACAAATTAAAAGAG TTAAGGACTCTGAAGATGTACCTATGGTCCTAGTAGGAAATAATTGTGATTTGCCTT CTAGAACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGAAGTTATGGAATTCCT
TTTATTGAAACATCAACAAAGACAAGACAGAGAGTGGAGGATGCTTTTTATACATTG
GTGAGAGAGATCCGACAATACAGATTGAAAAAAATCAGCAAAGAAGAAAAGACTC
CTGGCTGTGTGAAAATTAAAAAATGCATTATAATGTAAGCTCGCTTTCTTGCTGTCC
AATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTA
TGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCCA
ATAGGCCGAAATCGGCAAGCGCGATCGCAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAATTCCTCGAGGC
GCGCCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG
GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC
AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC
GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA
CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC
CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG
TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC
TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC
AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC
ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT
GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG
AACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG
TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA
GCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC
GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATT
ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAG
CCCAATCTGAATAATGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGA
GCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAA
AAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCA
AGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAAT
TTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGA
ATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCA GCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGA TTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAG GAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCT GAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTG AGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCAT AAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAGCGATA GATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATC AGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATGGCTCAT AACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATA TTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACGGGCCAGAGCTGCA (SEQ ID NO: 11) or an equivalent thereof.
|0258] In some embodiments, the RNA is a messenger RNA (mRNA).
|0259| In some embodiments, the GC content of the full-length RNA is about 35 % to about 70 % (including any percentage or any subranges within the range) of the total RNA content, such as about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%.
(0260] In some embodiments, the RNA is chemically modified. In some embodiments, the chemical modification comprises, or consists essentially or, or yet further consists of one or both of the incorporation of an Nl-methyl-pseudouridine residue or a pseudouridine residue. In some embodiments, at least about 50% to about 100% of the uridine residues in the RNA are Nl- methyl pseudouridine or pseudouridine. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at east about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of residues of the RNA is chemically modified by
one or more of modifications as disclosed herein. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of uridine residues of the RNA is chemically modified by one or more of modifications as disclosed herein. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of uridine residues of the RNA is N1 -methyl pseudouridine or pseudouridine.
[02611 In some embodiments, all or some of uridine residues are replaced by pseudouridines during in vitro transcription. This modification stabilizes the mRNA against enzymatic degradation in the cell, leading to enhanced translation efficiency of the mRNA. The pseudouridines used can be Nl-methyl-pseudouridine, or other modifications that are well known in the art such as N6m -ethyladenosine (m6A), inosine, pseudouridine, 5-methylcytidine (m5C), 5-hydroxymethylcytidine (hm5C), and N1 -methyladenosine (mlA). The modification optionally is made throughout the entire mRNA. The skilled artisan will recognize that other modified RNA residues can be used to stabilize the protein’s three-dimensional structure and increase protein translation.
[0262] Further provided is a polynucleotide encoding a DNA or an RNA as disclosed herein, or a polynucleotide complementary thereto, or both. In some embodiments, the polynucleotide is selected from the group of: a deoxyribonucleic acid (DNA), an RNA, a hybrid of DNA and
RNA, or an analog of each thereof. In further embodiments, the analog comprises, or consists essentially of, or yet further consists of a peptide nucleic acid or a locked nucleic acid or both.
[0263] In some embodiments, the polynucleotide further comprises a regulatory sequence directing the transcription thereof. In some embodiments, the regulatory sequence is suitable for use in an in vitro transcription system. In further embodiments, the regulatory sequence comprises, or consists essentially of, or yet further consists of a promotor. In yet further embodiments, the promoter comprises, or consists essentially of, or yet further consists of: a bacteriophage RNA polymerase promoter, such as a T7 promoter, or a SP6 promoter, or a T3 promoter. In some embodiments, the polynucleotide comprises a marker selected from a detectable marker, a purification marker, or a selection marker.
[0264] In a further aspect, provided is a vector comprising, or consisting essentially of, or yet further consisting of a polynucleotide as disclosed herein.
[0265] In some embodiments, the vector further comprises a regulatory sequence operatively linked to the polynucleotide to direct the transcription thereof. In some embodiments, the regulatory sequence is suitable for use in an in vitro transcription system. In further embodiments, the regulatory sequence comprises, or consists essentially of, or yet further consists of a promotor. In yet further embodiments, the promoter comprises, or consists essentially of, or yet further consists of: a bacteriophage RNA polymerase promoter, such as a T7 promoter, or a SP6 promoter, or a T3 promoter. In some embodiments, the vector further comprises a marker selected from a detectable marker, a purification marker, or a selection marker.
[0266] In some embodiments, the vector further comprises a regulatory sequence operatively linked to the polynucleotide to direct the replication thereof. In further embodiments, the regulatory sequence comprises, or alternatively consists essentially of, or yet further consists of one or more of the following: an origin of replication or a primer annealing site, a promoter, or an enhancer.
[0267] In some embodiments, the vector is a non-viral vector. In further embodiments, the non- viral vector is a plasmid, or a liposome, or a micelle. In some embodiments, the vector is pUC57,
or pSFVl, or pcDNA3, or pTK126, or another plasmid available at addgene or Standard European Vector Architecture (SEVA).
[0268] In some embodiments, the vector is a viral vector. In further embodiments, the viral vector is selected from the group consisting of an adenoviral vector, or an adeno-associated viral vector, or a retroviral vector, or a lentiviral vector, or a plant viral vector.
|0269| In yet a further aspect, provided is a cell comprising one or more of: a DNA or an RNA as disclosed herein, a polynucleotide as disclosed herein, or a vector as disclosed herein. In some embodiments, the cell is suitable for replicating any one or more of: the DNA, the RNA, the polynucleotide, or the vector, thereby producing the one or more of: the RNA, the polynucleotide, or the vector. In some embodiments, the cell is suitable for transcribing the polynucleotide or the vector to the RNA, thereby producing the RNA.
[0270] In some embodiments, the cell is a prokaryotic cell. In further embodiments, the prokaryotic cell is an Escherichia coli cell.
[0271 ] In some embodiments, the cell is a eukaryotic cell. In further embodiments, the eukaryotic cell is any one of a mammal cell, an insect cell, or a yeast cell.
[0272] In some embodiments, a cell as disclosed herein is suitable for producing (such as transcribing or expressing) an RNA as disclosed herein. Such production can be in vivo or in vitro. For example, the cell can be used to produce the RNA in vitro. Such RNA is then administrated to a subject in need thereof optionally with a suitable pharmaceutical acceptable carrier. Alternatively, the cell can be used as a cell therapy and directly administrated to a subject in need thereof optionally with a suitable pharmaceutical acceptable carrier. In further embodiments, the cell therapy can additionally deliver other prophylactic or therapeutic agent to the subject. In some embodiments, the cell used as a cell therapy is an immune cell, such as a T cell, a B cell, an NK cell, an NKT cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage.
|0273| In one aspect, provided is a composition comprising, or consisting essentially of, or yet further consisting of a carrier, and one or more of: an RNA as disclosed herein, a polynucleotide
as disclosed herein, a vector as disclosed herein, or a cell as disclosed herein. In some embodiments, the carrier is a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an additional anti-cancer therapy. Additionally or alternatively, the composition further comprises an adjuvant.
|0274| In a further aspect, provided is a method of producing an RNA, such as those as disclosed herein. In some embodiments, the method comprises, or consists essentially of, or yet further consists of culturing a cell as disclosed herein under conditions suitable for expressing the RNA (such as transcribing a DNA to the RNA). In some embodiments, the cell comprises the DNA encoding the RNA of the disclosure. In some embodiments, the method comprises, or consists essentially of, or yet further consists of contacting a polynucleotide as disclosed herein or a vector as disclosed herein with an RNA polymerase, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine-5'-triphosphate (GTP), and uridine triphosphate (UTP) or a chemically modified UTP under conditions suitable for expressing the RNA (such as transcribing a DNA to the RNA). In some embodiments, the method further comprises isolating the RNA. In some embodiments, the method further comprises storing the RNA.
[0275] In yet a further aspect, provided is an RNA produced by a method as disclosed herein, or a composition comprising, or consisting essentially of, or yet further consisting of the produced RNA.
Improving mRNA Vaccine Expression efficiency
[0276] To improve the mRNA vaccine expression efficiency in the mammalian cells, mRNA stability can be enhanced by partial chemical modification. To further increase the translation efficiency, short and double strand RNAs derived from aberrant RNA polymerase activities are removed. To improve the potency of mRNA vaccines, sequence optimization can be used, together with usage of modified nucleosides, such as pseudouridine ((p), 5-methylcytidine (5mC), Cap-1 structure and optimized codons, which in turn improve translation efficiency. During in vitro transcription of mRNA, immature mRNA can be produced as a contaminant which inhibits translation through stimulating innate immune activation. FPLC and HPLC purification can be used to remove these contaminants.
[0277| In a composition presented herein, the template for in vitro transcription of mRNA contains five cis-acting structural elements, namely from 5’ to 3’ end: (i) an optimized cap structure, (ii) an optimized 5’ untranslated region (UTR), (iii) a codon optimized coding sequence, (iv) an optimized 3’ UTR and (v) a stretch of repeated adenine nucleotides (poly A tail) (FIG. 12). These cis-acting structural elements are further optimized in the endeavor for better mRNA features. Provided herein, the 5'-UTR includes a start codon and some other elements, but does not encode polypeptide (i.e. it is non-coding). In some embodiments, a 5'-UTR of the present disclosure comprises, or consists essentially of, or yet further consists of a cap structure with 7-methylguanosine (7mG) sequences. The 3'-UTR is directly downstream (3') from the stop codon (the codon of an mRNA transcript representing a termination signal) and does not encode a polypeptide (is non-coding). A polyA tail is a special region of mRNA that is downstream from 3 '-UTR and contains multiple consecutive adenosine monophosphates.
|0278] A typical mRNA production cassette comprises, or consists essentially of, or yet further consists of a Cap structure at its 5 ’-UTR region, followed by an in-frame mRNA sequence coding for a corresponding protein or peptide. In some embodiments, 3 ’-UTR with polyA tail is required for efficient mRNA production. In some embodiments, an expression cassette is used not only for efficiency of mRNA production but also for the subsequent protein or peptide production (FIG. 12).
|0279| In some embodiments, mRNA is produced by in vitro transcription (IVT) from a linear DNA template containing a bacteriophage promoter, the optimized UTR’s and the codon optimized sequence by using an RNA polymerase (T7, T3 or SP6) and a mix of the different nucleosides. In other embodiments, the linear DNA template can be cloned into a plasmid DNA (pDNA) as a delivery vector. The plasmid vectors can be adapted for mRNA vaccine production. Commonly used plasmids include pSFVl, pcDNA3 and pTK126. One unique mRNA expression system is pEVL (see Grier et al. (2016) Mol Ther Nucleic Acids. 19;5:e306, “pEVU: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences,” the disclosure of which is incorporated herein by reference in its entirety).
[0280| In some embodiments, the vaccine comprises, or consists essentially of, or yet further consists of an effective amount of an RNA, which comprises, or consists essentially of, or yet further consists of an open reading frame encoding one or more of NYE-SO-1, a variant of NYE- SO-1, and a pharmaceutically acceptable carrier. The effective amount is an amount effective to induce in the subject a neoantigen-specific, such as NYE-SO-l-specific, immune response. In one embodiment, the carrier comprises, or consists essentially of, or yet further consists of a polymeric nanoparticle or a liposomal nanoparticle. In some embodiments, the carrier is a Histidine-Lysine-copolymer or a Spermine-Liposome Conjugate. In some embodiments, the carrier further comprises DOTAP or MC3 or both.
[02811 In some embodiments, the vaccine comprises, or consists essentially of, or yet further consists of an effective amount of an mRNA, which comprises, or consists essentially of, or yet further consists of an open reading frame encoding multiple neoantigens separated by selfcleaving 2A peptide sites, signal sequences to incorporate the neoantigen into the membrane and/or be secreted using different signal sequences, such as the albumin signal sequence.
|0282| In some embodiments, the vaccine comprises an effective amount of an mRNA, which comprises an open reading frame encoding one or more NYE-SO-1 -based neoantigens, and/or other neoantigens, together with a pharmaceutically acceptable carrier. The effective amount is an amount effective to induce in the subject a NYE- SOI -specific immune response. In one embodiment, the carrier comprises a polymeric nanoparticle or a liposomal nanoparticle. In one aspect of this embodiment, the carrier is a Histidine-Lysine-copolymer or a Spermine-Liposome Conjugate. In another aspect of this embodiment, the carrier further includes DOTAP or MC3.
Histidine-Lysine (HK) polypeptides as mRNA vaccine delivery systems
[0283J Despite significant progress in the rational design of mRNA vaccines and elucidation of their mechanism of action during the past few years, their widespread application is limited by the presence of ubiquitous ribonucleases (RNases), as well as the need to facilitate vaccine entry into cells and subsequent escape from endosomes, and to target them to lymphoid organs or particular cells. See, for example, Midoux and Pichon, Expert Rev Vaccines. 2015; 14(2): 221-
34. mRNA formulations with chemical carriers provide more specificity and internalization in dendritic cells (DCs) for better immune responses and dose reduction.
[0284] A non-viral delivery system is more advantageous than the viral delivery system. See, for example, Brito et al. Adv Genet. 2015; 89: 179-233. One non-limiting examples is that non-viral methods are preferred over viral delivery systems for their safety and cost-effectiveness. See, for example, Juliano et al. Nucleic Acids Res. 2008; 36: 4158-4171 . Non-viral methods for delivery of vaccines include naked mRNA vaccines, gene gun, protamine condensation, adjuvant based vaccines, and encapsulated mRNA vaccines. Positive-sense RNA viruses, alpha viruses can be used for the viral delivery system. The glycoproteins (El and E2) of alpha virus can be used for endosomal escape and cell targeting in the host. In addition to direct delivery by viral or non- viral mediated methods, ex vivo transfected mRNA is an alternative to naked mRNA vaccination. In this method, mRNAs are transfected into monocytes, macrophages, T cells, dendritic cells (DCs) and mesenchymal stem cells (MSC), see, for example, Sahin et al., Nat Rev Drug Discov. 2014; 13: 759-780, before administration. A strong immune response can be induced by ex vivo transfected mRNA vaccination when compared to naked mRNA vaccination, which offers only optimal expression.
[0285] As described herein, a series of branched Histidine-Lysine (HK) polypeptides (HKP) can be applied to encapsulate mRNAs by electrostatic action. The HKPs used herein are a group of linear and branched peptides that consist of histidine and lysine residues and these peptides, in most cases, form spherical nanoparticles when mixed with nucleic acids. Such polypeptides are disclosed in US Patent No. 7,070,807 B2, issued July 4, 2006, and in US Patent No. 7,163,695 B2, issued January 16, 2007. The disclosures of each of these patents are incorporated herein by reference in their entireties. Similar to other carriers, HKP carriers differ in their ability to carry various nucleic acids. For instance, the four-branched HK peptide (H2K4b) is a good carrier of plasmids (see, for example, Chen, et al., Nucleic Acids Res. 2001; 29: 1334-1340; and Zhang et al., Methods Mol Biol. 2004; 245: 33-52), but is a poor carrier for siRNA. In addition, H3K4b, H3K(+H)4b, and H3K8b are excellent carriers of siRNA (see, for example, Leng et al., J Gene Med. 2005;7: 977-986), but only H3K(+H)4b shows effectiveness in carrying mRNA into the
targeted cells. Furthermore, H3K(+H)4b is a more effective carrier of mRNA than DOTAP liposomes. In addition, a delivery carrier combination of H3K(+H)4b, MC3 and/or DOTAP can be used as described herein to enhance the efficacy of mRNA delivery. The results as described herein showed that the H3k(+H)4b, MC3 and/or DOTAP combination was the most effective carrier of mRNA. This combination was synergistic for its ability to carry mRNA into cells.
Formulation and related methods
[0286] Accordingly, in one aspect, provided is a composition (such as an immunogenic composition) comprising, or consisting essentially of, or yet further consisting of, for example an effective amount of, an RNA as disclosed herein formulated in a pharmaceutically acceptable carrier. In some embodiments, the composition comprises, or consists essentially of, or yet further consists of the RNA and the pharmaceutically acceptable carrier.
[0287] In some embodiments, the pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle. In some embodiments, the nanoparticle is a polymeric nanoparticle or a liposomal nanoparticle or both. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a polymeric nanoparticle or a liposomal nanoparticle or both.
[0288] In some embodiments, the polymeric nanoparticle carrier comprises, or consists essentially of, or yet further consists of a Histidine-Lysine co-polymer (HKP). In further embodiments, the HKP comprises, or consists essentially of, or yet further consists of H3K(+H)4b. In yet further embodiments, the HKP comprises, or consists essentially of, or yet further consists of H3k(+H)4b. In some embodiments, the HKP comprises a side chain selected from SEQ ID NOs: 14-23.
[0289] In some embodiments, the mass ratio of HKP and the RNA in the composition is about 10: 1 to about 1: 10, including any range or ratio there between, for example, about 5: 1 to 1 :5, about 5:1 to 1 : 1, about 10: 1, about 9.5:1, about 9: 1, about 8.5: 1, about 8: 1, about 7.5: 1, about 7: 1, about 6.5: 1, about 6: 1, about 5.5: 1, about 5: 1, about 4.5:1, about 4: 1, about 3.5: 1, about 3: 1, about 2:5 : 1, about 2: 1, about 1.5: 1, about 1 : 1, about 1 :1.5, about 1 :2, about 1 :2.5, about 1 :3,
about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1:6, about 1:6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, or about 1 : 10. In one embodiment, the mass ratio of HKP and the RNA in the composition is about 2.5:1. In another embodiment, the mass ratio of HKP and the RNA in the composition is about 4:1.
[0290] In some embodiments, the polymeric nanoparticle carrier further comprises a lipid. In further embodiments, the lipid is a cationic lipid. In yet further embodiments, the cationic lipid is ionizable.
[0291] In some embodiments, the cationic lipid comprises, or consists essentially of, or yet further consists of Dlin-MC3-DMA (MC3) or dioleoyloxy-3-(trimethylammonio)propane (DOTAP) or both.
[0292] In some embodiments, the lipid further comprises one or more of: a helper lipid, a cholesterol, or a PEGylated lipid. In some embodiments, the lipid further comprises PLA or PLGA.
[0293] In some embodiments, the HKP and the mRNA self-assemble into nanoparticles upon admixture.
[0294] In some embodiments, the liposomal nanoparticle carrier comprises, or consists essentially of, or yet further consists of a Spermine-Lipid Cholesterol (SLiC). In further embodiments, the SLiC is selected from the group consisting of TM1-TM5, the structures of which are illustrated in FIG. 13.
[0295] In some embodiments, the pharmaceutical acceptable carrier is a lipid nanoparticle (LNP). In some embodiments, the lipid is a cationic lipid. In further embodiments, the cationic lipid is ionizable. In some embodiments, the LNP comprises, or consists essentially of, or yet further consists of one or more of: 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}octanoate (SM-102), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), or an
equivalent of each thereof. In some embodiments, the LNP further comprises one or more of: a helper lipid, a cholesterol, or a PEGylated lipid.
[0296] In some embodiments, the mass ratio of LNP and the RNA in the composition is about 10:1 to about 1: 10, including any range or ratio there between, for example, about 5:1 to 1:5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10. In one embodiment, the mass ratio of LNP and the RNA in the composition is about 2.5:1. In another embodiment, the mass ratio of LNP and the RNA in the composition is about 4: 1.
[9297] In some embodiments, the helper lipid comprises, or consists essentially of, or yet further consists of one or more of: disteroylphosphatidyl choline (DSPC), Dipalmitoylphosphatidylcholine (DPPC), (2A)-3-(Hexadecanoyloxy)-2-{[(9Z)-octadec-9- enoyl] oxy} propyl 2-(trimethylazaniumyl)ethyl phosphate (POPC), or dioleoyl phosphatidylethanolamine (DOPE).
[0298] In some embodiments, the cholesterol comprises, or consists essentially of, or yet further consists of a plant cholesterol or an animal cholesterol or both.
[0299] In some embodiments, the PEGylated lipid comprises, or consists essentially of, or yet further consists of one or more of: PEG-c-DOMG (R-3-[(co-m ethoxy - poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine), PEG-DSG (1,2- Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) optionally PEG2000-DMG ((l,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N- [methoxy(polyethylene glycol)-2000)], or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
[9300] In some embodiments, the mass ratio of the cationic lipid and the helper lipid is about 10:1 to about 1: 10, including any range or ratio there between, for example, about 5: 1 to 1 :5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about
7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10. In one embodiment, the mass ratio of the cationic lipid and the helper lipid is about 1:1.
[03011 In some embodiments, the mass ratio of the cationic lipid and cholesterol is about 10:1 to about 1: 10, including any range or ratio there between, for example, about 5: 1 to 1 :5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10. In one embodiment, the mass ratio of the cationic lipid and cholesterol is about 1:1.
[0302] In some embodiments, the mass ratio of the cationic lipid and PEGylated lipid is about 10:1 to about 1: 10, including any range or ratio there between, for example, about 5:1 to 1:5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about6.5:l, about6:l, about5.5:l, about5:l, about 4.5:1, about 4:1, about3.5:l, about3:l, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10. In one embodiment, the mass ratio of the cationic lipid and PEGylated lipid is about 1:1.
[0303] The mass ratio of the cationic lipid, helper lipid, cholesterol and PEGylated lipid can be calculated by one of skill in the art based on the ratios of the cationic lipid and the helper lipid, the cationic lipid and the cholesterol and the cationic lipid and the PEGylated lipid as disclosed herein.
[0304] In some embodiments, the LNP comprises, or consists essentially of, or yet further consists of SM-102, DSPC, cholesterol and PEG2000-DMG. In some embodiments, the mass ratio of the SM-102, DSPC, cholesterol and PEG200-DMG is about 1 : 1 : 1 : 1. In some
embodiments, the molar ratio of the SM-102, DSPC, cholesterol and PEG2000-DMG is about 50:10:38.5:1.5.
[0305] In some embodiments, a mass ratio as provided here can be substituted with another parameter, such as a molar ratio, a weight percentage over the total weight, a component’s weight over the total volume, or a molar percentage over the total molar amount. Knowing the component and its molecular weight, one of skill in the art would have no difficulty in converting a mass ratio to a molar ratio or other equivalent parameters.
[0306] In a further aspect, provided is a method of producing a composition as disclosed herein. The method comprises, or consists essentially of, or yet further consists of contacting an RNA as disclosed herein with an HKP, thereby the RNA and the HKP are self-assembled into nanoparticles.
[0307] In some embodiments, the mass ratio of HKP and the RNA in the contacting step is about 10: 1 to about 1: 10, including any range or ratio there between, for example, about 5: 1 to 1 :5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10. In one embodiment, the mass ratio of HKP and the RNA in the contacting step is about 2.5:1. In another embodiment, the mass ratio of HKP and the RNA in the contacting step is about 4:1.
[0308] In some embodiments, the method further comprises contacting the HKP and RNA with a cationic lipid. In further embodiments, the cationic lipid comprises, or consists essentially of, or yet further consists of Dlin-MC3-DMA (MC3) or DOTAP (dioleoyloxy-3- (trimethylammonio)propane) or both. In yet further embodiments, the mass ratio of the cationic lipid and the RNA in the contacting step is about 10:1 to about 1: 10, including any range or ratio therebetween, for example, about 5:1 to 1:5, about 5:1 to 1:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2:5 :1, about 2:1, about 1.5:1, about 1:1,
about 1 :1.5, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1:4, about 1:4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1 :6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1:8.5, about 1 :9, about 1 :9.5, or about 1 : 10. In one embodiment, the mass ratio of the RNA and the cationic lipid in the contacting step is about 1: 1. Accordingly, the mass ratio of the HKP, the RNA and the cationic lipid in the contacting step can be calculated based on the ratio between the HKP and the RNA and the ratio between the RNA and the cationic lipid. For example, if the ratio of the HKP to the RNA is about 4: 1 and the ratio of the RNA to the cationic lipid is about 1 : 1, the ratio of the HKP to the RNA to the cationic lipid is about 4: 1 : 1.
|0309[ In yet a further aspect, provided is a method of producing a composition as disclosed herein. The method comprises, or consists essentially of, or yet further consists of contacting an RNA as disclosed herein with a lipid, thereby the RNA and the lipid are self-assembled into lipid nanoparticles (LNPs).
[0310] In some embodiments, the LNPs comprise, or consist essentially of, or yet further consist of one or more of: 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}octanoate (SM-102), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), or an equivalent of each thereof.
[0311] In some embodiments, the LNPs further comprise one or more of: a helper lipid, a cholesterol, or a PEGylated lipid. In some embodiments, the helper lipid comprises, or consists essentially of, or yet further consists of one or more of: disteroylphosphatidyl choline (DSPC), Dipalmitoylphosphatidylcholine (DPPC), (27?)-3-(Hexadecanoyloxy)-2-{[(9Z)-octadec-9- enoyl] oxy} propyl 2-(trimethylazaniumyl)ethyl phosphate (POPC), or dioleoyl phosphatidylethanolamine (DOPE). In some embodiments, the cholesterol comprises, or consists essentially of, or yet further consists of a plant cholesterol or an animal cholesterol or both. In some embodiments, the PEGylated lipid comprises, or consists essentially of, or yet further consists of one or more of: PEG-c-DOMG (R-3-[(co-m ethoxy - poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine), PEG-DSG (1,2-
Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) optionally PEG2000-DMG ((l,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N- [methoxy(polyethylene glycol)-2000)], or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
[03121 In some embodiments, the LNPs comprise, or consist essentially of, or yet further consist of SM-102, DSPC, cholesterol and PEG2000-DMG. In some embodiments, the mass ratio of the SM-102, DSPC, cholesterol and PEG200-DMG is about 1 : 1 : 1 : 1. Additionally or alternatively, the molar ratio of the SM-102, DSPC, cholesterol and PEG2000-DMG is about 50: 10:38.5: 1.5.
[0313] In some embodiments, the contacting step is performed in a microfluidic mixer. In further embodiments, the microfluidic mixer is a slit interdigitial micromixer, or a staggered herringbone micromixer (SHM).
[0314] Also provided is a composition produced by a method as disclosed herein.
Compositions and Carriers
[0315] Additional aspects of the disclosure relate to compositions comprising, or alternatively consisting essentially of, or yet further consisting of, a carrier and one or more of the products - e.g. an RNA comprising an ORF encoding one or more NYE-SO-1 derived peptides. The carrier may be a pharmaceutically acceptable carrier. In one aspect, provided herein is a composition comprising, or alternatively consisting essentially of, or yet further consisting of the RNA comprising an ORF encoding one or more NYE-SO-1 derived peptides disclosed herein, and, optionally, a pharmaceutically acceptable carrier.
[0316] In some embodiments, the pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle. In further embodiments, the carrier comprises a Histidine-Lysine co-polymer (HKP). In still further embodiments, the HKP may comprise H3K(+H)4b. In other embodiments, the polymeric nanoparticle carrier may comprise PLA or PLGA.
[0317] In some embodiments, the mRNA or the composition may comprise a plurality of mRNA molecules encoding the same or different polypeptide. In further embodiments, the the plurality
of mRNA are encapsulated in the same or different nanoparticle or liposome. In some embodiments, the mRNA in the composition is encapsulated in l,2-Dioleoyl-3- trimethylammonium propane (DOTAP). In other embodiments, the liposomal nanoparticle carrier comprises a Spermine-Lipid Cholesterol (SLiC). In further embodiments, the SLiC is selected from the group consisting of the structures TM1-TM5 shown in FIG. 13.
[0318] Briefly, pharmaceutical compositions of the present disclosure include but are not limited to any one of the claimed compositions as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure may be formulated for oral, intravenous, topical, enteral, and/or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intravenous administration.
[0319] Administration of the mRNA or compositions can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. In a further aspect, the cells and composition of the disclosure can be administered in combination with other treatments. The mRNA and compositions are administered to the host using methods known in the art.
[0320] In some embodiments, the carrier may be a polymeric nanoparticle or a liposomal nanoparticle. Non-limiting examples of polymeric nanoparticles include H3K(+H)4b, PLA or PLGA. A non-limiting example of a liposomal nanoparticles is Spermine-Lipid Cholesterol (SLiC).
Method of treatment
[03211 Also provided is a method of treating a subject having a cancer, or at risk of having a cancer, or suspect of having a cancer. In some embodiments, the cancer expresses NYE-SO-1. Methods to determine when the method is successful are known in the art and briefly described herein. The treatment method can be combined with an assay to determine if the cancer or a tumor cell expresses NYE-SO-1 .
[0322] Further provided is a method of inhibiting the growth of a tumor or cancer cell. The method comprises, or consists essentially of, or yet further consists of contacting an immune cell with any one or more of an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, or a composition as disclosed herein, thereby activating the immune cell, and contacting the tumor or cancer cell with the activated immune cell. In some embodiments, the cancer cell or tumor expresses NYE-SO-1. In one aspect, the method of treating a cancer subject with a tumor expressing NYE-SO-1, comprises, or alternatively consists essentially of, or yet further consists of contacting a cancer cell with an RNA described in this disclosure, optionally combined with a pharmaceutically acceptable carrier. The contacting can be in vitro or in vivo.
[0323] Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated or prevented. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials. In one aspect they are administered directly by direct injection or systemically such as intravenous injection or infusion.
[0324] The subject to be treated can be an animal, e.g., a mammal or a human patient. The cancer to be treated can be a liquid tumor or cancer or a solid tumor or cancer. In one aspect, the cancer expresses NYE-SO-1. The cancer can be a Stage I, II, III or IV. The treatment can be combined with other therapies as described herein. In one aspect, the treatment is adjuvant to tumor resection. The treatment can be first-line, second-line, third-line, fourth line, fifth line therapy.
[0325| Additionally, or alternatively, provided is a screening method or a screening step of a method as disclosed herein for personalized or precision method, or alternatively to test for new combination therapies. The method comprises, or consists essentially of, or yet further consists of detecting the expression of NYE-SO-1 as disclosed herein. In some embodiments, expression of NYE-SO-1 can be detected using sequencing, southern blots, or northern blots. In some embodiments, expression of NYE-SO-1 protein can be detected using flow cytometry or western blots. The method can be practiced in an animal to produce an animal model for treatment or to treat an animal, as determined by a treating veterinarian. Methods to determine when the method is successful are known in the art and briefly described herein.
|'0326| In some embodiments, the cancer is an adenocarcinoma, an adenocarcinoma, an adenoma, a leukemia, a lymphoma, a carcinoma, a melanoma, an angiosarcoma, a pancreatic cancer, a colon cancer, a colorectal cancer, a rectal cancer, or a seminoma. The cancer can be primary or metastatic. The subject in need thereof may be suffering from an active cancer or be in remission, or at risk of developing a cancer, primary or secondary.
|0327| Additionally or alternatively, provided is a method for inducing an immune response, for example to expressed NYE-SO-1 as disclosed herein, in a subject in need thereof. In some embodiments, the immune response comprises, or consists essentially of, or yet further consists of any one or more of: a Thl immune response, activation of CD8+ T cells, or production of a pro-inflammatory cytokine, such as interleukin-2 (IL-2), interferon-gamma (IFN-y), or tumor necrosis factor-beta (TNF-P). Methods to determine when the method is successful are known in the art and briefly described herein.
[0328] These methods comprise, or consist essentially of, or yet further consist of administering to the subject, for example an effective amount of (e.g., a pharmaceutically effective amount of), any one or more of a DNA or an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, or a composition as disclosed herein.
[0329] In some embodiments, the DNA encodes one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. In other embodiments, the RNA comprises one or more of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. In further embodiments, the
RNA further comprise a 5’UTR (for example comprising, or consisting essentially of, or yet further consisting of SEQ ID NO: 10) and a 3’UTR (for example comprising, or consisting essentially of, or yet further consisting of SEQ ID NO: 9). In some embodiments, the vector comprises, or consists essentially of, or yet further consists of SEQ ID NO: 11. In some embodiments, the composition comprises the RNA formulated in a carrier, such as an LNP or a HKP nanoparticle as disclosed herein.
[0330] In some embodiments, the administration is intratumoral, or intravenous, or intramuscular, or intradermal, or subcutaneous.
[0331] In some embodiments, the subject is a mammal, or a human.
[0332] In some embodiments, the method further comprises administering to the subject an additional anti-cancer therapy. In some embodiments, the anti-cancer therapy is administrated prior to, or concurrently with, or after the administration of any one or more of the following: the RNA as disclosed herein, the polynucleotide as disclosed herein, the vector as disclosed herein, the cell as disclosed herein, or the composition as disclosed herein.
[0333] In some embodiments, the administration was repeated for at least one time, at least two times, at least three times, at least four times, or more. In further embodiments, the interval between any two administrations can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer.
[0334] In some embodiments, the method further comprises detecting the expression of NYE- SO-1 as disclosed herein in a biological sample of the subject, such as a tumor biopsy or a circulating tumor DNA, prior to the administration.
[0335] In some embodiments, the method further comprises monitoring the expression of NYE- SO-1 as disclosed herein in a biological sample of the subject, such as a tumor biopsy or a circulating tumor DNA, after the administration.
[0336| In some embodiments, the method further comprises detecting antibodies recognizing and binding to NYE-SO-1 as disclosed herein in a biological sample of the subject, such as a blood sample, after the administration.
[0337] As used herein, an effective dose of an RNA, or polynucleotide, or vector, or cell or composition as disclosed herein is the dose required to produce a protective immune response in the subject to be administered. A protective immune response in the present context is one that treats a cancer in a subject. The RNA, or polynucleotide, or vector, or cell or composition as disclosed herein can be administered one or more times. An initial measurement of an immune response to the vaccine may be made by measuring production of antibodies in the subject receiving the RNA, or polynucleotide, or vector, or cell, or composition. Methods of measuring antibody production in this manner are also well known in the art, is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a cancer. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the formulated composition.
[0338] In some embodiments, the RNA compositions can be administered at dosage levels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one or more times a day, per week, per month, etc. to obtain the desired therapeutic or prophylactic effect. In some embodiments, the RNA composition is administered at a dosage of about 10 to about 500 pg/kg of body weight, or any dosage or subranges therein, such as about 28.5-285 pg/kg of body weight. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every 2 months,
every three months, every 6 months, etc. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein can be used. In some embodiments, the RNA compositions can be administered at dosage levels sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg. In some embodiments, the RNA compositions can be administered once or twice (or more) at dosage levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.
[0339] In some embodiments, the RNA compositions can be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages and frequency of administration are encompassed by the present disclosure. For example, the RNA composition can be administered three or four times.
[0340] In certain embodiments, the RNA or composition is further combined with another therapy, such as a purified protein drug. Non-limiting examples include a therapeutic mAb (for example Avastin™, Herceptin™, Yervoy™, Keytruda™, Opdivo™, Tecentriq™ etc ), or a therapeutic protein, such as GM-CSF, an interleukin, an interferon, thymosin A, hexarelin or adiponectin. In one embodiment, the combined treatment comprises administrating mRNA or
composition as disclosed herein and a combined therapy concurrently, sequentially and/or separately to a subject in need thereof. In one embodiment the mRNA or composition and combined therapy are administrated to the subject via the same administration route. In another embodiment, different administration route is used. In certain embodiments, there is an about 0.5 hour, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks or about 1 months gap in any of the two administrations.
[0341] In certain embodiments, any combination therapy as described herein achieves a synergistic effect in treating a disease, such as a cancer.
[0342] Also provided is a method of treating a cancer, comprising administering an effective amount of one or more of an RNA or composition as described herein to a subject in need thereof. In one embodiment, the method comprising, or alternatively consisting of, or yet further consisting essentially of administering an effective amount of an RNA or composition as described herein. In a further embodiment, the method further comprises administering an effective amount of a combined treatment, whereby a synergistic effect is achieved in treating cancer. Anti-cancer and immune enhancing therapies are known in the art, several of which are described herein.
[0343] Additionally provided is a method for delivering an RNA or composition as described herein to a subject in need thereof.
[0344] In one embodiment, the subject is suspected of having and/or is diagnosed with a cancer expressing NYE-SO-1.
(0345) In one embodiment, the cancer is a solid tumor (e.g., carcinoma or sarcoma) or a nonsolid cancer (for example, a blood cancer). Additionally or alternatively, the cancer or tumor is a primary or metastatic cancer or tumor. In a further embodiment, the cancer is selected from a gastric cancer, a colorectal cancer, a prostate cancer, a breast cancer, a triple negative breast cancer, an ovarian carcinoma, a renal cell carcinoma, an Ewing sarcoma, a melanoma, a mesothelioma, a lung cancer, a non-small cell lung cancer, a stage IV lung cancer, a brain
cancer, a glioblastoma, a lymphoma, a leukemia, or a multiple myeloma (MM). In another embodiment, the cancer affects the blood and/or bone marrow.
[0346] In one aspect, the methods further comprising identifying the subject for the therapy by assaying a cancer sample from the patient for a NYE-SO-1 expression. The therapy is then administered to the subject expressing NYE-SO-1.
103471 Any of the methods as disclosed herein further comprise monitoring efficacy prior to, concurrently with, and/or after performing the method or any step(s) recited in the method. In one embodiment, the efficacy is evaluated and/or quantified as a treatment of a disease (for instance a cancer) in a subject, an inhibition of a cancer cell growth, and/or a biomarker change.
[0348] In another embodiment, both efficacy outcomes and biomarker can be simultaneously determined in the cancer or tumor in a subject. In a non-limiting example, a rodent bearing a human tumor xenograft is used. Human tumor cells, preferably engineered to express a bioluminescent gene, such as Luciferase, are transplanted into a rodent (such as a mice or a rat), allowing visualization by injection of an activatable fluorescence optcial imaging agent in the rodent.
Kits
[0349] As set forth herein, the present disclosure provides methods for producing and administering an RNA comprising an ORF encoding NYE-SO-1 or a variant thereof. In one particular aspect, the present disclosure provides kits for performing these methods as well as instructions for carrying out the methods of the present disclosure such as administering to a subject an effective amount of mRNA described by the present disclosure.
[0350] In one aspect the kit comprises, or alternatively consists essentially of, or yet further consists of, any one of the RNA molecules disclosed herein. In another aspect, disclosed herein are kits comprising, or alternatively consisting essentially of, or yet further consisting of the composition as disclosed herein and optionally, instructions for use. Such a kit may also comprise, or alternatively consist essentially of, or yet further comprise media and other reagents appropriate for the administration of RNA molecules, such as those disclosed herein.
[03511 The kits of this disclosure can also comprise, e.g., a buffering agent, a preservative or a protein-stabilizing agent. The kits can further comprise components necessary for detecting the detectable label, e.g., an enzyme or a substrate. The kits can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of a kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present disclosure may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.
[03521 As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components may be provided in solution or as a liquid dispersion or the like.
[0353J The following examples are illustrative of procedures which can be used in various instances in carrying the disclosure into effect.
[0354] In some embodiments, the kit comprises, or alternatively consists essentially of, or yet further consist of instructions for use and one or more of an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, or a composition as disclosed herein. In further embodiments, the kit is suitable for use in a method of treatment as disclosed herein. In some embodiments, the kit further comprises an anti-cancer therapy.
[0355] In some embodiments, the kit comprises, or alternatively consists essentially of, or yet further consist of instructions for use and one or more of: an RNA as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, a cell as disclosed herein, a composition as disclosed herein, an HKP, or a lipid optionally a cationic lipid. In further embodiments, the kit is suitable for use in a method producing an RNA or a composition as disclosed herein.
[0356] In some embodiments, the kit comprises, or alternatively consists essentially of, or yet further consist of instructions of use, a polynucleotide or a vector as disclosed herein, an RNA
polymerase, ATP, CTP, GTP, and UTP or a chemically modified UTP. In further embodiments, the kit is suitable for use in an in vitro method producing an RNA or a composition as disclosed herein.
EXPERIMENTAL METHODS
The following examples are illustrative of procedures which can be used in various instances in carrying the disclosure into effect.
Example 1: In vitro transcription (IVT) assay
[0357] In vitro transcription was performed following RNAimmune’s In vitro transcription SOP. The NYESO-1 mRNA producing plasmid was linearized using Xhol (NEB) at 37°C for 4 hours and the linearized plasmid was purified using QIAquick PCR purification kit following manufacturer’s instruction (Qiagen). After purification, in vitro transcription reaction was performed in a 20 pl reaction with all the components added in the following order: 1 pg of linearized DNA; Ipl of ATP, CTP, GTP and Nl-methyl-pseudouri dine tri phosphate; 0.8pl CleanCap AG (3’0me); 2pl 10X IVT reaction buffer; 0.5 pl Murine RNase Inhibitor; 0.4pl E.coli Inorganic Pyrophosphatase; and 3.2pl T7 RNA polymerase. The IVT reaction was incubated at 37°C for 4 hours. The components of 10X Transcription Buffer include: 400 mM Tris-HCL (pH 8), 100 mM DTT, 20 mM Spermidine, 0.02% Triton X, 165 mM Magnesium Acetate, and DNase/RNase-Free Water. After in vitro transcription reaction, the mRNA was purified using RNeasy Mini Kit following manufacturer’s instruction (Qiagen).
[0358] The IVT reaction was adjusted to a volume of lOOpl with RNase- free water. Then, mRNA was mixed well with 350pl buffer RLT.250pl of 100% ethanol was then added to the mixture, mix well by pipetting, and 700pl of the sample was transferred to a RNeasy Mini spin column, centrifuged for 15 seconds at >8000 x g (>10,000 rpm). After discarding the flow- through, the spin column was washed twice with 500pl Buffer RPE. Then the spin column was transferred to a 1.5 ml collection tube (kit supplied), mRNA was eluted with 30pl RNase-free water. If the expected RNA yield is >30 pg, repeat elution step using another 30pl RNase-free water.
[0359| RNA gel electrophoresis was employed to determine mRNA quality. (FIGs. 1A-1B) The quality of purified mRNA was evaluated using 1% agarose gel in NorthernMax™-Gly Gel Prep with Sybr Gold (1 :20,000).The running buffer was prepared by diluting NorthernMax™-Gly Gel Prep from lOx stock solution in DEPC-treated ultrapure water. RNA sample was prepared by adding one volume of RNA (up to 30pg total RNA) to one volume NorthernMax®-Gly Sample Loading Buffer and incubated at 65C for 30 min to prevent formation of secondary structure. Gel markers also treated similarly. RNA sample and markers were run on 1% agarose gel at 5V/cm. Once the bromophenol blue dye front migrated approximately % the length of the gel, electrophoresis was stopped, and mRNA was visualized with blue light.
Example 2: Lipid Nanoparticle Formulation
[0360| To make a 4x stock solution of each component in ethanol, each component was prepared as following: lOOmg of SM-102 in 5.63ml 100% ethanol; 39.5mg of DSPC in 10ml 100% ethanol; 74.4mg of Cholesterol in 100% ethanol; and 18.8mg of DMG-PEG2000 in 100% ethanol. The final concentration of 4x stock is 25mM SM-102, 5mM DSPC, 19.5mM Cholesterol and 0.75mM DMG-PEG2000. Each lipid component was then added to a 5ml tube at a ratio of 1 : 1 : 1 : 1 to make a lx working solution. The final concentration of working concentration is 6.25mM SM-102, 1.25mM DSPC, 4.815mM Cholesterol, and 0.1875mM DMG-PEG2000. To make mRNA working concentration, the mRNA stock solution was diluted in 25 mM Sodium Acetate pH5.0 at a final concentration of 0.13 mg per ml.
| 03611 The formulation of lipid nanoparticles was done using NanoAssmblr Ignite (PNI) following manufacturer's instructions with an mRNA:lipid ratio of 3 : 1. After formulation, the formulated LNPs were transferred into pre-soaked dialysis cassette and dialyzed in 20mM Tris- HC1, 8% sucrose dialysis buffer for at least 18 hours at 4°C. After dialysis, LNPs were removed from dialysis cassette using a 3-5ml syringe, filter sterilized using an acrodisc filter (0.2 pM pore size) and poured into the top compartment of an Amicon® Ultra- 15 centrifugal filtration tube. The tube was spun at 2000 x g at 4°C until the solution was reconcentrated to desired volume. LNPs were transferred to a new tube and characterized by Dynamic Light Scattering to measure particle size, poly dispersity and zeta potential using Zetasizer Ultra (Malven Panalytical). The
encapsulation efficiency was measured using Ribogreen assay (Invitrogen). The mRNA concentration was measured using Ribogreen assay and Nanodrop. The final LNPs were stored at -80C for future use.
Example 3: Western Blot Analysis
[0362] To select the NYESO1 vaccine candidate, in vitro expression levels of vaccine candidates were examined. (FIG. 2) 293 T cell was transfected with various forms of NYESO1 mRNA using Lipofectamin MessengerMax transfection reagents. Forty-eight hours post transfection, cells were collected, cell lysates were prepared, and the protein concentration was determined using BCA assay. The expression level of each construct was compared using Western Blot analysis with anti-NYESOl antibody (anti-CTAGIB, Abeam).
|0363| To test the quality of formulated NYES01 vaccine, various amounts of mRNA-LNPs (0.5ug, lug and 2.5ug) were transfected into 293T cells. (FIG. 4) Forty -eight hours post transfection, cells were collected, cell lysates were prepared, and the protein concentration was determined using BCA assay. The expression level of each construct was compared using Western Blot analysis with anti-NYESOl antibody (anti-CTAGIB, Abeam).
Example 4: ELISA assay
[0364] Balb/c female mice were immunized at week 0 and week 3 with 1, 5, and 10 pg of NYESO1 vaccine. (FIG. 3) Sera were collected 2 weeks post-immunization. The total IgG and the ratio of IgG2a/IgGl were measured. Briefly, 96-well plates were coated with 0.1 pg of NYESO1 protein (RayBiotech) per well and incubated overnight at 4°C. For measuring total IgG, individual sera were diluted starting 1 :200 in lx ELISA diluent buffer (Biolegnd) with 3- fold dilution up to 1 :437400 dilution and incubated for 2 h at room temperature. Subsequently, 100 pl of horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Jackson ImmunoResearch) at dilution of 1 :5000 was added, followed by incubation for Ihr at room temperature. The color reaction was developed with TMB substrate (Biolgend) for 15min at room temperature and then stopped with IN HC1. The absorbance was detected at 450 nm.
(FIGs. 5A-5B)
[0365| For measuring IgGl and IgG2a subtypes, individual sera of 1st immunization were diluted at 1 :200 in lx ELISA diluent buffer, while sera of 2nd immunization were diluted starting at 1 : 1800 with 3-fold dilution up to 1:48600. 100 pl of diluted sera was added to NYESO1 coated 96-well plate and incubated for 2 hours at room temperature. After 2 hours incubation 100 pl of horseradish peroxidase (HRP) conjugated goat anti -mouse IgGl and IgG2a subtypes (Abeam) at dilutions of 1 :5000 was added, followed by incubation for 1 h at room temperature. The color reaction was developed with TMB substrate (Biolgend) for 15min at room temperature and then stopped with IN HC1. The absorbance was detected at 450 nm. The ratio of IgG2a/IgGl was calculated based on the O.D. reading. (FIGs. 7A-7B and 8A-8C)
|'0366| For IgG quantification, sera collected 35 days (FIGs. 6A-6B) post 2nd immunization were used for quantifying IgG amount. Anti-CTAGIB antibody (Abeam) was used as standard starting at 200 ng/pl with 3 -fold dilution. Following the procedure for measuring total IgG, the absorbance of standards was detected at 450 nm. The standard curve was plotted using Sigmoidal, 4PL, X function in GraphPad. The amount of IgG of each immunization dose was calculated by interpolate the standard curve.
Example 5: ELISpot assay
[0367] Eight weeks after the final boost immunization, the spleens of the mice were collected, dissociated into single cell suspension mechanically, passed through a 70-pm cell strainer (Miltenyi Biotec), and lysed by ACK lysis buffer (KD Medical). The cells were then resuspended in RPMI supplemented with 10% FBS, lx Pen/Strep and lx 2-Mercaptoethanol, counted and adjusted to 2 x 106 cells/ml. After this, 100 pl of splenocytes were added to a well of IFN-y coated 96-well ELISpot plate (Mabtech). Splenocytes were then incubated for 16 hours in cell culture incubator with (stimulated) or without (unstimulated) stimulation of either full set overlapping peptides pools of NYESO1 protein that was prepared according to manufacturer’s instruction (JPT peptide) or a single NYESO1 157-165 peptide (iba). After incubation, following manufacturer’s instruction, biotinylated anti-IFN-y R4-A2-biotin monoclonal antibody was used as detecting Ab; and streptavidin-ALP complex and BCIP/NBT-plus substrate (Mabtech) were used to reveal the presence of spots. Spots formed by fFN-y-secreting cells were counted using
Cytation 7 (Biotek) and results are presented as spot-forming cells per 2x105 splenocytes. (FIGs. 9 and 10)
Example 6: Intracellular Staining (ICS) assay
[0368] Induction of antigen-specific T cells was determined using ICS. Splenocytes which were prepared as described above. IxlO6 splenocytes were added in 12 x 75 mm plastic tubes in the presence of protein transport inhibitor, brefeldin A (BioLegend) with (stimulated) or without (unstimulated) stimulation of either full set overlapping peptides pools of NYESO1 protein that was prepared according to manufacturer’s instruction (JPT peptide) or single NYESO1 157-165 peptide (iba). After 16 hour incubation, FACS analysis was performed to determine cytokines splenocytes. Cell surface staining was performed using antibodies against CD3 (FITC), CD4 (BV421) and CD8 (BV650). Intracellular staining was performed using antibodies against IL-4 (APC), and IFN-y (PE). eFluor450 was used to distinguish live/dead cells (Invitrogen). Cells were acquired using BD Celesta Flow cytometer (BD Biosciences) and flow cytometry data were analyzed using Flow Jo software. (FIGs. 11A-11D)
Equivalents
[0369] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.
[0370] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5' to 3' direction.
[0371 ] The disclosures illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as
terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.
[0372] Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.
[0373] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
[0374] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0375] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
Sequence Listing
SEQ ID NO: 1 (WT NYESO1 RNA)
AUGCAGGCCGAAGGCCGGGGCACAGGGGGUUCGACGGGCGAUGCUGAUGGCCCAG
GAGGCCCUGGCAUUCCUGAUGGCCCAGGGGGCAAUGCUGGCGGCCCAGGAGAGGC
GGGUGCCACGGGCGGCAGAGGUCCCCGGGGCGCAGGGGCAGCAAGGGCCUCGGGG
CCGGGAGGAGGCGCCCCGCGGGGUCCGCAUGGCGGCGCGGCUUCAGGGCUGAAUG
GAUGCUGCAGAUGCGGGGCCAGGGGGCCGGAGAGCCGCCUGCUUGAGUUCUACCU
CGCCAUGCCUUUCGCGACACCCAUGGAAGCAGAGCUGGCCCGCAGGAGCCUGGCC
CAGGAUGCCCCACCGCUUCCCGUGCCAGGGGUGCUUCUGAAGGAGUUCACUGUGU
CCGGCAACAUACUGACUAUCCGACUGACUGCUGCAGACCACCGCCAACUGCAGCU
CUCCAUCAGCUCCUGUCUCCAGCAGCUUUCCCUGUUGAUGUGGAUCACGCAGUGC
UUUCUGCCCGUGUUUUUGGCUCAGCCUCCCUCAGGGCAGAGGCGCUAASEQ ID
NO: 2 (WT NYESO1 peptide)
MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPG
GGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPP
LPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPS GQRR
SEQ ID NO: 3 (Codon Optimized NYESO1 RNA)
AUGGCACAGGCCGAGGGCAGAGGCACCGGCGGCAGCACCGGCGACGCCGAUGGCC
CUGGAGGCCCUGGCAUCCCCGACGGCCCUGGCGGCAACGCCGGAGGCCCCGGCGA
GGCCGGCGCUACAGGAGGAAGAGGCCCCAGAGGCGCCGGCGCCGCCCGGGCCAGC
GGCCCAGGCGGCGGCGCCCCUAGAGGUCCCCACGGCGGAGCCGCUUCCGGCCUGA
ACGGCUGCUGCAGGUGUGGCGCUAGAGGACCUGAGAGCAGACUGCUGGAAUUCU
ACCUGGCCAUGCCUUUCGCCACACCUAUGGAAGCCGAGCUGGCUAGACGGAGCCU
GGCCCAGGACGCCCCUCCACUGCCUGUCCCCGGAGUGCUGCUGAAGGAAUUUACC
GUGUCUGGCAAUAUCCUGACCAUCCGCCUGACAGCCGCUGAUCACCGGCAGCUGC
AGCUCUCCAUCAGCAGCUGCCUGCAGCAGCUGUCUCUGCUGAUGUGGAUCACCCA
GUGCUUCCUGCCCGUGUUCCUGGCCCAACCUCCUAGCGGCCAGCGGAGAAUAAAG
CUCGCUUUCUUGCUGUCCAAUUUCUAUUAA
SEQ ID NO: 4 (Codon Optimized NYESO1 peptide)
MAQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGP
GGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAP PLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPS GQRRIKLAFLLSNFY
SEQ ID NO: 5 (WT NYESO1 with COVID signal RNA)
ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTCAGCAGCCAGCAGGCCGAAGGCCGG
GGCACAGGGGGTTCGACGGGCGATGCTGATGGCCCAGGAGGCCCTGGCATTCCTGA
TGGCCCAGGGGGCAATGCTGGCGGCCCAGGAGAGGCGGGTGCCACGGGCGGCAGA
GGTCCCCGGGGCGCAGGGGCAGCAAGGGCCTCGGGGCCGGGAGGAGGCGCCCCGC
GGGGTCCGCATGGCGGCGCGGCTTCAGGGCTGAATGGATGCTGCAGATGCGGGGCC
AGGGGGCCGGAGAGCCGCCTGCTTGAGTTCTACCTCGCCATGCCTTTCGCGACACCC
ATGGAAGCAGAGCTGGCCCGCAGGAGCCTGGCCCAGGATGCCCCACCGCTTCCCGT
GCCAGGGGTGCTTCTGAAGGAGTTCACTGTGTCCGGCAACATACTGACTATCCGACT
GACTGCTGCAGACCACCGCCAACTGCAGCTCTCCATCAGCTCCTGTCTCCAGCAGCT
TTCCCTGTTGATGTGGATCACGCAGTGCTTTCTGCCCGTGTTTTTGGCTCAGCCTCCC TCAGGGCAGAGGCGCTAA
SEQ ID NO: 6 (WT NYESO1 with CO VID signal peptide)
MFVFLVLLPLVSSQQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGP RGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAE
LARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQ CFLPVFLAQPPSGQRR
SEQ ID NO: 7 (Codon Optimized NYESO1 with CO VID signal RNA)
AUGGCAUUCGUGUUUCUGGUCCUGCUGCCUCUGGUGUCCAGCCAGCAGGCCGAGG
GCAGAGGUACAGGCGGCAGCACCGGCGAUGCCGAUGGCCCCGGAGGCCCCGGCAU
CCCCGACGGCCCAGGCGGCAACGCCGGCGGCCCUGGAGAGGCCGGAGCCACCGGC
GGAAGAGGCCCAAGAGGAGCCGGCGCCGCUCGGGCCUCUGGCCCUGGAGGCGGAG
CUCCUAGAGGCCCCCACGGCGGCGCUGCUUCUGGCCUGAACGGCUGCUGCAGAUG
CGGCGCCAGGGGCCCUGAGAGCAGACUGCUGGAGUUCUACCUGGCCAUGCCUUUC
GCCACACCUAUGGAAGCCGAACUGGCCCGCCGGAGCCUGGCUCAGGACGCCCCUC
CUCUGCCUGUGCCCGGCGUGCUGCUGAAGGAAUUCACCGUGUCUGGCAAUAUCCU
GACCAUCCGGCUGACAGCCGCCGACCACAGACAGCUGCAGCUGAGCAUCAGCAGC
UGCCUGCAACAGCUUUCCCUGCUGAUGUGGAUCACCCAGUGUUUUCUGCCCGUGU
UCCUGGCCCAACCUCCAAGCGGCCAGCGGAGAAUAAAGCUCGCUUUCUUGCUGUC CAAUUUCUAUUAA
SEQ ID NO: 8 (Codon Optimized NYESO1 with CO VID signal peptide)
MAFVFLVLLPLVSSQQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRG
PRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEA
ELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWIT QCFLP VFL AQPP SGQRRIKL AFLL SNF Y
SEQ ID NO: 9 (3’ UTR)
AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC
AACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUA
AUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAA GCGAUCGCAAGCUUCUCGAGC
SEQ ID NO: 10 (5’ UTR)
UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCA ACCUCAAACAGACACCGCCACC
SEQ ID NO: 11 (pUC57 plasmid)
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG
GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTC
AGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTG
TACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAA
TACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATC
GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC
GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA
GTGAATTCGAGCTCGGTACCTCGCGAATGCATCTAGATATCGGATCCCGGGCCCGTC
GACTGCAGAGGCCTGCATGCAAGCTTTAATACGACTCACTATAAGGACATTTGCTTC
TGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTTGTTTTT
CTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTATGACTGAATATAAACTTGTGGTAG
TTGGAGCTGATGACGTAGGCAAGAGTGCCTTTACGATACAGCTAATTCAGAATCATT
TTGTGGACGAATATGATCCAACAATAGAGGATTCCTACAGGAAGCAAGTAGTAATT
GATGGAGAAACCTGTCTCTTGGATATTCTCGACACAACAGATCACGAGGAGTACAG
TGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCAT
AAATAATACTAAATCATTTGAAGATATTCACCATTATAGAGAACAAATTAAAAGAG
TTAAGGACTCTGAAGATGTACCTATGGTCCTAGTAGGAAATAATTGTGATTTGCCTT
CTAGAACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGAAGTTATGGAATTCCT
TTTATTGAAACATCAACAAAGACAAGACAGAGAGTGGAGGATGCTTTTTATACATTG
GTGAGAGAGATCCGACAATACAGATTGAAAAAAATCAGCAAAGAAGAAAAGACTC
CTGGCTGTGTGAAAATTAAAAAATGCATTATAATGTAAGCTCGCTTTCTTGCTGTCC
AATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTA
TGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCCA
ATAGGCCGAAATCGGCAAGCGCGATCGCAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAATTCCTCGAGGC
GCGCCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG
GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC
AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC
GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA
CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC
CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG
TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC
TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC
AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC
ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT
GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG
AACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG
TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA
GCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC
GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATT
ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAG
CCCAATCTGAATAATGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGA
GCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAA
AAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCA
AGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAAT
TTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGA
ATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCA
GCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGA
TTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAG
GAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCT
GAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTG
AGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCAT
AAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT
ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAGCGATA
GATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATC
AGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATGGCTCAT
AACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATA
TTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACGGGCCAGAGCTGCA
SEQ ID NO: 12 (Poly A tail)
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAA
SEQ ID NO: 13 (Poly A tail)
CGGCAAUAAAAAGACAGAAUAAAACGCACGGUGUUGGGUCGUUUGUUC
SEQ ID NO: 14 (HK peptide branch)
KHKHHKHHKHHI<HHI<HHI<HI<
SEQ ID NO: 15 (HK peptide branch)
I<HHHI<HHHI<HHHKHHHK
SEQ ID NO: 16 (HK peptide branch)
KHHHKHHHKHHHHKHHHK
SEQ ID NO: 17 (HK peptide branch) kHHHkHHHkHHHHkHHHk
SEQ ID NO: 18 (HK peptide branch)
HKHHHKHHHKHHHHKHHHK
SEQ ID NO: 19 (HK peptide branch)
HHKHHHKHHHKHHHHKHHHK
SEQ ID NO: 20 (HK peptide branch)
KHHHHKHHHHKHHHHKHHHHK
SEQ ID NO: 21 (HK peptide branch)
KHHHKHHHKHHHKHHHHK
SEQ ID NO: 22 (HK peptide branch)
KHHHKHHHHKHHHKHHHK
SEQ ID NO: 23 (HK peptide branch)
KHHHKHHHHKHHHKHHHHK
References
[1] Almeida LG, Sakabe NJ, deOliveira AR, Silva MCC, Mundstein AS, Cohen T, et al. CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens. Nucleic Acids Res (2009) 37:D816-9. doi:10.1093/nar/gkn673.
[2] Simpson AJG, Caballero OL, Jungbluth A, Chen Y-T, Old LJ. Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer (2005) 5:615-25. doi: 10.1038/nrcl669.
[3] Caballero OL, Chen Y-T. Cancer/testis (CT) antigens: potential targets for immunotherapy. Cancer Sci (2009) 100:2014-21. doi: 10.1111/j.1349-7006. 2009.01303.x.
[4] Rooney MS, Shukla SA, Wu CJ, Getz G, Hacohen N. Molecular and genetic properties of tumors associated with local immune cytolytic activity. Cell (2015) 160:48-61. doi: 10.1016/j.cell.2014.12.03.
[5] Chen Y-T, Ross DS, Chiu R, Zhou XK, Chen Y-Y, Lee P, et al. Multiple cancer/testis antigens are preferentially expressed in hormone-receptor negative and high-grade breast cancers. PLoS One (2011) 6:el7876. doi: 10.1371/joumal. pone.0017876.
[6] Woloszynska-Read A, Mhawech-Fauceglia P, Yu J, Odunsi K, Karpf AR. Intertumor and intratumor NY-ESO-1 expression heterogeneity is associated with promoter-specific and global DNA methylation status in ovarian cancer. Clin Cancer Res (2008) 14:3283-90. doi: 10.1158/1078-0432. CCR-07-5279.
[7] van der Bruggen P, Traversari C, Chomez P, Lurquin C, Plaen ED, van den Eynde BV, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (1991) 254: 1643-7. doi: 10.1126/science.1840703.
[8] van der Bruggen P, Traversari C, Chomez P, Lurquin C, Plaen ED, van den Eynde BV, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (19SE) 254: 1643-7. doi: 10.1126/science,1840703.
[9] Whitehurst AW. Cause and consequence of cancer/testis antigen activation in cancer. Annu Rev Pharmacol Toxicol (2014) 54:251-72. doi: 10.1146/annurev-pharmtox-011112- 140326.
[10] Gjerstorff MF, Andersen MH, Ditzel HJ. Oncogenic cancer/testis antigens: prime candidates for immunotherapy. Oncotarget (2015) 6: 15772-87.doi: 10.18632/oncotarget.4694.
[11] Fratta E, Coral S, Covre A, Parisi G, Colizzi F, Danielli R, et al. The biology of cancer testis antigens: putative function, regulation and therapeutic potential. Mol Oncol (2011) 5: 164- 82. doi: 10.1016/j. mol onc.2011.02.001.
[12] Jungbluth AA, Chen YT, Stockert E, Busam KJ, Kolb D, Iversen K, et al. Immunohistochemical analysis of NY-ESO-1 antigen expression in normal and malignant human tissues. Int J Cancer (2001) 92:856-60. doi: 10.1002/ijc.1282.
[13] Sharma P, Gnjatic S, Jungbluth AA, Williamson B, Herr H, Stockert E, et al. Frequency of NY-ESO-1 and LAGE-1 expression in bladder cancer and evidence of a new NY-ESO-1 T- cell epitope in a patient with bladder cancer. Cancer Immun (2003) 3: 19. doi : 10.1186/2051- 1426-3-S2-O19..
[14] Kurashige T, Noguchi Y, Saika T, Ono T, Nagata Y, Jungbluth A, et al. Ny-ESO-1 expression and immunogenicity associated with transitional cell carcinoma: correlation with tumor grade. Cancer Res (2001) 61 :4671-4.
[15] Bolli M, Schultz- Thater E, Zajac P, Guller U, Feder C, Sanguedolce F, et al. NY-ESO- 1/LAGE-l coexpression with MAGE-A cancer/testis antigens: a tissue microarray study. IntJ Cancer (2005) 115:960-6.doi: 10.1002/ijc.20953
[16] Akcakanat A, Kanda T, Koyama Y, Watanabe M, Kimura E, Yoshida Y, et al. NY-ESO-
1 expression and its serum immunoreactivity in esophageal cancer. Cancer Chemother Pharmacol (2004) 54:95-100. doi: 10.1007/s00280-.
[17] Prasad ML, Jungbluth AA, Patel SG, Iversen K, Hoshaw-Woodard S, Busam KJ. Expression and significance of cancer testis antigens in primary mucosal melanoma of the head and neck. Head Neck (2004) 26: 1053-7. doi: 10.1002/hed.10384.
[18] Odunsi K, Jungbluth AA, Stockert E, Qian F, Gnjatic S, Tammela J, et al. NY-ESO-1 and LAGE-1 cancer-testis antigens are potential targets for immunotherapy in epithelial ovarian cancer. Cancer Res (2003) 63: 6076-83.
[19] Fossa A, Berner A, Fossa SD, Hemes E, Gaudemack G, Smeland EB. NY-ESO-1 protein expression and humoral immune responses in prostate cancer. Prostate (2004) 59:440-7. doi: 10.1002/pros.20025.
[20] Oba-Shinjo SM, Caballero OL, Jungbluth AA, Rosemberg S, Old LJ, Simpson AJG, et al. Cancer-testis (CT) antigen expression in medulloblastoma. Cancer Immun (2008) 8:7.
[21] Kim SH, Lee S, Lee CH, Lee MK, Kim YD, Shin DH, et al. Expression of cancer-testis antigens MAGE- A3 /6 and NY-ESO-1 in non-small-cell lung carcinomas and their relationship with immune cell infiltration. Lung (2009) 187:401-11. doi: 10.1007/s00408-009-9181-3.
[22] Wang Y, Wu X-J, Zhao A-L, Yuan Y-H, Chen Y-T, Jungbluth AA, et al. Cancer/testis antigen expression and autologous humoral immunity to NYESO-1 in gastric cancer. Cancer Immun (2004) 4:11.
[23] Aung PP, Liu Y-C, Ballester LY, Robbins PF, Rosenberg SA, Lee C-CR. Expression of New York esophageal squamous cell carcinoma- 1 in primary and metastatic melanoma. Hum Pathol (2014) 45:259-67. doi:10.1016/j.humpath.2013.05.029.
[24] Lai J-P, Robbins PF, Raffeld M, Aung PP, Tsokos M, Rosenberg SA, et al. NY-ESO-1 expression in synovial sarcoma and other mesenchymal tumors: significance for NY-ESO-1- based targeted therapy and differential diagnosis. Mod Pathol (2012) 25:854-8. doi : 10.1038/m odpathol .2012.31.
[25] Grigoriadis A, Caballero OL, Hoek KS, da Silva L, Chen Y-T, Shin SJ, et al. CT-X antigen expression in human breast cancer. Proc Natl Acad Set USA (2009) 106: 13493-8. doi: 10.1073/pnas.0906840106.
[26] Ademuyiwa FO, Bshara W, Attwood K, Morrison C, Edge SB, Ambrosone CB, et al. NY-ESO-1 cancer testis antigen demonstrates high immunogenicity in triple negative breast cancer. PLoS One (2012) 7:e38783. doi: 10.1371/journal. pone.0038783.
[27] Theurillat J-P, Ingold F, Frei C, Zippelius A, Varga Z, Seifert B, et al. NY-ESO-1 protein expression in primary breast carcinoma and metastases: correlation with CD8+ T-cell and CD79a+ plasmacytic/B-cell infiltration. Int J Cancer (2007) 120:2411-7. doi: 10.1002/ijc.22376.
[28] Hemminger JA, Ewart Toland A, Scharschmidt TJ, Mayerson JL, Kraybill WG, Guttridge DC, et al. The cancer-testis antigen NY-ESO-1 is highly expressed in myxoid and round cell subset of liposarcomas. Mod Pathol (2013) 26:282-8. doi:10.1038/modpathol.2012.133.
[29] Pollack SM, Jungbluth AA, Hoch BL, Farrar EA, Bleakley M, Schneider DJ, et al. NY- ESO-1 is a ubiquitous immunotherapeutic target antigen for patients with myxoid/round cell liposarcoma. Cancer (2012) 118:4564-70. doi:10. 1002/cncr.27446.
[30] Jungbluth AA, Antonescu CR, Busam KJ, Iversen K, Kolb D, Coplan K, et al.
Monophasic and biphasic synovial sarcomas abundantly express cancer/ testis antigen NY-ESO- 1 but not MAGE- Al or CT7. Int J Cancer (2001) 94: 252-6. doi: 10.1002/ijc.1451.
[31] Rodolfo M, Luksch R, Stockert E, Chen Y-T, Collini P, Ranzani T, et al. Antigenspecific immunity in neuroblastoma patients: antibody and T-cell recognition of NY-ESO-1 tumor antigen. Cancer Res (2003) 63:6948-55.
[32] Barrow C, Browning J, MacGregor D, Davis ID, Sturrock S, Jungbluth AA, et al. Tumor antigen expression in melanoma varies according to antigen and stage. Clin Cancer Res (2006) 12:764-71. doi: 10.1158/1078-0432.CCR-05-1544.
Claims
1. An isolated deoxyribonucleic acid (DNA) comprising an open reading frame (ORF) encoding at least one of the following:
1) the ribonucleic (RNA) sequence as set forth in SEQ ID NO: 1 or the peptide as set forth in SEQ ID NO: 2;
2) the ribonucleic (RNA) sequence as set forth in SEQ ID NO: 3 or the peptide as set forth in SEQ ID NO: 4;
3) the ribonucleic (RNA) sequence as set forth in SEQ ID NO: 5 or the peptide as set forth in SEQ ID NO: 6; or
4) the ribonucleic (RNA) sequence as set forth in SEQ ID NO: 7 or the peptide as set forth in SEQ ID NO: 8.
2. An isolated ribonucleic acid (RNA) comprising at least one of the following:
1) the ribonucleic (RNA) sequence as set forth in SEQ ID NO: 1;
2) the ribonucleic (RNA) sequence as set forth in SEQ ID NO: 3;
3) the ribonucleic (RNA) sequence as set forth in SEQ ID NO: 5; or
4) the ribonucleic (RNA) sequence as set forth in SEQ ID NO: 7.
3. The DNA of claim 1, further encoding one or more of: a 3 -UTR, a 5'-UTR, a cap structure, and a poly-adenine nucleotide tail (a polyA tail).
4. The DNA of claim 3, wherein the 5'-UTR comprises a cap structure and a start codon, optionally wherein the 5’-UTR encodes
UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCA ACCUCAAACAGACACCGCCACC (SEQ ID NO: 10) or an equivalent thereof.
5. The DNA of claim 3 or 4, wherein the 3'-UTR comprises a stop codon and a polyA tail, optionally wherein the 3’-UTR encodes
AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC
AACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUA
AUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAA
GCGAUCGCAAGCUUCUCGAGC (SEQ ID NO: 9) or an equivalent thereof.
6. The RNA of claim 2, further comprising one or more of: a 3 -UTR, a 5'-UTR, a cap structure, and a poly-adenine nucleotide tail (a polyA tail).
7. The RNA of claim 6, wherein the 5'-UTR comprises a cap structure and a start codon, optionally wherein the 5’-UTR comprises UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCA ACCUCAAACAGACACCGCCACC (SEQ ID NO: 10) or an equivalent thereof.
8. The RNA of claim 6 or 7, wherein the 3'-UTR comprises a stop codon and a polyA tail, optionally wherein the 3’-UTR comprises AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC AACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUA AUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAA GCGAUCGCAAGCUUCUCGAGC (SEQ ID NO: 9) or an equivalent thereof.
9. The RNA of any one of claims 2 or 6 to 8, prepared by transcribing a polynucleotide encoding the RNA in an in vitro transcription (IVT) system.
10. The RNA of any one of claims 2 or 6 to 9, prepared by transcribing a plasmid DNA (pDNA) vector, optionally a pUC57 plasmid, encoding the RNA, optionally wherein the plasmid comprises SEQ ID NO: 11 or an equivalent thereof.
11. The RNA of any one of claims 2 or 6 to 10, wherein the GC content of the full-length RNA is about 35 % to about 70 % of the total RNA content.
12. The RNA of any one of claims 2 or 6 to 11, wherein the RNA is chemically modified, optionally wherein the chemical modification comprising one or both of the incorporation of an Nl-methyl-pseudouridine residue or a pseudouridine residue, further optionally wherein at least about 50% to about 100% of the uridine residues in the RNA are N1 -methyl pseudouridine or pseudouridine.
13. A polynucleotide comprising the DNA of any one of claims 1 or 3 to 5, encoding the RNA of any one of claims 2 or 6 to 12, or a polynucleotide complementary thereto, or any combination thereof.
14. The polynucleotide of claim 13, wherein the polynucleotide is selected from the group of: a deoxyribonucleic acid (DNA), an RNA, a hybrid of DNA and RNA, or an analog of each thereof.
15. A vector comprising the polynucleotide of claim 13 or 14.
16. The vector of claim 15, further comprising a regulatory sequence operatively linked to the polynucleotide to direct the transcription thereof.
17. The vector of claim 16, wherein the regulatory sequence comprises a promotor.
18. The vector of claim 17, wherein the promoter comprises: a bacteriophage RNA polymerase promoter, a T7 promoter, a SP6 promoter, or a T3 promoter.
19. The DNA of any one of claims 1 or 3 to 5, the RNA of any one of claims 2 or 6 to 12, the polynucleotide of claim 13 or 14, or the vector of any one of claims 15 to 18, further comprising a marker selected from a detectable marker, a purification marker, or a selection marker.
20. The RNA of claim 6 wherein the ORF is SEQ ID NO: 1, the 3’ UTR is SEQ ID NO: 9, the 5’ UTR is SEQ ID NO: 10, the poly A tail is SEQ ID NO: 12, and the cap structure is a 7- methylguanosine (7mG) cap.
21. The vector of any one of claims 15 to 18, wherein the vector is a non-viral vector, optionally a plasmid, or a liposome, or a micelle.
22. The vector of claim 21, wherein the plasmid comprises or consists of SEQ ID NO: 11 or an equivalent thereof.
23. The vector of any one of claims 15 to 18, wherein the vector is a viral vector, optionally an adenoviral vector, or an adeno-associated viral vector, or a retroviral vector, or a lentiviral vector, or a plant viral vector.
24. A cell comprising one or more of: the DNA of any one of claims 1 or 3 to 5, the RNA of any one of claims 2 or 6 to 12, the polynucleotide of claim 13 or 14, or the vector of any one of claims 15 to 18 or 21 to 23.
25. The cell of claim 24, wherein the cell is a eukaryotic cell, optionally a mammal cell, an insect cell, or a yeast cell.
26. A composition comprising a carrier, optionally a pharmaceutically acceptable carrier, and one or more of: the DNA of any one of claims 1 or 3 to 5, the RNA of any one of claims 2 or 6 to 12, the polynucleotide of claim 13 or 14, the vector of any one of claims 15 to 18 or 21 to 23, or the cell of any one of claims 24 or 25.
27. A method of producing an RNA, comprising culturing the cell of any one of claims 24 or 25 under conditions suitable for transcribing a DNA encoding the RNA to the RNA.
28. A method of producing an RNA, comprising contacting the polynucleotide of claim 13 or 14 or the vector of any one of claims 15 to 18 or 21 to 23 with an RNA polymerase, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine-5'-triphosphate (GTP), and uridine triphosphate (UTP) or a chemically modified UTP under conditions suitable for transcribing the polynucleotide or the vector to the RNA.
29. The method of claim 27 or 28, further comprising isolating the RNA.
30. An RNA produced by the method of any one of claims 27 to 29.
31. An immunogenic composition comprising an effective amount of the DNA of any one of claims 1 or 3 to 5 or the RNA of any one of claims 2 or 6 to 12 formulated in a pharmaceutically acceptable carrier.
32. The composition according to claim 31, wherein the pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle or both.
33. The composition of claim 32, wherein the polymeric nanoparticle carrier comprises a Histidine-Lysine co-polymer (HKP).
34. The composition of claim 33, wherein the HKP comprises H3K(+H)4b or H3k(+H)4b or both.
35. The composition of claim 33 or 34, wherein the polymeric nanoparticle carrier further comprises a lipid, optionally a cationic lipid.
36. The composition of claim 35, wherein the cationic lipid is ionizable.
37. The composition of claim 35 or 36, wherein the cationic lipid comprises Dlin-MC3-DMA (MC3) or dioleoyloxy-3-(trimethylammonio)propane (DOTAP) or both.
38. The composition of any one of claims 35 to 37, wherein the lipid further comprises one or more of: a helper lipid, a cholesterol, or a PEGylated lipid.
39. The composition of any one of claims 35 to 38, wherein the lipid further comprises PLA or PLGA.
40. The composition of any one of claims 34 to 39, wherein the HKP and the mRNA selfassemble into nanoparticles upon admixture.
41. The composition of any one of claims 33 to 40, wherein the liposomal nanoparticle carrier comprises a Spermine-Lipid Cholesterol (SLiC).
42. The composition of claim 41, wherein the SLiC is selected from the group consisting of TM1-TM5.
43. The composition of claim 31, wherein the pharmaceutical acceptable carrier is a lipid nanoparticle (LNP).
44. The composition of claim 43, wherein the LNP comprises a lipid, optionally a cationic lipid, optionally wherein the cationic lipid is ionizable, and optionally wherein the LNP comprises one or more of: 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-
(undecyloxy)hexyl] mino} octanoate (SM-102), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), or an equivalent of each thereof.
45. The composition of claim 44, wherein the LNP further comprises one or more of: a helper lipid, a cholesterol, or a PEGylated lipid.
46. The composition of claim 38 or 45, wherein the helper lipid comprises one or more of: disteroylphosphatidyl choline (DSPC), Dipalmitoylphosphatidylcholine (DPPC), (27?)-3- (Hexadecanoyloxy)-2-{[(9Z)-octadec-9-enoyl]oxy}propyl 2-(trimethylazaniumyl)ethyl phosphate (POPC), or dioleoyl phosphatidylethanolamine (DOPE).
47. The composition of any one of claims 38, 45 or 46, wherein the cholesterol comprises a plant cholesterol or an animal cholesterol or both.
48. The composition of any one of claims 38 or 45 to 47, wherein the PEGylated lipid comprises one or more of: PEG-c-DOMG (R-3-[(co-methoxy- poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine), PEG-DSG (1,2- Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) optionally PEG2000-DMG ((l,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N- [methoxy(polyethylene glycol)-2000)], or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
49. The composition of any one of claims 43 to 48, wherein the LNP comprises SM-102, DSPC, cholesterol and PEG2000-DMG.
50. The composition of claim 49, wherein the mass ratio of the SM-102, DSPC, cholesterol and PEG200-DMG is about 1 : 1 : 1 : 1 and/or wherein the molar ratio of the SM-102, DSPC, cholesterol and PEG2000-DMG is about 50: 10:38.5:1.5.
51. A method of producing the composition of any one of claims 31 to 40 or 46 to 49, comprising contacting the DNA of any one of claims 1 or 3 to 5 or the RNA of any one of claims 2 or 6 to 12 with an HKP, thereby the DNA or RNA and the HKP are self-assembled into nanoparticles.
52. The method of claim 51, wherein the mass ratio of HKP and the DNA or RNA in the contacting step is about 10: 1 to about 1 : 10, optionally 2.5:1.
53. The method of claim 51 or 52, further comprising contacting the HKP and DNA or RNA with cationic lipid, optionally wherein the cationic lipid comprises Dlin-MC3-DMA (MC3) or DOTAP (dioleoyloxy-3-(trimethylammonio)propane) or both.
54. The method of claim 53, wherein the mass ratio of the cationic lipid and the DNA or RNA in the contacting step is about 10: 1 to about 1: 10, optionally 1 : 1.
55. The method of claim 53 or 54, wherein the mass ratio of the HKP, the DNA or RNA and the cationic lipid in the contacting step is about 4:1 :1.
56. A method of producing the composition of any one of claims 31 or 43 to 48, comprising contacting the DNA of any one of claims 1 or 3 to 5 or the RNA of any one of claims 2 or 6 to 12 with a lipid, thereby the DNA or RNA and the lipid are self-assembled into lipid nanoparticles (LNPs).
57. The method of claim 56, wherein the LNPs comprise one or more of: 9-Heptadecanyl 8- {(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 2,2-dilinoleyl-4- dimethyl aminoethyl -[1 ,3 ]-di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), or an equivalent of each thereof.
58. The method of claim 57, wherein the LNPs further comprise one or more of: a helper lipid, a cholesterol, or a PEGylated lipid, optionally wherein the helper lipid comprises one or more of: disteroylphosphatidyl choline (DSPC), Dipalmitoylphosphatidylcholine (DPPC), (27?)- 3-(Hexadecanoyloxy)-2-{ [(9Z)-octadec-9-enoyl]oxy}propyl 2-(trimethylazaniumyl)ethyl phosphate (POPC), or dioleoyl phosphatidylethanolamine (DOPE), optionally wherein the cholesterol comprises a plant cholesterol or an animal cholesterol or both, and optionally wherein the PEGylated lipid comprises one or more of: PEG-c-DOMG (R-3-[(o)-methoxy- poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine), PEG-DSG (1,2- Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) optionally PEG2000-DMG ((l,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N- [methoxy(polyethylene glycol)-2000)], or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
59. The method of any one of claims 56 to 58, wherein the LNPs comprise SM-102, DSPC, cholesterol and PEG2000-DMG, optionally wherein the mass ratio of the SM-102, DSPC, cholesterol and PEG200-DMG is about 1 : 1 : 1 : 1 or optionally wherein the molar ratio of the SM- 102, DSPC, cholesterol and PEG2000-DMG is about 50: 10:38.5: 1.5.
60. The method of any one of claims 51 to 59, wherein the contacting step is performed in a microfluidic mixer, optionally selected from a slit interdigital micromixer, or a staggered herringbone micromixer (SHM).
61. A method of treating a subject having a cancer, the method comprising administering to said subject a pharmaceutically effective amount of any one or more of: the DNA of any one of claims 1 or 3 to 5, the RNA of any one of claims 2 or 6 to 12, the polynucleotide of claim 13 or 14, the vector of any one of claims 15 to 18 or 21 to 23, the cell of any one of claims 24 or 25, or the composition according to any one of claims 26 or 31 to 39.
62. The method of claim 61, wherein the administration is intratumoral, or intravenous, or intramuscular, or intradermal, or subcutaneous.
63. The method of claim 61 or 62, wherein the subject is a mammal, or a human.
64. The method of any one of claims 61 to 63, wherein the cancer comprises an NY-ESO-1 expressing cancer including, but not limited to: neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancers.
65. The method of any one of claims 61 to 64, further comprising administering to the subject an additional anti-cancer therapy.
66. A kit for use in the method of any one of claims 61 to 65, comprising instructions for use and one or more of: the DNA of any one of claims 1 or 3 to 5, the RNA of any one of claims 2 or 6 to 12, the polynucleotide of claim 13 or 14, the vector of any one of claims 15 to 18 or 21 to 23, the cell of any one of claims 24 or 25, the composition according to any one of claims 26 or
31 to 50, or an anti-cancer therapy.
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020010321A1 (en) * | 1996-10-03 | 2002-01-24 | Yao-Tseng Chen | Truncated forms of NY-ESO-1 protein |
US20100221349A1 (en) * | 2005-02-01 | 2010-09-02 | Powderject Vaccines, Inc. | Nucleic acid constructs |
US20100291156A1 (en) * | 2007-10-09 | 2010-11-18 | Marijke Barner | Composition for treating lung cancer, particularly of non-small lung cancers (nsclc) |
WO2022155238A1 (en) * | 2021-01-12 | 2022-07-21 | Cornell University | Needle and rod proteins as inflammasome agonists for augmenting immune responses |
-
2023
- 2023-11-02 WO PCT/US2023/078543 patent/WO2024097894A1/en unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020010321A1 (en) * | 1996-10-03 | 2002-01-24 | Yao-Tseng Chen | Truncated forms of NY-ESO-1 protein |
US20100221349A1 (en) * | 2005-02-01 | 2010-09-02 | Powderject Vaccines, Inc. | Nucleic acid constructs |
US20100291156A1 (en) * | 2007-10-09 | 2010-11-18 | Marijke Barner | Composition for treating lung cancer, particularly of non-small lung cancers (nsclc) |
WO2022155238A1 (en) * | 2021-01-12 | 2022-07-21 | Cornell University | Needle and rod proteins as inflammasome agonists for augmenting immune responses |
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