CN116970614A - Compositions and methods for ribonucleic acid vaccines encoding NY-ESO-1 - Google Patents
Compositions and methods for ribonucleic acid vaccines encoding NY-ESO-1 Download PDFInfo
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Abstract
Compositions and methods are provided for effective NY-ESO-1 vaccines for cancer treatment. The compositions include pharmaceutical compositions comprising an RNA molecule encoding a NY-ESO-1 derived peptide and a pharmaceutically acceptable carrier. Methods of stimulating a systemic immune response and performing therapy are provided, including intratumoral injection, intravenous injection, intramuscular injection, intradermal injection, and subcutaneous injection.
Description
Technical Field
The present disclosure relates to the field of RNA-based vaccines that generate an immune response to epitopes expressed by a cancer of interest.
Background
Cancers belong to a family of genetic diseases in which changes in genetic material drive normal cells into a deregulated state, manifested as malignant growth of tumor tissue. Among all the different types of cancer, lung cancer is responsible for the most number of deaths, with 135,720 expected to die of the disease. This is almost three times the number of 53,200 deaths due to colorectal cancer, the second most common cause of cancer death. Pancreatic cancer ranks the most deadly cancer third leading to death of 47,050.
Cancer-testis antigens (CTA) form a family of antigens encoded by 276 genes, including more than 70 gene families, whose expression is typically limited to testis germ cells and placenta trophoblasts, with no or low expression in normal adult somatic cells (1, 2, 3).
In addition to lower expression in normal cells, several CTAs have been found to be re-expressed in a variety of epithelial cancers. For example, rooney et al identified 60 CTAs that are not expressed in normal tissue but are ectopically expressed in many tumor types, including melanoma, head and neck cancer, lung cancer, liver cancer, stomach cancer, and ovarian cancer (4).
Based on the frequency of CTA expression, tumors can be classified as CTA-rich, CTA-intermediate or CTA-lean. CTA-rich tumors include melanoma, ovarian cancer, lung cancer, and bladder cancer. The CTA-intermediate expressing tumor group includes breast cancer, bladder cancer and prostate cancer. Renal cell carcinoma, colorectal carcinoma and lymphoma/leukemia are all classified as CTA-lean tumors (3, 5). In addition, although CTA expression may increase in a large number of primary tumors, this may not necessarily be reflected on a single cell level. For example, microdissection of ovarian cancer specimens confirmed the considerable intratumoral heterogeneity of NY-ESO-1 expression (6).
Cancer-testis antigens are not only re-expressed in tumor tissue, but they may also be immunogenic proteins, as many members of this family have been shown to elicit spontaneous cellular and humoral immune responses in cancer patients.
The first identified CTA (MAGE-A1) was discovered by its ability to induce an autologous cytotoxic T lymphocyte response in melanoma patients (7). Since then, several other CTAs have been identified as immunogenic Tumor Associated Antigens (TAAs), including SSX-2, NY-ESO-1, and members of the bag, gap, and MAGE families (2, 8).
Although the function of CTA has been studied, it is still not clear. Transgenic mouse models have revealed that several CTA members play a key role in male fertility (9). Few CTAs are involved in cell metabolism during spermatogenesis, cytoskeletal dynamics, double strand break repair, maintenance of genomic integrity, and regulation of mRNA expression. Although more and more studies demonstrate the re-expression of CTA in cancer, their functional role in tumorigenesis has largely not been explored. More recent data indicate the role of CTA in regulating epithelial-mesenchymal transition and tumor cell survival (10).
NY-ESO-1 is a promising CTA for immune-based therapies because its tumor expression is markedly correlated with the induction of immune responses in many malignant tumors (11). In addition, NY-ESO-1 is a typical example of CTA, whose expression is limited to germ cells and placental cells and re-expressed in tumor cells.
NY-ESO-1 has been reported to be expressed 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). Among these tumor types, the expression frequency of NY-ESO-1 varies greatly, with the most commonly expressed tumors being mucoid and round cell liposarcoma (89% -100%), neuroblastoma (82%), synovial sarcoma (80%), melanoma (46%) and ovarian carcinoma (43%) (18, 47-51). Other tumor types showed protein expression of NY-ESO-1 in the range of 20% -40%.
It is important to note that most cancer types show heterologous expression of NY-ESO-1, which may limit the therapeutic response to NY-ESO-1 targeted therapies. The most uniform expression has been reported in mucoid and round cell liposarcoma (94%) and synovial sarcoma (70%), which may be correlated with promising results that have been obtained in adoptive cellular immunotherapy trials (28, 30).
Based on the above-mentioned cancer incidence, there is a need for effective treatment of cancer, and NY-ESO-1 provides a way to do so. The present disclosure meets this need and provides related advantages as well.
Disclosure of Invention
In one aspect, the disclosure relates to RNA for use as a vaccine that induces in vivo production of NY-ESO-1. This production of NY-ESO-1 in turn elicits an immune response that may prevent or treat cancer. DNA molecules encoding RNAs (such as mrnas) are also provided.
Vectors, host cells and vaccines comprising the mRNA and/or DNA molecules disclosed herein are also provided, as are compositions containing such vectors, host cells and vaccines, and in vitro and in vivo uses of such DNA, mRNA, vectors, host cells, vaccines and compositions.
Applicants have increased the overall activity and expression level of mRNA vaccines through the use of chemical modifications, such as m7G caps or polyA tails. Thus, in one aspect, the present disclosure provides mRNA that also includes one or more of an m7G cap and/or a polyA tail. In addition, applicants provide in vitro transcription techniques to increase mRNA vaccine production. Thus, the present disclosure also provides this.
mRNA and vector (vector) can be coupled to a vector (carrier) technology such as branched histidine-lysine (HK) polypeptide (HKP) for encapsulating mRNA, as well as methods for coupling mRNA to a vector technology. As disclosed herein, applicants' encapsulated mRNA exhibits enhanced specificity and cell penetration. The combination of mRNA vaccines encapsulated in H3K (+h) 4b and MC3/DOTAP liposome carriers was more effective than liposomes alone.
In one aspect, an isolated deoxyribonucleic acid (DNA) or an isolated ribonucleic acid (RNA) is provided, the DNA or RNA comprising, consisting essentially of, or still further consisting of an Open Reading Frame (ORF) encoding a NY-ESO-1 derived peptide.
In some embodiments, the isolated DNA encodes one or more of the following: 1) An RNA sequence shown as SEQ ID NO. 1 or a peptide shown as SEQ ID NO. 2; 2) An RNA sequence shown as SEQ ID NO. 3 or a peptide shown as SEQ ID NO. 4; 3) An RNA sequence shown as SEQ ID NO. 5 or a peptide shown as SEQ ID NO. 6; or 4) an RNA sequence as shown in SEQ ID NO. 7 or a peptide as shown in SEQ ID NO. 8.
In other embodiments, the isolated RNA comprises one or more of the following: 1) An RNA sequence shown as SEQ ID NO. 1; 2) An RNA sequence shown as SEQ ID NO. 3; 3) An RNA sequence shown as SEQ ID NO. 5; or 4) an RNA sequence shown in SEQ ID NO. 7.
In one aspect, the RNA molecule further comprises a 3'-UTR, a 5' -UTR, and optionally further comprises (a) stabilizing the molecule and (b) an additional element that enhances expression of a polypeptide encoded by the ORF. In a further aspect, the 5' -UTR comprises an m7G cap structure and/or an initiation codon. In a further aspect, the 3' -UTR comprises a stop codon and/or a polyA tail.
In another aspect, an immunogenic composition comprising an mRNA as disclosed herein and a pharmaceutically acceptable carrier is provided. In a further aspect, the pharmaceutically acceptable carrier is a polymer nanoparticle and/or a liposome nanoparticle. Still further, the vector comprises a branched histidine-lysine (HKP) polypeptide (HKP) to encapsulate mRNA, such as H3K (+h) 4b or MC3/DOTAP liposome vector. In one aspect, both carriers are used in a single composition.
In another aspect, the mRNA of the immunogenic composition further comprises a 3'-UTR, a 5' -UTR, and optionally further comprises (a) a stabilizing molecule and (b) additional elements that enhance expression of the polypeptide encoded by the ORF. In a further aspect, the 5' -UTR comprises an m7G cap structure and an initiation 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 nucleotide AAUAAA.
In another aspect, the mRNA of the composition can be expressed in a linear In Vitro Transcription (IVT) system or a plasmid DNA (pDNA) vector delivery system. Such a linear system is also provided herein.
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 mixing. In another aspect, the carrier comprises PLA or PLGA. In a further aspect, the composition contains 1, 2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP).
In another aspect, the carrier comprises spermine-lipid cholesterol (SLiC). In a further aspect, the SLiC is selected from the structures TM1-TM5 shown in FIG. 13.
In another aspect, there is provided the use of an mRNA or composition disclosed herein in the manufacture of a medicament for treating a tumor expressing NY-ESO-1 in a subject. The above-described compositions may be provided in any acceptable manner, as determined by the treating physician, researcher or veterinarian. Non-limiting modes of administration include: intratumoral, intravenous, intramuscular, intradermal or subcutaneous administration. In a further aspect, the subject is a mammal, e.g., a human.
Drawings
FIGS. 1A and 1B show in vitro transcription of NY-ESO-1 vaccine candidates. Four NY-ESO-1 vaccine candidates were designed and synthesized and sequence verified. Plasmids encoding NY-ESO-1 vaccine candidates were digested with XhoI for 4 hours at 37℃and then subjected to agarose gel validation (FIG. 1A) and in vitro transcription (FIG. 1B). The integrity of the mRNA was checked using agarose gel (fig. 1B).
FIG. 2 shows in vitro expression of NY-ESO-1 vaccine candidates in 293T cells. Various amounts of NY-ESO-1 vaccine candidate mRNA (0.5. Mu.g, 1. Mu.g, and 2.5. Mu.g) were transfected into 293T cells. 48 hours after transfection, western blot analysis was performed to detect in vitro expression of each candidate. Wild-type NY-ESO-1 showed the highest in vitro expression among all candidates.
Figure 3 shows an in vivo animal study plan. This animal study plan was intended to determine the vaccine dose for future studies. On days 0 and 21, three groups of mice (3 mice/group) were immunized with 1. Mu.g, 5. Mu.g, and 10. Mu.g of NY-ESO-1 vaccine. Two weeks after immunization, blood was collected and ELISA assays were performed to determine the immunogenicity of the NY-ESO-1 vaccine. At the end of the study, mice were sacrificed and spleens were removed to isolate spleen cells. Enzyme-linked immunosorbent assay (ELISPot), intracellular staining and cytokine profile analysis were then performed to measure the T cell response induced by the NY-ESO-1 vaccine.
FIG. 4 shows the in vitro expression of the NY-ESO-1 vaccine in 293T cells. The newly formulated NY-ESO-1 vaccine was transfected into 293T cells at 0.5. Mu.g, 1. Mu.g and 2.5. Mu.g. Western blot analysis was performed to detect in vitro expression of NY-ESO-1 delivered by LNP.
FIGS. 5A and 5B show immunogenicity of NY-ESO-1 as measured by ELISA assay. Serum was collected two weeks after immunization for testing immunogenicity. Serum was serially diluted, starting at 1:200 dilution 3-fold to 1:437400. Dose-dependent immunogenicity of NY-ESO-1 was observed 14 days (fig. 5A) and 35 days (fig. 5B) after immunization.
Fig. 6A and 6B show IgG quantification results of serum collected on day 35. The amount of IgG in serum collected from day 35 after immunization 1 was measured using an anti-CTAG 1B antibody as a standard. Standard curves were plotted using the S-mode (Sigmoidal), 4PL, X functions in GraphPad (fig. 6A). The amount of IgG per immunization dose was calculated by interpolating the standard curve (fig. 6B).
Fig. 7A and 7B show IgG subtype assays performed on day 14 serum samples using ELISA assays. The figure shows IgG subtypes in serum collected two weeks after immunization 1 (fig. 7A). Serum was diluted at a dilution of 1:200. The ratio of IgG2a/IgG1 was calculated based on the OD readings. The results indicate that immunization 1 induced a Th 1-biased response (fig. 7B).
Figures 8A-8C show IgG subtype assays performed on day 35 serum samples using ELISA assays. The figure shows the IgG subtype in serum collected two weeks after immunization 2. Serum was diluted from 1:1800 dilution to 1:48600 (fig. 8A-8B). The ratio of IgG2a/IgG1 was calculated based on OD readings (FIG. 8C). The results indicated that the 1. Mu.g NY-ESO-1 group had a Th1 biased response, while the 5. Mu.g and 10. Mu.g groups exhibited a Th1 and Th2 balanced response.
Figure 9 shows an enzyme-linked immunosorbent assay for T cell responses. At the end of the study, spleens from immunized mice were removed, spleen cells were isolated, and stimulated with two peptide pools. One is a whole set of NY-ESO-1 peptide mixtures and the other is a single NY-ESO-1 157-165 peptide. The number of ifnγ spots in stimulated spleen cells showed a dose-dependent increase, and the complete set of NY-ESO-1 peptide mixtures induced more ifnγ secreting spleen cells.
FIG. 10 shows the quantitative results of IFN gamma secreting cells in stimulated spleen cells.
Fig. 11A-11D show intracellular staining to determine T cell responses. At the end of the study, spleens from immunized mice were removed, spleen cells were isolated, and stimulated with two peptide pools. One is a whole set of NY-ESO-1 peptide mixtures and the other is a single NY-ESO-1 157-165 peptide. Sixteen hours after stimulation, FACS analysis was performed to detect ifnγ and IL4 secreting T cells. The results indicate that the NY-ESO-1 vaccine-induced T cell response was a CD8+ cell-mediated response (FIGS. 11C and 11D), rather than a CD4+ cell-mediated response (FIGS. 11A and 11B).
FIG. 12 provides a schematic representation of the optimized mRNA vaccine expression structure. Such structures may be included and/or transcribed in a linear In Vitro Transcription (IVT) expression system or plasmid DNA delivery vector.
Fig. 13 shows the structure of spermine-lipid conjugate (SLiC) species.
Detailed Description
Definition of the definition
As will be appreciated, the section or sub-section headings used herein are for organizational purposes only and are not to be construed as limiting and/or separating the described subject matter.
It is to be understood that the invention is not limited to the specific 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 the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which 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 of the techniques and patent publications cited herein are incorporated 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.
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, for example, sambrook and Russell et al (2001) 'Molecular Cloning: A Laboratory Manual', 3 rd edition; ausubel et al, editions (2007) "Current Protocols in Molecular Biology" series; a "Methods in Enzymology" series, (academic press, inc., new york); macPherson et al (1991) "PCR 1: a Practical Approach "(Oxford University Press IRL Press); macPherson et al (1995) "PCR 2: A Practical Approach"; harlow and Lane editions (1999) "Antibodies, A Laboratory Manual"; freshney (2005) "Culture of Animal Cells: A Manual of Basic Techique", 5 th edition; gait was edited (1984) "Oligonucleotide Synthesis"; U.S. Pat. nos. 4,683,195; hames and Higgins, editions (1984) "Nucleic Acid Hybridization"; anderson (1999) "Nucleic Acid Hybridization"; hames and Higgins, editions (1984) "Transcription and Translation"; "Immobilized Cells and Enzymes" (IRL Press (1986)); perbal (1984) "A Practical Guide to Molecular Cloning"; miller and Calos et al (1987) 'Gene Transfer Vectors for Mammalian Cells' (Cold Spring Harbor Laboratory); makrides et al (2003) "Gene Transfer and Expression in Mammalian Cells"; mayer and Walker editions (1987) "ImmunochemicalMethods in Cell and Molecular Biology" (Academic Press, london); herzenberg et al (1996) "Weir's Handbook of Experimental Immunology"; "Manipulating the Mouse Embryo: A Laboratory Manual", 3 rd edition (Cold Spring Harbor Laboratory Press (2002)); sohail et al (2004) "Gene Silencing by RNA Interference: technology andApplication" (CRC Press); and Plotkin et al, plotkin; "Human Vaccines", 7 th edition (Elsevier).
As used in this specification and the claims, the singular forms "a," "an," "the," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof.
As used herein, the term "comprising" is intended to mean that the compounds, compositions, and methods include the recited elements, but do not exclude other elements. When used to define compounds, compositions and methods, "consisting essentially of … …" shall mean excluding other elements having any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants, e.g., from isolation and purification methods, as well as pharmaceutically acceptable carriers, preservatives, and the like. "consisting of … …" shall mean excluding other ingredients beyond trace elements. Implementations defined by each of these transitional terms are within the scope of the present technology.
All numerical symbols (e.g., pH, temperature, time, concentration, and molecular weight, including ranges) are approximations, varying positively (+) or negatively (-) in 1%, 5%, or 10% increments. It should be understood that all numerical symbols are preceded by the term "about", although not always explicitly stated. It is also to be understood that the agents described herein are merely exemplary and that equivalents thereof are known in the art, although not always explicitly stated.
The term "about" as used herein when referring to a measurable amount, such as an amount or concentration, is meant to encompass a 20%, 10%, 5%, 1%, 0.5% or even 0.1% change in the specified amount.
As used herein, a comparative term, such as high, low, increasing, decreasing, or any grammatical variation thereof, may refer to certain variations relative to a reference. In some embodiments, such a change may 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-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or about 20-fold, or about 30-fold, or about 40-fold, or about 50-fold, or about 60-fold, or about 70-fold, or about 80-fold, or about 90-fold, or about 100-fold or more than the reference. In some embodiments, such a change may 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 9%, 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.
As will be understood by those of skill in the art, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof for any and all purposes. Furthermore, as will be appreciated by those skilled in the art, the scope includes each individual member.
"optional" or "optionally" means that the subsequently described circumstance may or may not occur, and thus that the description includes instances where the circumstance occurs and instances where it does not.
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 understood in the alternative ("or").
"substantially" or "essentially" means almost all or all, e.g., 95% or more of a given amount. In some embodiments, "substantially" or "essentially" means 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%.
When used to describe the selection of any component, range, dosage form, etc. disclosed herein, the term "acceptable", "effective" or "sufficient" means that the component, range, dosage form, etc. is suitable for the purpose disclosed.
The terms "subject," "host," "individual," and "patient" are used interchangeably herein to refer to human and veterinary subjects, such as humans, animals, non-human primates, dogs, cats, sheep, mice, horses, and cattle. In some embodiments, the subject is a human.
In some embodiments, the terms "first," "second," "third," "fourth," or similar terms in component names are used to distinguish and identify more than one component that has some correspondence in name. For example, "first RNA" and "second RNA" are used to distinguish between the two RNAs.
The phrase "first line" or "second line" or "third line" refers to the order in which the patient is treated. The first line treatment regimen is the treatment administered first, whereas the second line therapy or the third line therapy regimen is administered after the first line therapy or after the second line therapy, respectively. First-line therapy is defined by the national cancer institute as "first-line treatment for a disease or disorder". In cancer patients, the primary treatment may be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First line therapy is also known to those skilled in the art as "primary therapy and primary treatment". See national cancer institute website www.cancer.gov, last visit time was 5 months 1 day 2008. In general, subsequent chemotherapy regimens are administered to the patient because the patient does not exhibit a positive clinical or sub-clinical response to the first line therapy, or because the first line therapy has ceased.
"3' untranslated region" (3 ' UTR) refers to the region of an mRNA that is directly downstream (i.e., 3 ') of the stop codon (i.e., the codon in the mRNA transcript that signals the termination of translation) that does not encode a polypeptide.
The term "adenovirus" is synonymous with the term "adenovirus vector" and refers to a virus of the genus adenovirus. The term "adenovirus" refers generally to animal adenoviruses of the genus mammalian adenoviruses, including but not limited to, human, bovine, ovine, equine, canine, porcine, murine, and simian adenoviruses subgenera. In particular, human adenoviruses include the subgenera A-F and each serotype thereof, and each serotype and subgenera includes, but is not limited to, human adenovirus type 1, type 2, type 3, type 4a, type 5, type 6, type 7, type 8, type 9, type 10, type 11 (Ad 11A and Ad 11P), type 12, type 13, type 14, type 15, type 16, type 17, type 18, type 19a, type 20, type 21, type 22, type 23, type 24, type 25, type 26, type 27, type 28, type 29, type 30, type 31, type 32, type 33, type 34a, type 35P, type 36, type 37, type 38, type 39, type 40, type 41, type 42, type 43, type 44, type 45, type 46, type 47, type 48, and type 91. The term "bovine adenovirus" includes, but is not limited to, bovine adenovirus type 1, type 2, type 3, type 4, type 7, and type 10. The term "canine adenovirus" includes, but is not limited to, canine type 1 (strains CLL, glaxo, R1261, utrect, toronto 26-61) and type 2. The term "equine adenovirus" includes, but is not limited to, equine type 1 and type 2. The term "porcine adenovirus" includes, but is not limited to, porcine type 3 and type 4. In one embodiment of the invention, the adenovirus is derived from human adenovirus serotype 2 or 5. For the purposes of the present invention, an adenovirus vector may have replication capacity or replication defects in a target cell. In some embodiments, the adenovirus vector is a conditionally or selectively replicating adenovirus, wherein genes required for viral replication are operably linked to a cell and/or environment specific promoter. Examples of selective replication or conditional replication viral vectors are known in the art (see, e.g., U.S. patent No. 7,691,370).
Retroviruses such as gamma retrovirus and/or lentivirus comprise (a) an envelope containing lipids and glycoproteins, (b) a vector genome, which is an RNA delivered to a target cell (typically a dimeric RNA comprising a cap at the 5 'end and LTR flanking the polyA tail at the 3' end), (c) a capsid, and (d) a protein, such as a protease. U.S. patent No. 6,924,123 discloses that certain retroviral sequences facilitate integration into the target cell genome. The patent teaches that each retroviral genome contains genes called gag, pol and env, which encode virion proteins and enzymes. These genes are flanked by regions called Long Terminal Repeats (LTRs). The LTR is responsible for proviral integration and transcription. They also function as enhancer-promoter sequences. In other words, the LTR may control the expression of viral genes. Encapsidation of retroviral RNA occurs through the psi sequence located at the 5' end of the viral genome. The LTRs themselves are identical sequences and can be divided into three elements, which are referred to as U3, R and U5. U3 is derived from a sequence unique to the 3' end of RNA. R is derived from the 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 these three elements can vary widely 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 LTR. U3 contains most of the transcriptional control elements of provirus, including promoters and multiple enhancer sequences that are responsive to the cell, and in some cases to viral transcriptional activator proteins.
Regarding the structural genes gag, pol and env themselves, gag encodes the internal structural proteins of the virus. The gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes a Reverse Transcriptase (RT) which contains a DNA polymerase that mediates genome replication, an associated RNase H and an Integrase (IN).
To produce viral vector particles, the vector RNA genome is expressed in a host cell by a DNA construct encoding the same. The components of the particle not encoded by the vector genome are provided in trans (trans) by an additional nucleic acid sequence expressed in the host cell ("packaging system", which typically includes one or both of the gag/pol and env genes). 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 mixed manner. The techniques involved are known to those skilled in the art.
The term "adeno-associated virus" or "AAV" as used herein refers to the genus "adeno-associated virus" or "AAV" associated with the name and belonging to the Paramyxoviridae familyParvoviridae) Dependent parvovirus genusdependoparvovirus) Is a member of the virus class. A variety of serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from a variety of tissue types. At least 11 sequentially numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes for use in the methods disclosed herein include any of 11 serotypes, such as AAV2, AAV8, AAV9, or variants or synthetic serotypes, such as AAV-DJ and AAV php.b. AAV particles comprise three major viral proteins: VP1, VP2 and VP3 alternatively consist essentially of, or still further consist of. In one embodiment, AAV refers to serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, aavphp.b, or AAV rh74. These vectors are commercially available or have been described in the patent or technical literature.
As used herein, "plant viruses" refers to a group of viruses that have been identified as pathogenic to plants. These viruses rely on plant hosts for replication because they lack the molecular mechanism to replicate without a plant host. Thus, plant viruses can be used as vectors for safely delivering genes of interest to non-plant animal subjects. Plant viruses include, but are not limited to, tobacco mosaic virus (tobacco mosaic virus), maize chlorotic mottle virus (Maize chlorotic mottle virus), maize Rakagaku's virus (Maize rayado fino virus), oat chlorotic dwarf virus (Oat chlorotic stunt virus), chayote flower She Wujing yellow mosaic virus (Chayote mosaic tymovirus), grape star mosaic associated virus (Grapevine asteroid mosaic-associated virus), grape mottle virus (Grapevine fleck virus), grape red virus (Grapevine Red Globe virus), sand grape vein eclosion virus (Grapevine rupestris vein feathering virus), melon necrotic mottle virus (Melon necrotic spot virus), physalis alkohexa yellow mosaic virus (Physalis mottle tymovirus), plum necrosis ring spot virus (Prunus necrotic ringspot), nigria tobacco latent virus (Nigerian tobacco latent virus), tobacco light green mosaic virus (Tobacco mild green mosaic virus), tobacco necrosis virus (Tobacco necrosis virus), eggplant mosaic virus (Eggplant mosaic virus), kennedy yellow mosaic virus (Kennedya yellow mosaic virus), tomato TVM-like virus (Lycopersicon esculentum TVM viroid), blue mosaic virus (Oat blue dwarf virus), presbya pepper virus (Obuda pepper virus), olive mosaic virus type 1 (Olive latent virus), red pepper virus (Paprika mild mottle virus), tomato mosaic virus (Tomato mosaic virus), light mosaic virus (3595), turnip vein-clear virus (Turnip vein-clear virus), carnation mottle virus (Carnation mottle virus), duck mottle virus (Cocksfoot mottle virus), achyranthes mosaic virus (Galinsoga mosaic virus), sorghum chlorosis stripe mosaic virus (Johnsongrass chlorotic stripe mosaic virus), dental ring spot virus (Odontoglossum ringspot virus), formononetia yellow mosaic virus (Ononis yellow mosaic virus), broomcorn mosaic virus (Panicum mosaic virus), poinsettia mosaic virus (Poinsettia mosaic virus), scindapsus aureus latent virus (Pothos latent virus) or plantain mosaic virus (Ribgrass mosaic virus).
Gene delivery vehicles also include DNA/liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes that also contain a targeting antibody or fragment thereof can be used in the methods disclosed herein. In addition to delivery of polynucleotides to a cell or cell population, the proteins described herein may be introduced directly into the cell or cell population by non-limiting protein transfection techniques, alternatively, culture conditions that may enhance expression and/or promote activity of the proteins disclosed herein are other non-limiting techniques.
As used herein, the term "animal" refers to a living multicellular vertebrate organism, i.e., a class that includes, for example, mammals and birds. The term "mammal" includes both human and non-human mammals.
As used herein, "cancer" is a disease state characterized by the presence of cells in a subject that exhibit abnormal uncontrolled replication, and is used interchangeably with the term "tumor. In some embodiments, the cancer is a cell expressing NY-ESO-1.
The terms "chemically modified" and "chemically modified" refer to modification of adenosine (a), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides in at least one of their position, pattern, percentage, or population. In some embodiments, the term refers to ribonucleotide modification in the cap portion of a naturally occurring 5' -terminal mRNA. In a further embodiment, the chemical modification is selected from the group consisting of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methyluridine, 5-methoxy-uridine or 2' -O-methyl-uridine. In some embodiments, the degree of incorporation of the chemically modified nucleotide has been optimized to improve the immune response to the vaccine formulation. In other embodiments, the term does not include ribonucleotide modifications in the cap portion of the naturally occurring 5' -terminal mRNA.
In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) includes non-naturally modified nucleotides that are introduced during or after synthesis of the polynucleotide to achieve a desired function or property. Modifications may be present on internucleotide linkages, purine or pyrimidine bases or sugars. Modifications may be introduced at the end of the chain or anywhere else in the chain, either by chemical synthesis or by polymerase. Any of the regions of the polynucleotide may be chemically modified.
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 more of the residues of the RNAs are modified chemically by one or more of the modifications 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 more of the uridine residues are modified by one or more of the modification chemistries disclosed herein.
As used herein, "complementary" sequences refer to two nucleotide sequences that contain multiple, mutually paired, individual nucleotide bases when aligned antiparallel to each other. Pairing of nucleotide bases forms hydrogen bonds, thereby stabilizing the double-stranded structure formed by the complementary sequences. Each nucleotide base in the two sequences need not pair with each other to be considered a "complementary" sequence. For example, a sequence may be considered complementary if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the nucleotide bases in the two sequences pair with each other. In some embodiments, the term complementary refers to 100% of the nucleotide bases in the two sequences being paired with each other. Furthermore, 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 if its individual nucleotide bases pair with nucleotide bases in the longer polynucleotide when aligned antiparallel to a specific region of the longer polynucleotide containing hundreds of nucleotides. Nucleotide base pairing is known in the art, such as in DNA, the pairing of purine adenine (a) with pyrimidine thymine (T), and pyrimidine cytosine (C) with purine guanine (G) at all times; in RNA, adenine (A) is paired with uracil (U), and guanine (G) is paired with cytosine (C). Furthermore, nucleotide bases that are aligned antiparallel to each other in two complementary sequences, but are not a pair, are referred to herein as mismatches.
As used herein, "NY-ESO-1" refers to an 18 kDa protein of 180 amino acids having a glycine-rich N-terminal region and a strongly hydrophobic C-terminal region with a Pcc-1 domain. The sequence of the wild type NY-ESO-1 is provided in SEQ ID NO. 2. NY-ESO-1 is a member of a family of antigens known as cancer-testis antigens (CTAs). NY-ESO-1 has been reported to be expressed 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.
"composition" is intended to mean a combination of an active agent with another inert (e.g., a detectable agent or label) or active compound or composition (such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc.), and includes a carrier, such as a pharmaceutically acceptable carrier. In some embodiments, the carrier, such as a pharmaceutically acceptable carrier, comprises, consists essentially of, or still further consists of a nanoparticle, such as a polymeric nanoparticle carrier (e.g., 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-oligosaccharides, tri-oligosaccharides, tetra-oligosaccharides and other oligosaccharides; derivatized sugars such as sugar alcohols, aldonic acids, esterified sugars, etc., and polysaccharides or sugar polymers), which may be present alone or in combination in amounts of 1% to 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 may also act as buffering agents) 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 within the scope of the present 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, glucans, starches, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), and inositol.
The compositions disclosed herein may be pharmaceutical compositions. "pharmaceutical composition" is intended to include a combination of an active agent and an inert or active carrier, such that the composition is suitable for in vitro, in vivo, or ex vivo diagnostic or therapeutic use.
The term "culturing" refers to the in vitro or ex vivo propagation of cells or organisms on or in various media. It is understood that the progeny of a cell grown in culture may not be identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.
In some embodiments, the cells disclosed herein are eukaryotic cells or prokaryotic cells. 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), 293T cell, or a549 cell. In some embodiments, the cell is a host cell.
"detectable label", "detectable marker" or "marker" are used interchangeably and include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes and proteins (including enzymes). The detectable label may also be linked to a polynucleotide, polypeptide, protein, or composition described herein.
As used herein, "dioleoyloxy-3- (trimethylammonio) propane" (DOTAP) refers to cationic lipid surfactants that are typically involved in the lipofection of DNA and RNA molecules.
An "effective amount" refers to an amount of an agent or a combined amount of two or more agents that is sufficient to effect such treatment of a disease when administered to treat a mammal or other subject. The "effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject to be treated.
The term "encode" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if the polynucleotide, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce an mRNA of the polypeptide or fragment thereof, and optionally translated to produce a polypeptide or fragment thereof. The antisense strand is the complement of such a nucleic acid and the coding sequence can be deduced therefrom. Furthermore, as used herein, an amino acid sequence coding sequence refers to a nucleotide sequence that encodes the amino acid sequence.
In some embodiments, the term "engineered" or "recombinant" refers to a cell that has at least one modification that is not normally present in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain, or parent host strain of the referenced species. In some embodiments, the term "engineered" or "recombinant" refers to synthesis by human intervention. As used herein, the term "recombinant protein" refers to a polypeptide produced by recombinant DNA techniques, wherein DNA encoding the polypeptide is typically inserted into a suitable expression vector, which in turn is used to transform a host cell to produce a heterologous protein.
As used herein, the term "enhancer" refers to a sequence element that enhances, increases, or improves transcription of a nucleic acid sequence, regardless of its position and orientation relative to the nucleic acid sequence to be expressed. Enhancers can increase transcription from a single promoter or from more than one promoter simultaneously. Any truncated, mutated or otherwise modified variant of a wild-type enhancer sequence is also within the above definition, provided that such transcription enhancing functionality is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of the wild-type activity, i.e., the activity of the full-length sequence).
"eukaryotic cells" include all life forms except the prokaryote. They can be readily distinguished by membrane bound nuclei. Animals, plants, fungi and protozoa are eukaryotes or organisms whose cells are organized into complex structures by the endomembrane and cytoskeletal tissues. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term "host" includes eukaryotic hosts including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include apes, dogs, cattle, pigs, mice, rats, birds, reptiles, and humans.
As used herein, the term "excipient" refers to a natural or synthetic substance formulated with a pharmaceutically active ingredient for the purpose of long-term stability, increasing the volume of a solid formulation, or added for the purpose of providing therapeutic enhancement (such as promoting drug absorption, reducing viscosity, or enhancing solubility) of the active ingredient in the final dosage form.
The term "expression" refers to the production of a gene product, such as an mRNA, peptide, polypeptide, or protein. As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.
As used herein, the term "F2A self-cleaving peptide" or "T2A self-cleaving peptide" refers to a class of 18-22 aa long peptides that can induce ribosome jump during protein translation in a cell. The apparent cleavage is caused by ribosomal skipping of the peptide bond between proline (P) and glycine (G) at the C-terminus of the 2A peptide.
"Gene" refers to a polynucleotide comprising at least one Open Reading Frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
"Gene product" or, alternatively, "gene expression product" refers to an amino acid (e.g., peptide or polypeptide) that is produced when a gene is transcribed and translated. In some embodiments, a gene product may refer to mRNA or other RNA, such as interfering RNA, that is produced when a gene is transcribed.
As used herein, "H3K (+H2) 4b" refers to a particular type of HKP in which the primary repeat sequence in its terminal branches is-HHK-, there are four-HHK-motifs in each branch, and an additional histidine is inserted in the second-HHK-motif of the terminal branch of H3K4 b.
As used herein, "histidine-lysine copolymer" (HKP) refers to a polymeric nanoparticle that utilizes a polymer of histidine and lysine to deliver a biologically relevant molecule to a target cell. The HK polymer contains four short peptide branches attached to a trilysine amino acid core. 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, wherein R represents a peptide branch and K is the amino acid L-lysine.
In formula I, wherein K is L-lysine and R 1 、R 2 、R 3 And R is 4 Independently a histidine-lysine peptide. In the HK polymers of the present invention, R 1-4 The branches may be identical or different. When the R branches are "different," the amino acid sequence of the branch is different from each of the other R branches in the polymer.
"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or homologous positions that the sequences have. An "unrelated" or "non-homologous" sequence has less than 40% identity, or alternatively less than 25% identity, to one of the sequences of the present disclosure. In some embodiments, the identity between two polypeptides or polynucleotides is calculated based on their full length, or based on the shorter sequence of both, or based on the longer sequence of both.
A polynucleotide or polynucleotide region (or polypeptide region) has a certain percentage (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that when aligned, the percentage of bases (or amino acids) is the same when the two sequences are compared. Such alignments and percent homology or sequence identity may be determined using software programs known in the art, such as those described in Ausubel et al, 2007, "Current Protocols in Molecular biology. Preferably, the alignment is performed using default parameters. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect=10; matrix = BLOSUM62; descriptive = 50 sequences; sortby=high SCORE; databases = non-redundant, genbank+embl+ddbj+pdb+ GenBank CDS translations +swissprotein+pir. For details of these programs, see the following internet addresses: blast.ncbi.nlm.nih.gov/blast.cgi, last visit date was 2021, 8 months 1.
In some embodiments, the polynucleotides disclosed herein are RNA or an analog thereof. In some embodiments, the polynucleotides disclosed herein are DNA or an analog thereof. In some embodiments, the polynucleotides disclosed herein are hybrids of DNA and RNA or analogs thereof.
In some embodiments, the equivalent of a reference nucleic acid, polynucleotide, or oligonucleotide encodes the same sequence encoded by the reference. In some embodiments, the equivalent of a reference nucleic acid, polynucleotide, or oligonucleotide hybridizes to a reference, a complementary reference, a reverse reference, or a reverse complementary reference, optionally under high stringency conditions.
Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide is a nucleic acid, polynucleotide or oligonucleotide 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 identity, or alternatively at least 99% sequence identity to a reference nucleic acid, polynucleotide or oligonucleotide, or alternatively, the equivalent nucleic acid hybridizes to the reference polynucleotide or its complement under high stringency conditions. In one aspect, the equivalent must encode the same protein or a functional equivalent of a protein, which optionally can be identified by one or more assays described herein. Additionally or alternatively, an equivalent of a polynucleotide will encode a protein or polypeptide having the same or similar function as the reference or parent polynucleotide.
"host cell" refers not only to a particular subject cell, but also 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 may be a prokaryotic or 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), 293T cell, or a549 cell. Cultured cell lines are commercially available from, for example, the American type culture Collection.
"hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between bases of nucleotide residues. Hydrogen bonding may occur through watson-crick base pairing, hophattan binding (Hoogstein binding), or any other sequence-specific manner. A complex may comprise two strands forming a duplex structure, three or more strands forming a multi-strand complex, a single self-hybridizing strand, or any combination of these. Hybridization reactions may constitute a step in a broader process, such as the initiation of a PCR reaction or the cleavage of a polynucleotide by a ribozyme.
Hybridization reactions can be performed under conditions of varying "stringency". Typically, the low stringency hybridization reaction is performed in 10 XSSC or a solution of equal ionic strength/temperature at about 40 ℃. Medium stringency hybridization reactions are typically performed in 6 XSSC at about 50℃and high stringency hybridization reactions are typically performed in 1 XSSC at about 60 ℃. Hybridization reactions can also be carried out under "physiological conditions" well known to those skilled in the art. Non-limiting examples of physiological conditions are temperature, ionic strength, pH and Mg, which are normally present in cells 2+ Concentration.
Examples of stringent hybridization conditions include: an incubation temperature of about 25 ℃ to about 37 ℃; hybridization buffer concentrations of about 6 XSSC to about 10 XSSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4 XSSC to about 8 XSSC. Examples of medium hybridization conditions include: an incubation temperature of about 40 ℃ to about 50 ℃; buffer concentrations of about 9 XSSC to about 2 XSSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5 XSSC to about 2 XSSC. Examples of high stringency conditions include: an incubation temperature of about 55 ℃ to about 68 ℃; buffer concentrations of about 1 XSSC to about 0.1 XSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1 XSSC, 0.1 XSSC, or deionized water. Typically, the hybridization incubation time is from 5 minutes to 24 hours, with 1, 2 or more wash steps, and the wash incubation time is about 1 minute, 2 minutes or 15 minutes. SSC was 0.15M NaCl and 15 mM citrate buffer. It should be appreciated that equivalents of SSC using other buffer systems may be employed.
When hybridization occurs between two single stranded polynucleotides in an antiparallel configuration, the reaction is referred to as "annealing" and these polynucleotides are described as "complementary". A double-stranded polynucleotide may be "complementary" or "homologous" to another polynucleotide if hybridization can occur between one strand of the first polynucleotide and one strand of the second polynucleotide. "complementarity" or "homology" (the degree to which one polynucleotide is complementary to another polynucleotide) can be quantified in terms of the proportion of bases in opposite strands that are expected to form hydrogen bonds with each other according to commonly accepted base pairing rules.
"host cell" refers not only to a particular subject cell, but also 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 may be a prokaryotic or 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), 293T cell, or a549 cell. Cultured cell lines are commercially available from, for example, the American type culture Collection.
As used herein, "immune cells" include, for example, 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)) that may be derived from Hematopoietic Stem Cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural Killer (NK) cells, and NKT cells), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). In some embodiments, the immune cells are derived from one or more of the following: progenitor cells, embryonic stem cells, cells derived from embryonic stem cells, embryonic germ stem cells, cells derived from embryonic germ stem cells, cells derived from stem cells, pluripotent stem cells, induced pluripotent stem cells (iPSc), hematopoietic Stem Cells (HSC), or immortalized cells. In some embodiments, the HSCs are derived from umbilical cord blood of the subject, peripheral blood of the subject, or bone marrow of the subject. In some embodiments, the subject from which the immune cells are obtained directly or indirectly is the same subject to be treated. In some embodiments, the subject from which the immune cells are obtained directly or indirectly is different from the subject to be treated. In further embodiments, the subject from which the immune cells are obtained directly or indirectly is different from the subject to be treated, and these subjects are from the same species, such as a human.
An "immune response" broadly refers to an antigen-specific response of lymphocytes to a foreign substance. The terms "immunogen" and "immunogenic" refer to molecules that have the ability to elicit an immune response. All immunogens are antigens, but not all antigens are immunogenic. The immune response disclosed herein may be humoral (by antibody activity) or cell-mediated (by T cell activation). The response may occur in vivo or in vitro. Those skilled in the art will appreciate 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 also appreciate that nucleic acids encoding molecules capable of eliciting an immune response necessarily encode immunogens. The skilled artisan will also appreciate that immunogens are not limited to full-length molecules, but may also include partial molecules.
As used herein, the term "immunoconjugate" comprises an antibody or antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radiopharmaceutical, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
As used herein, an "immunogenic composition" refers to a composition that induces clinically significant effector T cell activity against a protein target associated with cancer cell activity. In general, an "immune response" refers broadly to an antigen-specific response of lymphocytes to a foreign substance. The terms "immunogen" and "immunogenic" refer to molecules that have the ability to elicit an immune response. All immunogens are antigens, but not all antigens are immunogenic. The immune response disclosed herein may be humoral (by antibody activity) or cell-mediated (by T cell activation). The response may occur in vivo or in vitro. Those skilled in the art will appreciate 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 also appreciate that nucleic acids encoding molecules capable of eliciting an immune response necessarily encode immunogens. The skilled artisan will also appreciate that immunogens are not limited to full-length molecules, but may also include partial molecules. The immune response elicited may be determined by one skilled in the art, e.g., antibodies may be generated in the immune response as illustrated in the examples; such antibodies may bind to immunogens; thus, enzyme-linked immunosorbent assays (ELISA) can be used to detect and/or quantify immunogen specific antibodies.
As used herein, the term "immunoregulatory molecule" may refer to any molecule that may modulate or directly affect an immune response, 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 α, β and γ, factors that stimulate cell growth (e.g., GM-CSF), and other factors (e.g., tumor necrosis factor, DC-SIGN, mlPlα, mlPlβ, TGF- β, or TNF); factors that provide costimulatory signals for T cell activation, such as B7 molecules and CD40; auxiliary molecules such as CD83; proteins involved in antigen processing and presentation, such as TAP1/TAP2 transporter, proteosome molecules (such as LMP2 and LMP 7), heat shock proteins (such as gp96, HSP70 and HSP 90), and MHC or HLA molecules; and their biological equivalents.
As used herein, an "in vitro transcription system" (IVT) refers to a protein production system that occurs outside of living cells, which generally involves a composition comprising molecules necessary for transcription and/or translation of a target protein. IVT involves template directed synthesis of RNA molecules of almost any sequence. RNA molecules that can be synthesized using the IVT method range in size from short oligonucleotides to long nucleic acid polymers of several kilobases. IVT Methods allow for The synthesis of large amounts (e.g., from micrograms to milligrams) of RNA transcripts (Beckert et al Methods Mol biol., volume 703: pages 29-41 (2011); rio et al, "RNA: A Laboratory Manual.", cold Spring Harbor: cold Spring Harbor Laboratory Press,2011, pages 205-220; and Cooper, geoffey M., "The Cell: A Molecular applications.", 4 th edition, washington D.C.: ASM Press,2007, pages 262-299). Typically, IVT utilizes a DNA template with a promoter sequence upstream of the sequence of interest. The promoter sequence is most commonly of phage origin (e.g., a T7, T3 or SP6 promoter sequence), but many other promoter sequences are also acceptable, including those designed de novo. Transcription of the DNA template is generally best accomplished by using RNA polymerase corresponding to the specific phage promoter sequence. Exemplary RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, or SP6RNA polymerase, among others. IVT usually starts with dsDNA, but can be performed on a single strand.
The term "isolated" as used herein with respect to nucleic acids such as DNA or RNA refers to molecules that are isolated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated nucleic acid" is intended to include nucleic acid fragments that do not occur naturally as fragments and do not occur in the natural state. The term "isolated" is also used herein to refer to polypeptides, proteins, and/or host cells isolated from other cellular proteins, and is intended to include both purified and recombinant polypeptides. In other embodiments, the term "isolated" means separated from components, cells, and other substances with which the cell, tissue, polynucleotide, peptide, polypeptide, or protein is normally associated in nature. For example, an isolated cell is a cell isolated from a tissue or cell having a dissimilar phenotype or genotype. As will be apparent to those of skill in the art, non-naturally occurring polynucleotides, peptides, polypeptides, or proteins do not need to be "isolated" to distinguish them from their naturally occurring counterparts.
As used herein, the term "tag" or detectable label means a compound or composition that is directly or indirectly detectable, e.g., an N-terminal histidine tag (N-His), a magnetically active isotope (e.g. 115 Sn、 117 Sn and Sn 119 Sn), non-radioactive isotopes (such as 13 C and C 15 N), polynucleotide or protein (such as an antibody) that is conjugated directly or indirectly to the composition to be detected to produce a "labeled" composition. The term also includes sequences conjugated to polynucleotides that provide a signal upon expression of the inserted sequence, such as Green Fluorescent Protein (GFP) and the like. The label itself may be detectable (e.g. radioisotope labels or fluorescent labels), or in the case of enzymatic labels, may be catalytically detectableChemical changes of the substrate compound or composition being measured. The label may be suitable for small scale detection or more suitable for high throughput screening. Thus, suitable labels include, but are not limited to, magnetically active isotopes, nonradioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes). The label may be simply detected or it may be quantified. A simple detected response typically includes a response whose presence is only confirmed, while a quantitative response typically includes a response having a quantifiable (e.g., numerically reportable) value such as intensity, polarization, or other property. In luminescence or fluorescence assays, a detectable response may be generated directly using a fluorophore or fluorophore that binds to a detection component that is actually involved in binding, or indirectly using a fluorophore or fluorophore that binds to another (e.g., reporter or indicator) component. Examples of luminescent labels that generate a signal include, but are not limited to, bioluminescence and chemiluminescence. The detectable luminescent response typically includes a change or occurrence of a luminescent signal. Suitable methods and luminophores for luminescent labelling assay components are known in the art and are described, for example, in Haugland, richard p. (1996) "Handbook of Fluorescent Probes and Research Chemicals" (6 th edition). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.
Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescein Yellow (Lucifer Yellow), cascade Blue ™, and Texas Red (Texas Red). Other suitable optical dyes are described in Haugland, richard p. (1996) "Handbookof Fluorescent Probes and Research Chemicals" (6 th edition).
In some embodiments, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component, such as a cell surface marker, present in or on the surface of a cell or tissue. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to the second molecule. The choice of the fluorescent-labeled functional group will depend on the site of attachment to the linker, agent, marker or second labeling agent.
As used herein, a purification tag or marker refers to a tag that can be used to purify a molecule or component to which the tag is conjugated, such as an epitope tag (including but not limited to Myc tag, human influenza Hemagglutinin (HA) tag, FLAG tag), an affinity tag (including but not limited to glutathione-S transferase (GST), polyhistidine (His) tag, calmodulin Binding Protein (CBP), or Maltose Binding Protein (MBP)), or a fluorescent tag.
"selectable marker" refers to a protein or gene encoding a protein necessary for survival or growth of cells grown in a selective culture regimen. Typical selectable markers include sequences encoding proteins that confer resistance to a selective agent, such as an antibiotic, herbicide, or other toxin. Examples of selectable markers include genes that confer resistance to antibiotics such as spectinomycin, streptomycin, tetracycline, ampicillin, kanamycin, G418, neomycin, bleomycin, hygromycin, methotrexate, dicamba, glufosinate or glyphosate.
As used herein, "liposome nanoparticle" (LNP) refers to a nanoparticle that generally comprises, or alternatively consists essentially of, or still further consists of, a lipid (particularly an ionizable cationic lipid).
As used herein, "liposome" refers to one or more lipids that form a complex, which is typically surrounded by an aqueous solution. Liposomes are generally spherical structures comprising lipid fatty acids, lipid bilayer structures, unilamellar vesicles and amorphous lipid vesicles. Typically, liposomes are fully enclosed lipid bilayer membranes containing an entrapped volume of water. Liposomes can be unilamellar vesicles (with a single bilayer membrane), oligolayers, or multilamellar (onion-like structures featuring multiple membrane bilayers, each membrane bilayer separated from the next by a layer of water).
As used herein, "messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring or modified amino acid polymer) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. In some embodiments, the mRNA disclosed herein comprises, consists essentially of, or still further consists of at least one coding region, 5' untranslated region (UTR), 3' UTR, 5' cap, and poly-a tail.
As used herein, the term "micelle" refers to a polymer assembly consisting of a hydrophilic shell (or corona) and a hydrophobic and/or ionic interior. Furthermore, the term "micelle" may refer to any polyionic complex assembly consisting of a multiblock copolymer having a net positive charge and a suitable negatively charged polynucleotide.
As used herein, "neoantigen" refers to a new protein formed on cancer cells due to oncogenic DNA mutations.
As used herein, "normal cells corresponding to a cancerous tissue type" refers to normal cells from the same tissue type as the cancerous tissue. Non-limiting examples are normal white blood cells from a patient, such as a leukemia patient.
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 equivalents, derivatives, variants and analogues of RNA or DNA prepared from nucleotide analogues, as well as single-stranded (sense or antisense) and double-stranded polynucleotides suitable for use in the embodiments. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For clarity, when referring herein to a nucleotide of a nucleic acid, it may be DNA or RNA, the terms "adenosine", "cytidine", "guanosine" and "thymidine" are used. It will be appreciated that if the nucleic acid is RNA, the nucleotide having a uracil base is uridine.
As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, the term includes, but is not limited to, single, double or multiple stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers comprising purine and pyrimidine bases or other natural, chemical or biochemically modified, non-natural or derivatized nucleotide bases.
The term "oligonucleotide" or "polynucleotide" or "portion" or "fragment" thereof refers to a stretch of polynucleotide residues that are long enough to be used in PCR or various hybridization methods to identify or amplify the same or related portions of an mRNA or DNA molecule. The polynucleotide compositions of the invention include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, including 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 of skill in the art. Such modifications include, for example, labeling, methylation, substitution of one or more nucleotides in a naturally occurring nucleotide with an analog, internucleotide modifications (such as uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendant groups (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified linkages (e.g., alpha anomeric nucleic acids, etc.)). Also included are synthetic molecules that mimic the ability of polynucleotides to bind to a specified sequence through hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, molecules in which peptide bonds replace phosphoester bonds in the backbone of the molecule.
As used herein, an "open reading frame" (ORF) refers to a nucleotide sequence encoding a polypeptide or portion thereof. In some embodiments, the ORF is mRNA.
"operably linked" refers to polynucleotides arranged in a manner that allows them to function in a cell.
In some embodiments, the RNAs disclosed herein are "optimized". In some embodiments, optimization can be used to match codon frequencies in the target and host organisms to ensure proper folding; biasing GC content to increase mRNA stability or decrease secondary structure; minimizing tandem repeat codons or base strings (base run) that may impair gene construction or expression; custom transcription and translation control regions; insertion or removal of protein trafficking sequences (trafficking sequence); removal/addition of post-translational modification sites (e.g., glycosylation sites) in the encoded protein; adding, removing or shuffling protein domains; insertion or deletion of restriction sites; modifying the ribosome binding site and the mRNA degradation site; regulating the rate of translation so that the individual domains of the protein fold correctly; or to reduce or eliminate problematic secondary structures within polynucleotides.
As used herein, the term "PD-1" refers to a particular protein fragment associated with that name as well as any other molecule having similar biological functions that has at least 70%, or alternatively at least 80% amino acid sequence identity, or alternatively 90% sequence identity, or alternatively at least 95% sequence identity to a PD-1 sequence and/or a suitable binding partner for PD-L1 as shown herein. Non-limiting example sequences of PD-1 are provided herein, such as, but not limited to, the sequence with the following reference numbers-GCID: GC02M241849; HGNC 8760; entrez Gene 5133; ensembl: ENSG00000188389; OMIM 600244; and UniProtKB: Q15116— and sequence:
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL and equivalents thereof.
Non-limiting examples of commercially available antibodies include pamphlezumab (merck), nal Wu Liyou mab (nivolumab, beziram), pidilizumab (Cure Tech), AMP-224 (GSK), AMP-514 (GSK), PDR001 (nova) and cimeproof Li Shan anti (cemiplimab, regenerator and celecoxib).
As used herein, the term "PD-L1" refers to a particular protein fragment associated with that name as well as any other molecule having similar biological functions that has at least 70%, or alternatively at least 80% amino acid sequence identity, or alternatively 90% sequence identity, or alternatively at least 95% sequence identity, to the PD-L1 sequence and/or a suitable binding partner for PD-1 as shown herein. Non-limiting example sequences of PD-L1 are provided herein, such as, but not limited to, the sequence with the following reference numbers-GCID: GC09P005450; HGNC, 17635; entrez Gene 29126; ensembl: ENSG00000120217; OMIM 605402; and UniProtKB: Q9NZQ 7-and sequence:
MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET and equivalents thereof. Non-limiting examples of commercially available antibodies include atilizumab (roteizumab, roger gene tek), avistuzumab (avelumab, mergraranol and pyroxene), devaluzumab (durvalumab, aliskiren), BMS-936559 (Bai Messa nobody), and CK-301 (Chekpoint Therapeutics).
"pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier useful in the compositions disclosed herein. In some embodiments, the pharmaceutically acceptable carrier comprises, consists essentially of, or still further consists of a nanoparticle, such as a polymeric nanoparticle carrier (e.g., HKP nanoparticle) or a Lipid Nanoparticle (LNP). Additionally or alternatively, the pharmaceutically acceptable carrier includes an ion exchanger; alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin; buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate; a partial glyceride mixture 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; polyvinylpyrrolidone; a cellulose-based material; polyethylene glycol; sodium carboxymethyl cellulose; a polyacrylate; a wax; polyethylene-polyoxypropylene-block polymers; polyethylene glycol; and lanolin. Suitable drug carriers are described in "Remington's Pharmaceutical Sciences", mack Publishing Company, standard references in this field. They may be selected according to the intended form of administration (i.e., oral tablets, capsules, elixirs, syrups, and the like) and in accordance with conventional pharmaceutical practices.
A "plasmid" is an extrachromosomal DNA molecule separated from chromosomal DNA that is capable of replication independent of chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and generally provide selective advantages under given environmental conditions. The plasmid may carry a gene that provides resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the produced protein may function as a toxin in a similar environment. Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of a plasmid containing the gene that confers resistance to the particular antibiotic on the cell and a multiple cloning site (MCS or polylinker) that is a short region containing several commonly used restriction sites, so that the DNA fragment is conveniently inserted at that location. Another major use of plasmids is in the production of large quantities of proteins. In this case, researchers culture bacteria containing plasmids carrying the genes of interest. Just as bacteria produce proteins to confer antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted genes. This is an inexpensive and simple method for mass production of genes or proteins encoded thereby.
As used herein, "plasmid DNA vector delivery system" (pDNA) refers to an extrachromosomal DNA molecule separated from chromosomal DNA that is capable of replication independent of chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and generally provide selective advantages under given environmental conditions. The plasmid may carry a gene that provides resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the produced protein may function as a toxin in a similar environment. Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of a plasmid containing the gene that confers resistance to the particular antibiotic on the cell and a multiple cloning site (MCS or polylinker) that is a short region containing several commonly used restriction sites, so that the DNA fragment is conveniently inserted at that location. Another major use of plasmids is in the production of large quantities of proteins. In this case, researchers culture bacteria containing plasmids carrying the genes of interest. Just as bacteria produce proteins to confer antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted genes. This is an inexpensive and simple method for mass production of genes or proteins encoded thereby.
In some embodiments, the mRNA further comprises a polyA tail. A "polyA tail" is a region of mRNA downstream, e.g., immediately downstream (i.e., 3 '), of the 3' UTR that contains multiple consecutive adenosine monophosphates. The polyA tail may contain from 10 to 300 adenosine monophosphates. Additionally or alternatively, in a relevant biological environment (e.g., in a cell, in vivo), the polyA tail has the function of protecting mRNA from enzymatic degradation (e.g., within the cytoplasm) and assisting in transcription termination, mRNA transport out of the nucleus, and translation. In some embodiments, the polyA tail as used herein comprises, consists essentially of, or still further consists of one or more of the following:
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 12); or (b)
CGGCAAUAAAAAGACAGAAUAAAACGCACGGUGUUGGGUCGUUUGUUC(SEQ ID NO: 13)。
As used herein, poly (lactic-co-glycolic acid) or polylactic acid (PLGA or PGA) refers to a particular type of polymer nanoparticle consisting of glycolic acid and lactic acid in various proportions. These molecules are known to be biocompatible and biodegradable.
As used herein, "polymeric nanoparticle" refers to a nanoparticle composed of a polymeric compound (e.g., a compound composed of repeatedly linked units or monomers), including any organic polymer, such as histidine-lysine (HKP) polypeptide (HKP), which is a polymer-based particle that can transport biologically relevant molecules within the particle or in a surface-adsorbed manner.
"prokaryotic cells" generally lack a nucleus or any other membrane-bound organelle and are divided into two areas, bacterial and archaeal. In addition, these cells are free of chromosomal DNA, the genetic information of which is in a circular loop called a plasmid. The bacterial cells are very small, approximately the size of the animal's mitochondria (about 1-2 μm in diameter and 10 μm in length). Prokaryotic cells have three main shapes: rod-like, spherical, and spiral. Unlike eukaryotes, which undergo a complex replication process, bacterial cells divide by two divisions. Examples include, but are not limited to, bacillus bacteria, escherichia coli bacteria, and salmonella bacteria. Cultured cell lines are commercially available from, for example, the American type culture Collection.
The terms "protein," "peptide," and "polypeptide" are used interchangeably and refer in their broadest sense to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. Subunits (also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunits may be linked by other linkages, such as ester linkages, ether linkages, and the like. The protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can comprise the sequence of the protein or peptide. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids (including glycine, D and L optical isomers), amino acid analogs, and peptidomimetics.
As used herein, an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest that "corresponds to" or "aligns" an identified position in a reference sequence refers to the alignment of that residue position with that identified position in the sequence alignment between the sequence of interest and the reference sequence. Various programs can be used to perform such sequence alignments, such as Clustal Omega and BLAST. In one aspect, the equivalent polynucleotides, proteins, and corresponding sequences can be determined using BLAST (accessible BLAST. Ncbi. Lm. Nih. Gov/BLAST. Cgi, last access date is 2021, month 8, 1).
As used herein, an amino acid mutation refers herein to two letters, such as L19F, separated by an integer. The first letter provides a single letter code for the original amino acid residue to be mutated; while the last letter provides a mutation, such as a single letter code indicating the missing delta, or the amino acid residue after mutation. In some embodiments, the integer is the number of amino acid residues to be mutated in the amino acid sequence that have not been mutated, optionally counted from N-terminus to C-terminus. In some embodiments, the integer is the number of mutated amino acid residues in the mutated amino acid sequence, optionally counted from N-terminus to C-terminus.
Unless otherwise indicated, it is to be inferred that no explicit recitation is required and that when the present disclosure relates to polypeptides, proteins, polynucleotides, equivalents or biological equivalents thereof are also within the scope of the present disclosure. As used herein, the term "biological equivalent thereof" when referring to a reference protein, polypeptide or nucleic acid is intended to be synonymous with "equivalent thereof" and refers to a protein, polypeptide or nucleic acid having minimal homology while still retaining the desired structure or function. Unless specifically recited herein, any polynucleotide, polypeptide, or protein mentioned herein is intended to include equivalents thereof. For example, the equivalent has 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 to a reference protein, polypeptide, or nucleic acid (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 a polynucleotide, its equivalent is a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide or its complement.
An equivalent of a reference polypeptide comprises, consists essentially of, or alternatively consists of a 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 (in one aspect, as determined using the Clustal Omega alignment program), or consists of a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions to the complement of a polynucleotide encoding the reference polypeptide, optionally wherein the high stringency conditions comprise: an incubation temperature of about 55 ℃ to about 68 ℃; buffer concentrations of about 1 XSSC to about 0.1 XSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1 XSSC, 0.1 XSSC, or deionized water.
In some embodiments, a first sequence (nucleic acid sequence or amino acid) is compared to a second sequence, and the percent identity between the two sequences can be calculated. In further embodiments, the first sequence may be referred to herein as an equivalent, and the second sequence may be referred to herein as a reference sequence. In yet a further embodiment, the percent identity is calculated based on the full length sequence of the first sequence. In other embodiments, the percent identity is calculated based on the full length sequence of the second sequence.
In some embodiments, the equivalent of the reference polypeptide comprises, consists essentially of, or still further consists of the reference polypeptide with conservative substitutions of one or more amino acid residues. Substitutions may be "conservative", i.e. substitutions within the same amino acid family. Naturally occurring amino acids can be divided into the following four families, and conservative substitutions will be made within these 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, and cysteine.
The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or analogs thereof) of any length. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), 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. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise indicated or required, any embodiment of the present disclosure, i.e., a polynucleotide, encompasses both the double stranded form as well as each of the two complementary single stranded forms known or predicted to constitute the double stranded form.
A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (a); cytosine (C); guanine (G); thymine (T); when the polynucleotide is RNA, uracil (U) represents thymine. Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule. Such alphabetical representations may be entered into a database in a computer having a central processing unit and used for bioinformatic applications such as functional genomics and homology searches.
As used herein, the term "purification marker" refers to at least one marker for purification or identification. A non-exhaustive list of such markers 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 fluorescent markers include FLAG, GFP, YFP, RFP, dTomato, cherry, cy, cy5, cy 5.5, cy 7, DNP, AMCA, biotin, digoxin, tamra, texas red, rhodamine, alexafluors, FITC, TRITC, or any other fluorescent dye or hapten.
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 complex or other active compound is a nucleic acid, peptide, protein, biological complex or other active compound that is isolated in whole or in part from a protein or other contaminant. Typically, a substantially purified peptide, protein, biocomplex or other active compound for use in the present disclosure comprises more than 80% of all macromolecular species present in the formulation prior to the peptide, protein, biocomplex or other active compound being mixed or formulated with a pharmaceutical carrier, excipient, buffer, absorption enhancer, stabilizer, preservative, adjuvant or other adjunct ingredient into an intact pharmaceutical formulation for therapeutic administration. More typically, peptides, proteins, biocomposites or other active compounds are purified to represent more than 90%, typically more than 95%, of all macromolecular species present in the purified preparation prior to mixing with other preparation ingredients. In other cases, the purified preparation may be substantially homogeneous, wherein other macromolecular species are undetectable by conventional techniques.
The term "regulatory sequence", "expression control element" or "promoter" as used herein means a polynucleotide that is operably linked to a target polynucleotide to be transcribed or replicated and that facilitates expression or replication of the target polynucleotide.
Promoters are examples of expression control elements or regulatory sequences. Promoters may be located 5' or upstream of a gene or other polynucleotide, which provide a control point for regulating transcription of the gene. In some embodiments, the promoter used herein corresponds to an RNA polymerase. In further embodiments, the promoters used herein comprise, consist essentially of, or still further consist of a T7 promoter, or an SP6 promoter, or a T3 promoter. Non-limiting examples of suitable promoters are provided in WO2001009377 A1.
The term "RNA" as used herein refers to its generally accepted meaning in the art. In general, the term "RNA" refers to a polynucleotide comprising at least one ribofuranosyl nucleoside moiety. The term can include double-stranded RNA, single-stranded RNA, isolated RNA (such as partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such changes may include the addition of non-nucleotide material (e.g., at one or more nucleotides of the RNA). Nucleotides in a nucleic acid molecule may also include non-standard nucleotides (such as non-naturally occurring nucleotides or chemically synthesized nucleotides) or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNAs. In some embodiments, the RNA is messenger RNA (mRNA).
"messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring or modified amino acid polymer) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. In some embodiments, the mRNA disclosed herein comprises, consists essentially of, or still further consists of at least one coding region, 5' untranslated region (UTR), 3' UTR, 5' cap, and poly-a tail.
Vaccination is the most successful medical method of preventing and controlling disease. Successful development and use of vaccines has saved thousands of people's lives and saved significant amounts of money. A key advantage of RNA vaccines is that RNA can be produced from DNA templates in the laboratory using readily available materials, which is cheaper and faster than conventional vaccine production that may require the use of eggs or other mammalian cells. In addition, mRNA vaccines have the potential to simplify vaccine discovery and development and promote rapid responses to emerging infectious diseases (see, e.g., marugi et al, mol Ther.,2019, volume 27, phase 4: pages 757-772).
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 can 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, e.g., pari et al, nat Rev Drug discovery, 2018, volume 17: pages 261-279. The production process to produce mRNA vaccines is cell-free, simple and rapid compared to the production of whole-microorganism, attenuated live and subunit vaccines. This rapid and simple production method makes mRNA a promising biological product, which potentially fills the gap between emerging infectious diseases and urgent need for effective vaccines.
As used herein, "separate use" refers to the simultaneous administration of two compounds of a composition according to the invention in different pharmaceutical forms.
As used herein, "sequentially" refers to the sequential administration of two compounds of a composition according to the invention, each compound being in a different pharmaceutical form. A "subject" for diagnosis or treatment is a cell or animal, such as a mammal or human. The subject is not limited to a particular species and includes non-human animals that are subject to diagnosis or treatment, and are non-human animals that are subject to infection or animal models, e.g., apes, mice (such as rats, mice, hairmice), dogs (such as dogs), rabbits (such as rabbits), livestock, sports animals (sport animals), and pets. Human patients are also included in this term.
As used herein, "simultaneous use" refers to administration of two compounds of a composition according to the invention in a single and identical pharmaceutical form or in two different pharmaceutical forms simultaneously.
As used herein, "solid tumor" refers to an abnormal mass of tissue that does not typically contain cysts or liquid areas. Solid tumors may 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.
As used herein, spermine-lipid cholesterol (SLiC) refers to a particular type of liposome nanoparticle. Exemplary molecules can be seen in fig. 13.
"therapeutic agent" encompasses biological agents such as antibodies, peptides, proteins, enzymes, and chemotherapeutic agents. Therapeutic agents also encompass immunoconjugates of Cell Binding Agents (CBA) and chemical compounds, such as antibody-drug conjugates (ADCs).
In one aspect, the additional therapeutic agent is a chemotherapeutic agent. As used herein, "chemotherapeutic agent" refers to a substance that, when administered to a subject, treats or prevents the development of cancer in the subject.
Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, inhibitors of chromatin function, anti-angiogenic agents, antiestrogens, antiandrogens, or immunomodulators.
As used herein, "cytoreductive therapy" (Cytoreductive therapy) includes, but is not limited to, chemotherapy, cryotherapy, and radiation therapy. Agents that act to reduce cell proliferation are known in the art and are widely used. Chemotherapy drugs that kill cancer cells only when they divide are called cell cycle specific. These include agents that act in the S phase, including topoisomerase inhibitors and antimetabolites.
Topoisomerase inhibitors are drugs that interfere with the action of topoisomerase enzymes (topoisomerase I and II). During chemotherapy, topoisomerase controls the manipulation of DNA structures necessary for replication and is therefore cell cycle specific. Examples of topoisomerase I inhibitors include the camptothecin analogs, irinotecan, and topotecan listed above. Examples of topoisomerase II inhibitors include amsacrine, etoposide phosphate and teniposide.
Antimetabolites are generally analogs of normal metabolic substrates that often interfere with processes involved in chromosomal replication. They attack cells at very specific stages in the cycle. Antimetabolites include folic acid antagonists such as methotrexate; pyrimidine antagonists such as 5-fluorouracil, fluorouridine (foxuridine), cytarabine, capecitabine and gemcitabine; purine antagonists, such as 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitors such as cladribine, fludarabine, nelarabine and pravastatin; etc.
Plant alkaloids are derived from certain types of plants. The vinca alkaloid is prepared from herba Catharanthi rosei plantCatharanthus rosea) Is prepared by the method. The Taxus medicine is prepared from bark of Taxus Pacifica (Taxus). Vinca alkaloids and taxanes are also known as antimicrotubule agents. The podophyllotoxin is derived from Podophyllum plant. The camptothecin analogues are derived from Asian "camptotheca acuminata" Camptotheca acuminata). Podophyllotoxins and camptothecin analogs are also classified as topoisomerase inhibitors. Plant alkaloids are typically cell cycle specific.
Examples of such agents include vinca alkaloids, such as vincristine, vinblastine, and vinorelbine; taxanes, such as paclitaxel and docetaxel; podophyllotoxins, such as etoposide and teniposide; and camptothecin analogs, such as irinotecan and topotecan.
Cryotherapy includes, but is not limited to, therapies involving reduced temperature, such as cryotherapy.
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, dosages of ionizing radiation in the range of at least about 2 Gy to no more than about 10 Gy, and/or in the range of at least about 5J/m 2 Up to about 50J/m 2 Within a range of typically about 10J/m 2 Is used to control the radiation dose of ultraviolet radiation.
As used herein, a biological sample or sample may be obtained from a subject, cell line, or cultured cells or tissue. Exemplary samples include, but are not limited to, cell samples, tissue samples, tumor biopsy tissue, liquid samples such as blood and other liquid samples of biological origin, including, but not limited to, ocular fluid (aqueous humor and vitreous humor), peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculated semen, female tidal fluid, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menstrual blood, pus, sebum, vomiting fluid, vaginal secretions/rinse, synovial fluid, mucosal secretions, fecal water, pancreatic juice, sinus cavity lavage, bronchopulmonary aspirates, blastocyst cavity fluid, or umbilical cord blood. In some cases, the sample is a tumor/cancer biopsy.
The term "transduction" refers to the process of introducing an exogenous nucleotide sequence into a cell. In some embodiments, such transduction is performed by a viral vector or a non-viral vector.
As used herein, "treating" a disease in a subject refers to (1) preventing a symptom or disease from occurring in a subject that is predisposed to or has not yet exhibited symptoms of the disease; (2) inhibiting or arresting the development of the disease; or (3) improve or cause regression of the disease or disease symptoms. As understood in the art, "treatment" is a method for achieving a beneficial or desired result, including clinical results. For the purposes of this technology, a beneficial or desired result can include one or more of, but is not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disorder (including disease), stabilized (i.e., not worsening) state of the disorder (including disease), delay or slowing of disorder (including disease), progression, amelioration or palliation of the disorder (including disease), state and remission (whether partial or total), whether detectable or undetectable. Treatment containing the disclosed compositions and methods can be first line, second line, third line, fourth line, fifth line therapy, and is intended for use as monotherapy or in combination with other suitable therapies. In one aspect, "treating" does not include prophylaxis. When the disease is cancer, the following clinical endpoints are non-limiting examples of treatments: reduced tumor burden, reduced tumor growth, longer overall survival, longer tumor progression time, inhibition of metastasis, or reduction of tumor metastasis.
"under transcriptional control" (also used herein as "directing expression of … …") or any grammatical variant thereof is a term well known in the art and means that transcription and optionally translation of a polynucleotide sequence (typically a DNA sequence) depends on its operative linkage to an element that contributes to transcription initiation or promotes transcription.
"RNA polymerase" refers to an enzyme that produces a polyribonucleotide sequence that is complementary to a pre-existing template polynucleotide (DNA or RNA). In some embodiments, the RNA polymerase is a phage RNA polymerase, optionally a T7 RNA polymerase or SP6 RNA polymerase or a T3 RNA polymerase. Non-limiting examples of suitable polymerases are further detailed in US10526629B 2.
In some embodiments, the term "vector" means a recombinant vector that retains the ability to infect and transduce non-dividing cells and/or slowly dividing cells and optionally integrate into the genome of the target cell. Non-limiting examples of vectors include plasmids, nanoparticles, liposomes, viruses, cosmids, phages, BACs, YACs, and the like. In some embodiments, plasmid vectors may be prepared from commercially available vectors. In other embodiments, the viral vector may be produced from baculovirus, retrovirus, adenovirus, AAV, and the like 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 carrier is a nanoparticle, optionally a polymer nanoparticle or a lipid nanoparticle.
Vectors containing both promoters and cloning sites into which polynucleotides may be operably 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 (lajose, california) and Promega Biotech (madison, wisconsin). To optimize expression and/or in vitro transcription, it may be desirable to remove, add, or alter the 5 'and/or 3' untranslated portions of the clone to eliminate additional, potentially inappropriate translation initiation codons or other sequences that may interfere with or reduce expression at the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted directly 5' of the start codon to enhance expression.
A "viral vector" is defined as a recombinantly produced virus or viral particle comprising a polynucleotide that is to be delivered into a host cell in vivo, ex vivo, or in vitro. As known to those skilled in the art, there are 6 classes of viruses. DNA viruses constitute class I and class II. RNA viruses and retroviruses constitute the remaining classes. Class III viruses have double stranded RNA genomes. Class IV viruses have a positive single stranded RNA genome, which itself serves as mRNA. Class V viruses have a negative single stranded RNA genome that serves as a template for mRNA synthesis. Class VI viruses have a positive single stranded RNA genome, but have DNA intermediates not only in replication but also in mRNA synthesis. Retrovirus carries its genetic information in the form of RNA; however, once a virus infects a cell, the RNA is reverse transcribed into a DNA form that is integrated into the genomic DNA of the infected cell. The integrated DNA form is called provirus. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, and the like. Alphavirus vectors, such as those based on Semliki forest virus (Semliki Forest virus) and those based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) curr. Opin. Biotechnol, volume 5: pages 434-439 and Ying et al (1999) nat. Med., volume 5 (7): pages 823-827. As used herein, multiplicity of infection (MOI) refers to the number of viral particles added per cell during infection.
Compositions for use according to the present disclosure may be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dose" refers to physically discrete units suitable for a subject, each unit containing a predetermined amount of a composition calculated to produce a desired response associated with its administration (i.e., the appropriate route and regimen). Depending on the number of treatments and unit dose, the amount administered will depend on the desired outcome and/or protection. The precise amount of the composition will also depend on the judgment of the practitioner and will be unique to each individual. Factors that affect the dosage include the physical and clinical status of the subject, the route of administration, the intended therapeutic goal (symptomatic relief and cure), and the efficacy, stability and toxicity of the particular composition. After formulation, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically or prophylactically effective amount. The formulations are readily administered in a variety of dosage forms, such as the types of injectable solutions described herein.
As used herein, in combination means that each active ingredient of the composition is formulated separately for use in combination and may or may not be packaged separately in a specific dosage. The combined active ingredients may be administered simultaneously or sequentially.
Four branched histidine-lysine (HK) peptide polymer H2K4b has been shown to be a good vector for large molecular weight DNA plasmids (Leng et al, nucleic Acids Res, 2005; vol.33: page e 40), but a poor vector for relatively low molecular weight siRNA (Leng et al, J Gene Med, 2005; vol.7: pages 977-986). Two histidine-rich peptide analogues of H2K4b, H3K4b and H3K (+H2) 4b, proved to be effective vectors for siRNA (Leng et al, J Gene Med, 2005; volume 7: pages 977-986; chou et al, biomaterials, 2014; volume 35: pages 846-855), while the effectiveness of H3K (+H2) 4b appeared to be slightly higher (Leng et al, molTher, 2012; volume 20: pages 2282-2290). In addition, H3K4b vectors of siRNA induced cytokines to a significantly higher extent in vitro and in vivo than H3K (+H) 4b siRNA polyplex (polyplex) (Leng et al Mol Ther, 2012; vol. 20: pages 2282-2290). Suitable HK polypeptides are described in WO/2001/047496, WO/2003/090719 and WO/2006/060182, the contents of each of these patents being incorporated herein in their entirety. These polypeptides have a lysine backbone (three lysine residues) in which the epsilon-amino group of the lysine side chain and the N-terminus are coupled to various HK sequences. HK polypeptide vectors may be synthesized by methods well known in the art, including, for example, solid phase synthesis.
Such histidine-lysine peptide polymers ("HK polymers" or "HKP") were found to be unexpectedly effective as mRNA vectors, and they can be used alone or in combination with liposomes to provide efficient delivery of mRNA into target cells. Similar to PEI and other vectors, initial results indicate that HK polymers have different capacities to carry and release nucleic acids. However, because HK polymers can be reproducibly prepared on a peptide synthesizer, their amino acid sequences can be easily altered to achieve fine control of RNA binding and release, as well as stability of multimeric complexes containing HK polymers and RNA (Chou et al, biomaterials, 2014; volume 35: pages 846-855; midoux et al, bioconjug Chem, 1999; volume 10: pages 406-411; henig et al, journal of American Chemical Society, 1999; volume 121: pages 5123-5126). When the mRNA molecules are mixed with one or more HKP carriers, the components self-assemble into nanoparticles.
Advantageously, the HK polymer comprises four short peptide branches linked to a trilysine amino acid core, as described herein. 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, wherein R represents a peptide branch and K is the amino acid L-lysine.
In formula I, wherein K is L-lysine and R 1 、R 2 、R 3 And R is 4 Independently a histidine-lysine peptide. In the HK polymers of the present invention, R 1-4 The branches may be identical or different. When the R branches are "different," the amino acid sequence of the branch is different from each of the other R branches in the polymer. Suitable R-branches for use in the HK polymers of the present invention shown in formula I include, but are not limited to, the following R-branches R A -R J :
R A = KHKHHKHHKHHKHHKHHKHK- (SEQ ID NO: 14)
R B = KHHHKHHHKHHHKHHHK- (SEQ ID NO: 15)
R C = KHHHKHHHKHHHHKHHHK- (SEQ ID NO: 16)
R D = kHHHkHHHkHHHHkHHHk- (SEQ ID NO: 17)
R E = HKHHHKHHHKHHHHKHHHK- (SEQ ID NO: 18)
R F = HHKHHHKHHHKHHHHKHHHK- (SEQ ID NO: 19)
R G = KHHHHKHHHHKHHHHKHHHHK- (SEQ ID NO: 20)
R H = KHHHKHHHKHHHKHHHHK- (SEQ ID NO: 21)
R I = KHHHKHHHHKHHHKHHHK- (SEQ ID NO: 22)
R J = KHHHKHHHHKHHHKHHHHK- (SEQ ID NO: 23)
Specific HK polymers useful in mRNA compositions include, but are not limited to, those wherein R 1 、R 2 、R 3 And R is 4 Each of which is the same and is selected from R A -R J Is shown in Table 1. These HK polymers are referred to as H2K4b, 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, the capital letter "K" represents L-lysine and the lowercase letter "K" represents D-lysine. The additional histidine residues are underlined in the branched-chain sequence compared to H3K 4b. The HK polymer is named as follows:
1) For H3K4b, the main repeat in the branch is-HHK- (SEQ ID NO: 82), so "H3K" is part of the name; "4b" refers to the number of branches;
2) four-HHHK- (SEQ ID NO: 82) motifs in each of the branches of H3K4b and analogs thereof; the first HHK motif (SEQ ID NO: 82) ("1") is closest to the lysine core;
3) H3K (+H) 4b is an analog of H3K4b in which an additional histidine is inserted in the second HHK motif (SEQ ID NO: 82) of H3K4b (motif 2);
4) For the H3K (1+h) 4b and H3K (3+H) 4b peptides, additional histidines were present in the first (motif 1) and third (motif 3) motifs, respectively;
5) For H3K (1, 3+h) 4b, two additional histidines are present in both the first and third motifs of the branches.
TABLE 1
Polymer | Branched sequences | Sequence identifier |
H2K4b | R A = KHKHHKHHKHHKHHKHHKHK- | (SEQ ID NO: 14) |
H3K4b | R B = KHHHKHHHKHHHKHHHK- | (SEQ ID NO: 15) |
H3K(+H)4b | R C = KHHHKHHHKHHHHKHHHK- | (SEQ ID NO: 16) |
H3k(+H)4b | R D = kHHHkHHHkHHHHkHHHk- | (SEQ ID NO: 17) |
H-H3K(+H)4b | R E =HKHHHKHHHKHHHHKHHHK- | (SEQ ID NO: 18) |
HH-H3K(+H)4b | R F =HHKHHHKHHHKHHHHKHHHK- | (SEQ ID NO: 19) |
H4K4b | R G = KHHHHKHHHHKHHHHKHHHHK- | (SEQ ID NO: 20) |
H3K(1+H)4b | R H = KHHHKHHHKHHHKHHHHK- | (SEQ ID NO: 21) |
H3K(3+H)4b | R I = KHHHKHHHHKHHHKHHHK- | (SEQ ID NO: 22) |
H3K(1,3+H)4b | R J = KHHHKHHHHKHHHKHHHHK- | (SEQ ID NO: 23) |
Methods well known in the art, including gel blocking assays, heparin replacement assays, and flow cytometry, can be performed to evaluate the performance of different formulations containing HK polymer plus liposomes in successful mRNA delivery. Suitable methods are described, for example, in Gujrate et al, mol. Pharmaceuticals, volume 11: pages 2734-2744 (2014) and P ä rnasite et al Mol Ther Nucleic acids, volume 7: pages 1-10 (2017).
SmartFlare cube technology (Millipore Sigma) can also be used to detect cellular uptake of mRNA. These smart lights (smart flares) are beads with attached sequences that produce an increase in fluorescence that can be analyzed by fluorescence microscopy when recognizing RNA sequences in cells.
Other methods include measuring protein expression of mRNA, e.g., mRNA encoding luciferase can be used to measure transfection efficiency. See, e.g., he et al (J Gene Med.,2021, month 2; volume 23 (phase 2): page e 3295), which demonstrates the effectiveness of using HKP and liposome formulations to deliver mRNA.
In studies involving other RNA techniques, the combination of H3K (+H) 4b and DOTAP (cationic lipid) surprisingly has a synergistic effect in terms of the ability to carry mRNA into MDA-MB-231 cells (H3K (+H) 4 b/liposome vs. liposome, P < 0.0001). The combination was about 3-fold and 8-fold more effective as an mRNA vector than the polymer and cationic lipid vector alone, respectively. Not all HK peptides showed synergistic activity with DOTAP lipids. For example, the combination of H3K4b and DOTAP is less effective as a carrier for luciferase mRNA than DOTAP liposomes. In addition to DOTAP, other cationic lipids that can be used with HK peptide include Lipofectin (ThermoFisher), lipofectamine (ThermoFisher), and DOSPER.
The D-isomer of H3K (+H) 4b, in which the L-lysine in the branch is replaced by D-lysine, is the most efficient polymer carrier (H3K (+H) 4b versus H3K (+H) 4b, P < 0.05). The D-isomer of mRNA/liposome vector was approximately 4-fold and 10-fold more effective than H3k (+h) 4b and liposome vector alone, respectively. Although the D-H3K (+H) 4 b/lipid combination was slightly more effective than the L-H3K (+H) 4 b/lipid combination, the comparison was statistically not different.
Both H3K4b and H3K (+H) 4b can be used as vectors for nucleic acids in vitro, see e.g.Leng.et al, J Gene Med, 2005; roll 7: pages 977-986; and Chou et al, cancer gene ter, 2011; roll 18: pages 707-716. Despite these previous findings, H3K (+h) 4b was significantly better as an mRNA vector than other analogues (table 2).
TABLE 2
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In particular, under the condition of different weight ratios (HK: mRNA), the mRNA transfection efficiency is higher than that of H3K4b. At a 4:1 ratio, luciferase expression of H3K (+H) 4b was 10-fold higher in MDA-MB-231 cells than in H3K4b, with no apparent cytotoxicity. Furthermore, since the percentages of histidine (by weight) in H3K4b and H3K (+h4b) are 68.9% and 70.6%, respectively, buffering capacity does not appear to be a significant factor in their transfection differences.
Gel blocking assays showed that HK polymers delayed the electrophoretic mobility of mRNA. The higher the peptide to mRNA weight ratio, the stronger the blocking effect. However, at a ratio of 2:1, H3K (+H2) 4b blocks mRNA completely, whereas H3K4b does not block mRNA completely. This suggests that H3K (+h) 4b may form a more stable multimeric complex, which favors its ability to become a suitable carrier for mRNA delivery.
The H3K (+h) 4b peptide was further confirmed to bind more tightly to mRNA using heparin replacement assay. Various concentrations of heparin were added to the multimeric complex formed by mRNA and HK, and it was observed that H3K4b polymers released mRNA more readily than H3K (+h4b polymers, especially at lower heparin concentrations. These data indicate that H3K (+h4b) is able to bind mRNA and form a more stable multimeric complex than H3K4b.
Uptake of H3K4b and H3K (+H) 4b multimeric complexes by MDA-MB-231 cells was compared using flow cytometry using cyan-5 labeled mRNA. At different time points (1, 2 and 4 hours), the H3K (+h) 4b multimeric complex was introduced into cells more efficiently than the H3K4b multimeric complex. Similar to these results, fluorescence microscopy showed that there was significantly more H3K (+H) 4b multimeric complex located in the acidic endosomal vesicles than H3K4b multimeric complex (H3K 4b vs H3K (+H) 4b, P < 0.001). Interestingly, irregularly shaped H3K4b multimeric complexes that do not overlap with intracellular vesicles may be extracellular and no H3K (+h4b multimeric complexes were observed.
Both HK polymer and cationic lipid (i.e., DOTAP) are known to significantly and independently increase plasmid transfection. See, e.g., chen et al, gene ter, 2000; roll 7: pages 1698-1705. Thus, it was investigated whether these lipids together with HK polymer enhance mRNA transfection. Notably, the H3K (+h) 4b and H3K (+h) 4b vectors are significantly better mRNA vectors than DOTAP liposomes. The combination of H3K (+H) 4b and DOTAP lipids has a synergistic effect in carrying mRNA into MDA-MB-231 cells. The combination was about 3-fold and 8-fold more effective as mRNA vector than the polymer and liposome vector alone, respectively (H3K (+h) 4 b/lipid versus liposome or H3K (+h) 4 b). Notably, not all HK peptides showed an increase in activity with DOTAP lipids. The combination of H3K4b and DOTAP vector was less effective as a vector for luciferase mRNA than DOTAP liposome. The combination of DOTAP and H3K (+h) 4b vectors was found to be synergistic in their ability to carry mRNA into cells. See, e.g., he et al, J Gene med., 11/10/2020: e3295.
In some embodiments, the carrier, such as HKP nanoparticles, further comprises cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids. In some embodiments, the cationic lipid is an ionizable cationic lipid, and the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from 2, 2-diimine-4-dimethylaminoethyl- [1,3 ]]Dioxolane (DLin-KC 2-DMA), diiodo-methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA or MC 3) and di (-)Z) -non-2-en-1-yl) 9- ((4- (dimethylamino) butyryl) oxy) heptadecanedioate (L319).
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 a size small enough to allow their uptake by eukaryotic cells. Typically, the nanoparticles have a longest straight line dimension (e.g., diameter) of 200 nm or less. In some embodiments, the nanoparticle has a diameter of 100 nm or less. In some embodiments, smaller nanoparticles are used, for example, having a diameter of 50 a nm a or less, such as a diameter of 5 a nm a to 30 a nm a.
In some embodiments, the carrier is a polymeric nanoparticle. The term "polymeric nanoparticle" refers to a nanoparticle composed of a polymeric compound (e.g., a compound composed of repeatedly linked units or monomers) including any organic polymer such as histidine-lysine (HK) polypeptide (HKP).
As used herein, "liposome" refers to one or more lipids that form a complex, which is typically surrounded by an aqueous solution. Liposomes are generally spherical structures comprising lipid fatty acids, lipid bilayer structures, unilamellar vesicles and amorphous lipid vesicles. Typically, liposomes are fully enclosed lipid bilayer membranes containing an entrapped volume of water. Liposomes can be unilamellar vesicles (with a single bilayer membrane), oligolayers, or multilamellar (onion-like structures featuring multiple membrane bilayers, each membrane bilayer separated from the next by a layer of water).
In some embodiments, the carrier is a lipid nanoparticle (LNP, also referred to herein as a liposome nanoparticle). In some embodiments, the LNP has an average diameter of about 50 nm to about 200 nm. In some embodiments, the lipid nanoparticle carrier/formulation generally comprises a lipid, particularly an ionizable cationic lipid, such as SM-102, 2-diiodo-4-dimethylaminoethyl- [1,3 disclosed herein ]Dioxolane (DLin-KC 2-DMA), diiodo-methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA) or di (-)Z) -non-2-en-1-yl) 9- ((4- (dimethylamino) butyryl) oxy) heptadecanedioate (L319), or alternatively consists essentially of, or still further consists of. In some embodiments, the LNP carrier/formulation further comprises neutral lipids, sterols (such as cholesterol), and molecules capable of reducing particle aggregation, such as PEG or PEG-modified lipids (also referred to herein as PEGylated lipids). Additional exemplary lipid nanoparticle compositions and methods for their preparation are described, for example, in Semple et al @, et al2010) nat. biotechnol, volume 28: pages 172-176; jayarama et al (2012) Angew.chem.int.ed., volume 51: pages 8529-8533; and Maier et al (2013) Molecular Therapy, volume 21: pages 1570-1578, the contents of each of these documents are incorporated herein by reference in their entirety.
In one embodiment, the term "disease" or "disorder" as used herein refers to cancer, a state diagnosed with cancer, a state suspected of having cancer, or a state at risk of having cancer.
As used herein, "cancer" is a disease state characterized by the presence of cells in a subject that exhibit abnormal uncontrolled replication, and in some aspects, the term is used interchangeably with the term "tumor. The term "cancer or tumor antigen" or "neoantigen" refers to an antigen known to be associated with and expressed in cancer cells or tumor cells or tissues (such as on the cell surface), and the term "cancer or tumor-targeting antibody" refers to an antibody that targets such an antigen. In some embodiments, the neoantigen is not expressed in non-cancerous cells or tissues. In some embodiments, the neoantigen is expressed in a non-cancerous cell or tissue at a significantly lower level than the cancerous cell or tissue.
In some embodiments, the cancer is selected from: circulatory systems such as the heart (sarcomas [ hemangiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma ], mucinous tumors, rhabdomyomas, fibromas and lipomas), mediastinum and pleura, other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, e.g. nasal and middle ear, paranasal sinus, larynx, trachea, bronchi and lungs, 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, chomatoid hamartoma, mesothelioma; gastrointestinal systems such as esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), stomach, pancreas (ductal adenocarcinoma, insulinoma, glucagon tumor, gastrinoma, carcinoid tumor, vasoactive intestinal peptide tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, karposi's sarcoma, smooth myoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, smooth myoma); gastrointestinal stromal tumors and neuroendocrine tumors occur anywhere; genitourinary tract, such as kidney (adenocarcinoma, wilm's tumor) [ nephroblastoma ], lymphoma, leukemia), bladder and/or urinary tract (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, e.g., liver cancer (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, insulinoma, and glucagon tumor); bones such as osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, ewing's sarcoma, malignant lymphoma (reticulocytosoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondral tumor (osteochondral exochoma), benign chondrioma, chondroblastoma, cartilage mucinous fibroma, osteoid osteoma and giant cell tumor; neoplasms of the nervous system, such as the Central Nervous System (CNS), primary CNS lymphomas, skull cancers (bone tumors, hemangiomas, granulomas, xanthomas, malformed osteositis), meninges (meningiomas, glioma diseases), brain cancers (astrocytomas, medulloblastomas, gliomas, ependymomas, embryonal histiomas [ pineal tumor ], glioblastoma multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas); the reproductive system, such as gynaecology, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-neoplastic cervical dysplasia), ovary (ovarian carcinoma [ serous cyst adenocarcinoma, mucinous cyst adenocarcinoma, unclassified carcinoma ], granulosa-follicular cytoma, supporting stromal cytoma (seltoli-Leydig cell tumors), asexual cytoma, 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 genitalia; penile, prostate, testis, and other parts related to male genitals, blood systems such as blood ((myelogenous leukemia [ acute and chronic ], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), hodgkin's disease, non-Hodgkin's lymphoma; oral cavity such as lips, tongue, gums, bottom and other parts of the mouth, parotid gland and other parts of the salivary gland, tonsils, oropharynx, nasopharynx, pyriform fossa, hypopharynx and other parts of the lips, oral cavity and pharynx; skin such as malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, cutaneous fibroma and keloids; adrenal gland: neuroblastoma; and other tissues, these tissues include connective tissue and soft tissue, retroperitoneal and peritoneal, ocular, intraocular melanoma and appendages, breast, head or neck, anal region, thyroid, parathyroid, adrenal glands and other endocrine glands and related structures, secondary and unspecified malignant neoplasms of the lymph nodes, secondary malignant neoplasms of the respiratory and digestive systems, and secondary malignant neoplasms of other sites. In some embodiments, the cancer is colon, colorectal or rectal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is an adenocarcinoma, adenoma, leukemia, lymphoma, carcinoma, melanoma, angiosarcoma, or seminoma.
In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is not a solid tumor. In a further embodiment, the cancer is a leukemia cancer. In some embodiments, the cancer is from a carcinoma, sarcoma, myeloma, leukemia, or lymphoma. In some embodiments, the cancer is colon, colorectal or rectal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer.
In some embodiments, the cancer is a primary cancer or a metastatic cancer. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the cancer achieves remission, but can recur. In some embodiments, the cancer is unresectable.
In some embodiments, the cancer expresses NY-ESO-1 disclosed herein, such as lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, colorectal carcinoma, rectal carcinoma, follicular thyroid carcinoma, autoimmune lymphoproliferative syndrome, noonan syndrome (Noonan syndrome), juvenile myelomonocytic leukemia, bladder carcinoma, follicular thyroid carcinoma, and oral squamous cell carcinoma. Expression can be detected by sequencing biopsies of cancer, southern blotting (Southern Blotting), northern blotting (northern blotting), or by contact with antibodies that specifically bind to NY-ESO-1.
As used herein, the term "animal" refers to a living multicellular vertebrate organism, i.e., a class that includes, for example, mammals and birds. The term "mammal" includes human and non-human mammals such as non-human primates (e.g., apes, gibbons, chimpanzees, gorillas, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, bats, rats, rabbits, guinea pigs).
The terms "subject," "host," "individual," and "patient" are used interchangeably herein to refer to an animal, typically a mammal. Any suitable mammal can be treated by the methods described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, bats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal may be any age or at any stage of development (e.g., adult, adolescent, pediatric, infant or intrauterine mammal). The mammal may be male or female. In some embodiments, the subject is a human. In some embodiments, the subject has a disease or is diagnosed as 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 complete (such as cancer-free) cancer remission. In further embodiments, the subject is at risk for cancer recurrence or recurrence. In some embodiments, the subject is in partial cancer remission. In some embodiments, the subject is at risk of cancer metastasis.
As used herein, "treating" a disease in a subject refers to (1) preventing a symptom or disease from occurring in a subject that is predisposed to or has not yet exhibited symptoms of the disease; (2) inhibiting or arresting the development of the disease; or (3) improve or cause regression of the disease or disease symptoms. As understood in the art, "treatment" is a method for achieving a beneficial or desired result, including clinical results. For the purposes of this technology, a beneficial or desired result can include one or more of, but is not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disorder (including disease), stabilized (i.e., not worsening) state of the disorder (including disease), delay or slowing of disorder (including disease), progression, amelioration or palliation of the disorder (including disease), state and remission (whether partial or total), whether detectable or undetectable. When the disease is cancer, the following clinical endpoints are non-limiting examples of treatments: reduced tumor burden, reduced tumor growth, longer overall survival, longer tumor progression time, inhibition of metastasis, or reduction of tumor metastasis. In one aspect, the treatment does not include prophylaxis.
In some embodiments, the term "treating" as used herein means ameliorating a disease, so as to reduce, ameliorate, or eliminate one or more of its etiology, its progression, its severity, or its symptoms, or otherwise beneficially alter the disease in a subject. References to "treatment" of a patient are intended to include prophylaxis. Treatment may also be preemptive in nature, i.e., treatment may include preventing a disease in a subject exposed to or at risk of the disease. Prevention of a disease may involve preventing the disease entirely, for example in the case of preventing infection by a pathogen, or may involve preventing disease progression. For example, prevention of a disease may not mean to completely exclude any effects associated with the disease at any level, but may mean to prevent symptoms of the disease to clinically significant or detectable levels. Prevention of a disease may also refer to preventing the disease from progressing to an advanced stage of the disease.
When the disease is cancer, the following clinical endpoints are non-limiting examples of treatments: (1) Elimination of cancer in a subject or in a tissue/organ of a subject or in a cancer site; (2) A decrease in tumor burden (such as the number of cancer cells, the number of cancer lesions, the number of cancer cells in a lesion, the size of solid cancer, the concentration of liquid cancer in body fluids, and/or the amount of cancer in vivo); (3) Stabilization or delay or slowing or inhibition of cancer growth and/or development, including but not limited to cancer cell growth and/or division, growth of the size of a solid tumor or cancer site, progression of cancer, and/or metastasis (such as time to form new metastasis, total number of metastases, size of metastasis, and various tissues/organs housing metastatic cells); (4) there is less risk of cancer growth and/or development; (5) Inducing an immune response in the patient to the cancer, such as a higher number of tumor-infiltrating immune cells, a higher number of activated immune cells, or a higher number of cancer cells expressing the immunotherapeutic target, or a higher level of expression of the immunotherapeutic target in the cancer cells; (6) Higher probability of survival and/or increased duration of survival, such as increased overall survival (OS, which may be shown as a 1 year, 2 years, 5 years, 10 years, or 20 years survival), increased Progression Free Survival (PFS), increased Disease Free Survival (DFS), increased tumor recurrence time (TTR), and increased tumor progression time (TTP). In some embodiments, the subject after treatment experiences one or more endpoints selected from tumor remission, reduction in tumor size, reduction in tumor burden, increase in overall survival, increase in progression-free survival, inhibition of metastasis, improvement in quality of life, minimization of drug-related toxicity, and avoidance of side effects (e.g., reduction in treatment emergent adverse events). In some embodiments, the improvement in quality of life includes regression 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 mental health (e.g., the extent of irritability, depression, amnesia, tension, and anxiety); improvement or maintenance of social health (e.g., reducing the need for eating, dressing, or assistance with a restroom; improvement or maintenance of the ability to conduct normal recreational activities, hobbies, or social activities; improvement or maintenance of relationships with family members). In some embodiments, the improved patient quality of life is measured qualitatively by patient recitation, or quantitatively using validated quality of life tools known to those skilled in the art, or by a combination thereof. Other non-limiting examples of endpoints include reduced admission, reduced drug use for treatment of side effects, longer periods of discontinuation of treatment, and earlier return to work or care responsibility. In one aspect, prophylaxis (prophylaxis) is excluded from treatment.
As used herein, a biological sample or sample is obtained from a subject. Exemplary samples include, but are not limited to, cell samples, tissue samples, biopsy tissue, liquid samples such as blood and other liquid samples of biological origin, including, but not limited to, anterior nasal swabs, ocular fluids (aqueous humor and vitreous humor), peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid, or pre-ejaculated semen, female tidal fluid, sweat, tears, cyst fluid, pleural and peritoneal fluids, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menstrual blood, pus, sebum, vomiting fluid, vaginal secretion/flushing fluid, synovial fluid, mucosal secretion, fecal water, pancreatic fluid, sinus cavity lavage, bronchopulmonary aspirate, blastocyst cavity fluid, or umbilical cord blood. In some embodiments, the biological sample is a tumor biopsy.
In some embodiments, the sample comprises a fluid from a subject, including but not limited to blood or a blood product (e.g., serum, plasma, etc.), umbilical cord blood, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, stomach, peritoneum, catheter, ear, arthroscope), wash of the female genital tract, urine, stool, sputum, saliva, nasal mucus, prostatic fluid, lavage fluid, semen, lymph, bile, tears, sweat, breast milk, breast fluid, etc., or a combination thereof. In some embodiments, the liquid biological sample is a 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 products or any fraction of blood, such as serum, plasma, buffy coat, etc., as conventionally defined. In some embodiments, the term "blood" refers to peripheral blood. Plasma refers to the whole blood fraction obtained by centrifugation of blood treated with an anticoagulant. Serum refers to the watery portion of the fluid that remains after the blood sample has coagulated. Fluid samples are typically collected according to standard protocols commonly followed by hospitals or clinics. For blood, an appropriate amount of peripheral blood (e.g., 3 milliliters to 40 milliliters) is typically collected and may be stored according to standard methods either before or after preparation.
The term "adjuvant" refers to a substance or mixture that enhances the immune response to an antigen. As non-limiting examples, adjuvants may include dioctadecyl dimethyl ammonium bromide, dioctadecyl dimethyl ammonium chloride, dioctadecyl dimethyl ammonium phosphate or dioctadecyl dimethyl ammonium acetate (DDA) and the non-polar fraction of the total lipid extract of mycobacteria or a part of said non-polar fraction (see e.g. US 8,241,610). In another embodiment, the synthetic nanocarriers may comprise at least one polynucleotide and an adjuvant. As a non-limiting example, synthetic nanocarriers comprising a polynucleotide and an adjuvant may be formulated by the methods described in WO2011150240 and US20110293700, each of which is incorporated herein by reference in its entirety.
The term "contacting" means a direct or indirect bond or interaction between two or more. A specific example of direct interaction is binding. A specific example of an indirect interaction is the action of one entity on an intermediate molecule which in turn acts on the second mentioned entity. Contact as used herein includes in solution, in solid phase, in vitro, ex vivo, cell neutralization, and in vivo. In vivo contact may be referred to as administration.
The "administration" or "delivery" of cells or carriers or other agents and compositions containing them may be performed continuously or intermittently at a single dose throughout the course of treatment. Methods of determining the most effective mode and dosage of administration are known to those skilled in the art and will vary with the composition used for the treatment, the purpose of the treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be carried out, with the dosage level and mode selected by the treating physician or, in the case of animals, by the treating veterinarian. In some embodiments, administration or grammatical variations thereof also refers to more than one dose with a particular 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 more. In some embodiments, one dose is repeated one, two, three, four, five, six, seven, eight, nine, ten or more times. Suitable dosage formulations and methods of administering the agents are known in the art. The route of administration can also be determined, and the method of determining the most effective route of administration is known to those skilled in the art and will vary with the composition used for the treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, administration is infusion (e.g., into the peripheral blood of a subject) over a 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 more.
The term "administration" shall include, but is not limited to, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intraventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection or implantation), by inhalation spray, nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppositories), or topical routes of administration (e.g., gels, ointments, creams, aerosols, etc.), and may be formulated alone or together into suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The present disclosure is not limited by the route of administration, formulation, or dosing regimen.
In some embodiments, the RNA, polynucleotides, vectors, cells, or compositions disclosed herein are administered in an effective amount. An "effective amount" is an amount sufficient to produce a beneficial or desired result. The effective amount may be administered in one or more administrations, applications or administrations. Such delivery depends on many variables including the time of use of the individual dosage units, bioavailability of the therapeutic agent, route of administration, and the like. However, it will be appreciated that the specific dosage level of a therapeutic agent disclosed herein for any particular subject depends on a variety of factors including the activity of the particular agent employed, the bioavailability of the agent, the route of administration, the age and weight of the animal, the general health, sex, diet of the animal, the time of administration, the rate of excretion, drug combination and the severity and form of the particular condition being treated. In general, it will be desirable to administer an amount of the agent that is effective to achieve serum levels comparable to those found in vivo to be effective. These considerations, as well as effective formulations and methods of administration, are well known in the art and are described in standard textbooks.
In some embodiments, the RNA, polynucleotide, vector, cell, or composition 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 sufficient to obtain a pharmacological response; or alternatively, is an amount of a drug or agent that, when administered to a patient suffering from a particular condition or disease, is sufficient to have a desired effect, e.g., treatment, alleviation, amelioration, palliation, or elimination of one or more manifestations of the particular condition or disease in the patient. This effect does not necessarily occur by administering one dose, and may only occur after administering a series of doses. Thus, a therapeutically or pharmaceutically effective amount may be administered in one or more administrations.
In some embodiments, the methods of treatment 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 in which the patient is treated. The first line treatment regimen is the treatment administered first, whereas the second line therapy or the third line therapy regimen is administered after the first line therapy or after the second line therapy, respectively. First-line therapy is defined by the national cancer institute as "first-line treatment for a disease or disorder". In cancer patients, the primary treatment may be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First line therapy is also known to those skilled in the art as "primary therapy and primary treatment". See national cancer institute website www.cancer.gov, last visit time was 5 months 1 day 2008. In general, subsequent chemotherapy regimens are administered to the patient because the patient does not exhibit a positive clinical or sub-clinical response to the first line therapy, or because the first line therapy has ceased.
As used herein, "anti-cancer therapy" includes, but is not limited to, surgical excision, chemotherapy, cryotherapy, radiation therapy, immunotherapy, and targeted therapies. Agents that act to reduce cell proliferation are known in the art and are widely used. Chemotherapy drugs that kill cancer cells only when they divide are called cell cycle specific. These include agents that act in the S phase, including topoisomerase inhibitors and antimetabolites.
Topoisomerase inhibitors are drugs that interfere with the action of topoisomerase enzymes (topoisomerase I and II). During chemotherapy, topoisomerase controls the manipulation of DNA structures necessary for replication and is therefore cell cycle specific. Examples of topoisomerase I inhibitors include the camptothecin analogs, irinotecan, and topotecan listed above. Examples of topoisomerase II inhibitors include amsacrine, etoposide phosphate and teniposide.
Antimetabolites are generally analogs of normal metabolic substrates that often interfere with processes involved in chromosomal replication. They attack cells at very specific stages in the cycle. Antimetabolites include folic acid antagonists such as methotrexate; pyrimidine antagonists such as 5-fluorouracil, fluorouridine (foxuridine), cytarabine, capecitabine and gemcitabine; purine antagonists, such as 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitors such as cladribine, fludarabine, nelarabine and pravastatin; etc.
Plant alkaloids are derived from certain types of plants. Vinca peanut alkaloid is prepared from Catharanthus roseus plantCatharanthus rosea) Is prepared. The Taxus medicine is prepared from bark of Taxus Pacifica (Taxus). Vinca alkaloids and taxanes are also known as antimicrotubule agents. The podophyllotoxin is derived from Podophyllum plant. The camptothecin analogues are derived from Asian "camptotheca acuminata"Camptotheca acuminata). Podophyllotoxins and camptothecin analogs are also classified as topoisomerase inhibitors. Plant alkaloids are typically cell cycle specific.
Examples of such agents include vinca alkaloids, such as vincristine, vinblastine, and vinorelbine; taxanes, such as paclitaxel and docetaxel; podophyllotoxins, such as etoposide and teniposide; and camptothecin analogs, such as irinotecan and topotecan.
In some embodiments wherein the cancer is an immune cell cancer, the anti-cancer therapy may comprise, consist essentially of, or consist of hematopoietic stem cell transplantation.
In some embodiments, a therapeutic agent, such as a cell disclosed herein, can treat cancer in combination with another anti-cancer therapy or therapy that clears immune cells. For example, lymphocyte removal chemotherapy is performed followed by administration of the cells disclosed herein, such as four infusions per week. In further embodiments, these steps may be repeated one, two, three or more times until a partial or complete effect is observed or a clinical endpoint is reached.
Cryotherapy includes, but is not limited to, therapies involving reduced temperature, such as cryotherapy.
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, dosages of ionizing radiation in the range of at least about 2 Gy to no more than about 10 Gy, or at least about 5J/m 2 Up to about 50J/m 2 Within a range of typically about 10J/m 2 Is used to control the radiation dose of ultraviolet radiation.
In some embodiments, the immunotherapy modulates an immune checkpoint. In further embodiments, the immunotherapy comprises, consists essentially of, or still further consists of an immune checkpoint inhibitor, such as a cytotoxic T lymphocyte-associated protein 4 (CTLA 4) inhibitor, or a programmed cell death 1 (PD-1) inhibitor, or a programmed death ligand 1 (PD-L1) inhibitor. In still further embodiments, the immune checkpoint inhibitor comprises an antibody or equivalent thereof that recognizes and binds to an immune checkpoint protein, such as an antibody or equivalent thereof that recognizes and binds to CTLA4 (e.g., yervoy (ipilimumab)), CP-675,206 (tremeliumab), AK104 (california Li Shan anti (cadonilimab)) or AGEN1884 (zenferimab (zalifrelimab)), or an antibody or equivalent thereof that recognizes and binds to PD-1 (e.g., keytruda (pambrizumab), opdivo (nano Wu Liyou mab (nivolumab)), libtayo (cimipblock Li Shan mab (cetirimab)), tyvyt (sindilimab), BGB-a317 (tirelizumab), JS001 (terlipressizumab), SHR1210 (karilizumab), GB226 (terlipressizumab), JS001 (terlipressin Li Shan mab) AB122 (Hiberelimab), AK105 (Pa An Puli mab), HLX10 (St Lu Lishan mab (serplulimab)), BCD-100 (Palo Li Shan mab), AGEN2034 (Balstlilimab), MGA012 (Rafford Li Shan mab), AK104 (Cardontimab Li Shan mab), HX008 (Pratelimab), PF-06801591 (Sasanlimab)), PF-Li Shan mab, and, JNJ-63723283 (cetrilizumab), MGD013 (teborlizumab), CT-011 (pilyizumab) or jempeli (dorsalizumab)) or antibodies recognizing and binding PD-L1 or equivalents thereof (e.g., tecantriq (atizolizumab), imfinzi (durvalumab)), bavelio (avilomab), CS1001 (Shu Geli mab (sugemanimab)) or KN035 (en Wo Lishan antibody (envaranimab)) or essentially consist of, or still further consist of, the same.
As used herein, "targeted therapy" refers to cancer therapy using drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") involved in the growth, progression, recurrence and spread of cancer, such as T cells or NK cells or other immune cells that express Chimeric Antigen Receptors (CARs) that specifically target and bind to a neoantigen. In some embodiments, the neoantigen targeted by such targeted therapies may be the same as the neoantigen encoded by the RNAs disclosed herein. In other embodiments, the neoantigen targeted by such targeted therapies is different from the neoantigen encoded by the RNAs disclosed herein.
As used herein, cleavable peptide, also referred to as cleavable linker, means a peptide that can be cleaved by, for example, an enzyme. A translated polypeptide comprising such a cleavable peptide may yield two end products, thus allowing the expression of more than one polypeptide from one open reading frame. One example of a cleavable peptide is a self-cleaving peptide, such as a 2A self-cleaving peptide. 2A self-cleaving peptides are a class of 18-22 aa long peptides that can induce cleavage of recombinant proteins in cells. In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of P2A, T2A, E2A, F a and BmCPV2A. See, e.g., wang Y et al, sci rep, 2015, 5:16273, 2015, 11, 5.
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 essential amino acids in a relatively short peptide sequence (about 20 amino acids long depending on the viral source) containing the consensus polypeptide motif D-V/I-E-X-N-P-G-P, wherein X refers to any amino acid (SEQ ID NO: 99) that is normally considered self-cleaving.
In some embodiments, the term "linker" refers to any amino acid sequence comprising 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, which amino acid residues may be repeated 1 to 10 times, or alternatively 1 to about 8 times, or alternatively 1 to about 6 times, or alternatively 1 to about 5 times, or alternatively 1 to about 4 times, or alternatively 1 to about 3 times, or alternatively 1 to about 2 times. For example, the linker may comprise up to 15 amino acid residues consisting of a pentapeptide that is repeated three times. In one embodiment, the linker sequence is (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).
As used herein, the phrase "derived" means isolated, purified, mutated or engineered, or any combination thereof. For example, a NY-ESO-1 derived peptide refers to a peptide engineered from the NY-ESO-1 gene or the NY-ESO-1 protein (such as wild-type). In some embodiments, the NY-ESO-1 derived peptide is a NY-ESO-1 mutant or fragment thereof.
In some embodiments, a "signal peptide" refers to a peptide sequence that directs the transport and localization of a protein into a cell, for example, a specific organelle (such as the endoplasmic reticulum) and/or to the cell surface and/or to be secreted out of the cell. In some embodiments, the signal peptide is located 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 acids long.
As used herein, an Open Reading Frame (ORF) refers to a nucleotide sequence encoding a polypeptide or portion thereof. In some embodiments, the ORF is RNA.
Modes of carrying out the disclosure
Cancer treatments have traditionally included surgery, chemotherapy, and radiation therapy. Recently, with a deep understanding of cancer molecular pathology, targeted therapies and immunotherapies have been developed. Both therapies have shown promising results in cancer control. Cancer targeted therapies use sequence information to inhibit the activity of protein products of cancer-driven mutations. Because most somatic mutations exceed a single anatomical site or cancer type, targeted therapies can be applied to different tumors with the same potential mutation, regardless of their tissue location. Since 2017, the U.S. Food and Drug Administration (FDA) has approved several treatments for specific genetic defects regardless of tissue distribution. Examples include palbociclib, which is approved for patients with unresectable or metastatic high microsatellite instability (MSI-H) or solid tumors with defective mismatch repair function (dMMR), and emtrictinib (entrectinib), which is used for patients with NTRK (neurotrophic tyrosine receptor kinase) gene fusion.
Like targeted therapies, cancer immunotherapy has the potential to treat more than one type of cancer. Cancer immunotherapy utilizes the patient's immune system to combat tumor cells. Some cancer immunotherapy is mainly focused on humoral components of the immune system, antibodies, to kill cancer cells by inhibiting the function of proteins expressed by the cancer cells. Other cancer immunotherapy functions by cytotoxic T cells that have the ability to directly destroy tumor cells. The human immune system monitors and kills abnormal cells by recognizing mutated gene products that are not present in normal cells, as part of its normal function, thereby preventing or inhibiting the growth of cancer. Mutant forms of proteins produced by cancer cells are often referred to as tumor-associated antigens, also referred to as neoantigens. By exposing the immune system to cancer neoantigens, the ability of the human immune system to target and kill tumor cells can be enhanced. This approach is called cancer therapeutic vaccine. Human tumor cell lysates or purified tumor neoantigens can be used to stimulate tumor-specific immune responses from cancer patients. Many different cellular components of the immune system can be used to produce cancer vaccines. Fusion proteins consisting of tumor neoantigen, prostatectomy and the adjuvant granulocyte-macrophage colony stimulating factor were loaded into the patient's own dendritic cells as a first FDA-approved cancer therapeutic vaccine. Dendritic cells function as primary Antigen Presenting Cells (APCs) responsible for displaying neoantigens to be recognized by cytotoxic cells. Other cells may also function as APCs.
Despite the great promise of cancer therapeutic vaccines, there are a number of technical challenges from immune epitope discovery to vaccine manufacture. RNA-based vaccines have been proposed as a possible solution to these challenges and have shown promise in preclinical and clinical studies. A key advantage of mRNA vaccines is that mRNA can be produced from DNA templates in the laboratory using readily available materials, which is cheaper and faster than conventional vaccine production that may require the use of chicken eggs or other mammalian cells. In addition, mRNA vaccines have the potential to simplify vaccine discovery and development and promote rapid responses to emerging infectious diseases (see, e.g., marugi et al, mol Ther.,2019, volume 27, phase 4: pages 757-772).
Over the last two decades there has been a broad interest in RNA-based technologies for developing prophylactic and therapeutic vaccines. In this field, mRNA vaccines have been widely studied for infectious disease prevention and for cancer prevention and treatment. Preclinical and clinical trials have shown that mRNA vaccines provide a safe and durable 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 discovery, 2018, vol.17: pp.261-279). As previously mentioned, the production process to produce mRNA vaccines is completely cell-free, simple and rapid if compared to the production of whole-microorganism vaccines, attenuated live vaccines and subunit vaccines. This rapid and simple production method makes mRNA a promising biological product, which potentially fills the gap between emerging infectious diseases and urgent need for effective vaccines.
In contrast to traditional plasmid and virus based methods, this method allows the design of patient-personalized mRNAs that also benefit from not having to cross the nuclear membrane (as opposed to DNA), and therefore the risk of genomic integration is little or no. Furthermore, mRNA vaccines are safe, simple and inexpensive, and have maximum flexibility. In particular, they have self-adjuvanting properties, lack MHC haplotype restriction, and do not require entry into the nucleus (Schlake et al, RNA biol.,2012, volume 9, 11: pages 1319-1330). mRNA does not integrate into the genome, so it avoids tumorigenesis 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, any protein can be encoded for the development of therapeutic and prophylactic vaccines without affecting the properties of the mRNA.
Recently, self-amplifying mRNA vaccines have been demonstrated to be safe and effective against human viral pathogens (e.g., influenza). Influenza mRNA vaccines hold great promise, a platform that does not require eggs, and results in high fidelity antigen production in mammalian cells. Recently published results indicate that loss of glycosylation sites caused by mutations in Hemagglutinin (HA) of egg-adapted H3N2 vaccine strains results in poor neutralization of circulating H3N2 virus in vaccinated humans and ferrets (Zost et al, proc Natl Acad Sci usa, 2017, volume 114: pages 12578-12583). In contrast, the mRNA vaccine production process is egg-free and after vaccine administration, the mRNA encoded protein is properly folded and glycosylated in the host cell, avoiding the risk of producing incorrect antigens.
Generating a strong immune response in infants and elderly has been a problem with influenza vaccines. However, mRNA vaccines can be beneficial in that they have been demonstrated to induce balanced, long-term, and protective immunity to influenza a virus infection in even very young and very old mice. mRNA or RNA replicon-based vaccines have also been shown to be immunogenic in a variety of animal models, including non-human primates (Marugi et al, vaccine.,2017, vol.35, phase 2: pages 361-368).
As described herein, several NY-ESO-1 vaccines have been developed using an immunogenic composition comprising, consisting essentially of, or further consisting of a deoxyribonucleic acid or a messenger ribonucleic acid (mRNA) comprising, consisting essentially of, or further consisting of an Open Reading Frame (ORF) encoding one or more peptides of the different NY-ESO-1 constructs, formulated in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises, consists essentially of, or still further consists of, a polymeric nanoparticle or a liposomal nanoparticle, or both. The composition may be administered to a subject in an amount effective to induce a specific immune response against NY-ESO-1 expression in the subject.
Thus, in one aspect, there is provided an isolated deoxyribonucleic acid (DNA) or an isolated ribonucleic acid (RNA) comprising, consisting essentially of, or still further consisting of an Open Reading Frame (ORF) encoding a NY-ESO-1 derived peptide.
In some embodiments, the isolated DNA encodes one or more of the following: 1) An RNA sequence shown as SEQ ID NO. 1 or a peptide shown as SEQ ID NO. 2; 2) An RNA sequence shown as SEQ ID NO. 3 or a peptide shown as SEQ ID NO. 4; 3) An RNA sequence shown as SEQ ID NO. 5 or a peptide shown as SEQ ID NO. 6; or 4) an RNA sequence as shown in SEQ ID NO. 7 or a peptide as shown in SEQ ID NO. 8.
In other embodiments, the isolated RNA comprises one or more of the following: 1) An RNA sequence shown as SEQ ID NO. 1; 2) An RNA sequence shown as SEQ ID NO. 3; 3) An RNA sequence shown as SEQ ID NO. 5; or 4) an RNA sequence shown in SEQ ID NO. 7. In some embodiments, the RNA is formulated in a carrier (such as a pharmaceutical carrier). In a further embodiment, the RNA is encapsulated in a nanoparticle.
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 peptide encoded by the ORF.
In some embodiments, the 5' -UTR comprises, consists essentially of, or still further comprises an m7G cap structure and an initiation codon. In some embodiments, the 5' -UTR comprises, consists essentially of, or still further comprises UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACC (SEQ ID NO: 10) or an equivalent thereof.
In some embodiments, the 3' -UTR comprises, consists essentially of, or still further comprises a stop codon and a polyA tail. In some embodiments, the 3' -UTR comprises, consists essentially of, or still further consists of AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAAGCGAUCGCAAGCUUCUCGAGC (SEQ ID NO: 9) or an equivalent thereof. In some embodiments, the polyA tail comprises, consists essentially of, or still further consists of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 12) or CGGCAAUAAAAAGACAGAAUAAAACGCACGGUGUUGGGUCGUUUGUUC (SEQ ID NO: 13).
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 pSFV1, or pcDNA3, or pTK126. In some embodiments, the carrier comprises
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(SEQ ID NO: 11) or an equivalent thereof, or essentially consisting of, or still further consisting of.
In some embodiments, the RNA is messenger RNA (mRNA).
In some embodiments, the GC content of the full-length RNA is about 35% to about 70% (including any percentage or any subrange 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%.
In some embodiments, the RNA is chemically modified. In some embodiments, the chemical modification comprises, consists essentially of, or still further consists of one or both of incorporating an N1-methyl-pseudouridine residue or incorporating a pseudouridine residue. In some embodiments, at least about 50% to about 100% of the uridine residues in the RNA are N1-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 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 more of the residues of the RNAs are modified chemically by one or more of the modifications 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 more of the uridine residues are modified by one or more of the modification chemistries 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 more of the uridine residues are N1-methyl pseudouridine or pseudouridine.
In some embodiments, all or some of the uridine residues are replaced with pseudouridine during in vitro transcription. This modification stabilizes the mRNA in the cell against enzymatic degradation, resulting in enhanced mRNA translation efficiency. The pseudouridine used may be N1-methyl-pseudouridine, or other modifications well known in the art, such as N6-methyladenosine (m 6A), inosine, pseudouridine, 5-methylcytidine (m 5C), 5-hydroxymethylcytidine (hm 5C) and N1-methyladenosine (m 1A). Optionally, the modification is performed in the entire mRNA. The skilled artisan will recognize that other modified RNA residues can be used to stabilize the three-dimensional structure of the protein and increase protein translation.
Also provided are polynucleotides encoding the DNA or RNA disclosed herein, or polynucleotides complementary to the polynucleotides, or both. In some embodiments, the polynucleotide is selected from the group consisting of: deoxyribonucleic acid (DNA), RNA, hybrids of DNA and RNA, or analogs of each of them. In further embodiments, the analog comprises, consists essentially of, or still further consists of a peptide nucleic acid or a locked nucleic acid, or both.
In some embodiments, the polynucleotide further comprises regulatory sequences that direct its transcription. In some embodiments, the regulatory sequences are suitable for use in an in vitro transcription system. In further embodiments, the regulatory sequence comprises, consists essentially of, or still further consists of a promoter. In yet further embodiments, the promoter comprises, consists essentially of, or still further consists of a bacteriophage RNA polymerase promoter, such as a T7 promoter, or an 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.
In a further aspect, there is provided a vector comprising, consisting essentially of, or still further consisting of a polynucleotide disclosed herein.
In some embodiments, the vector further comprises regulatory sequences operably linked to the polynucleotide to direct its transcription. In some embodiments, the regulatory sequences are suitable for use in an in vitro transcription system. In further embodiments, the regulatory sequence comprises, consists essentially of, or still further consists of a promoter. In yet further embodiments, the promoter comprises, consists essentially of, or still further consists of a bacteriophage RNA polymerase promoter, such as a T7 promoter, or an 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.
In some embodiments, the vector further comprises regulatory sequences operably linked to the polynucleotide to direct its replication. In further embodiments, the regulatory sequence comprises one or more of the following: an origin of replication or primer annealing site, a promoter or enhancer, or alternatively consists essentially of, or still further consists of.
In some embodiments, the vector is a non-viral vector. In further embodiments, the non-viral vector is a plasmid, or a liposome or micelle. In some embodiments, the vector is pUC57, or pSFV1, or pcDNA3, or pTK126, or other plasmids available in addgene or european standard plasmid backbone system (Standard European Vector Architecture, SEVA).
In some embodiments, the vector is a viral vector. In a further embodiment, the viral vector is selected from an adenovirus vector, or an adeno-associated virus vector, or a retrovirus vector, or a lentivirus vector, or a plant virus vector.
In yet another aspect, there is provided a cell comprising one or more of the following: DNA or RNA as disclosed herein, polynucleotides as disclosed herein, or vectors as disclosed herein. In some embodiments, the cell is suitable for replicating any one or more of the following: DNA, RNA, polynucleotide, or vector, thereby producing one or more of the following: RNA, polynucleotide or vector. In some embodiments, the cell is suitable for transcription of a polynucleotide or vector into RNA, thereby producing RNA.
In some embodiments, the cell is a prokaryotic cell. In a further embodiment, the prokaryotic cell is an E.coli cell.
In some embodiments, the cell is a eukaryotic cell. In further embodiments, the eukaryotic cell is any one of a mammalian cell, an insect cell, or a yeast cell.
In some embodiments, the cells disclosed herein are suitable for producing (such as transcribing or expressing) an RNA disclosed herein. Such production may be in vivo or in vitro. For example, the cells may be used to produce RNA in vitro. Such RNA is then administered to a subject in need thereof, optionally together with a suitable pharmaceutically acceptable carrier. Alternatively, the cells may be used as a cell therapy and administered directly to a subject in need thereof, optionally together with a suitable pharmaceutically acceptable carrier. In further embodiments, the cell therapy may additionally deliver other prophylactic or therapeutic agents to the subject. In some embodiments, the cells used as cell therapies are immune cells, such as T cells, B cells, NK cells, NKT cells, dendritic cells, bone marrow cells, monocytes or macrophages.
In one aspect, a composition is provided comprising a carrier and one or more of the following: the RNA disclosed herein, the polynucleotide disclosed herein, the vector disclosed herein, or the cell disclosed herein, or a combination thereof. 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.
In a further aspect, methods of producing RNAs (such as those disclosed herein) are provided. In some embodiments, the method comprises, consists essentially of, or still further of culturing the cells disclosed herein under conditions suitable for expression of RNA (such as transcription of DNA into RNA). In some embodiments, the cell comprises DNA encoding an RNA of the present disclosure. In some embodiments, the method comprises, consists essentially of, or still further consists of contacting a polynucleotide disclosed herein or a vector 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 RNA, such as transcription of DNA into RNA. In some embodiments, the method further comprises isolating RNA. In some embodiments, the method further comprises storing the RNA.
In yet another aspect, there is provided an RNA produced by the methods disclosed herein, or a composition comprising, consisting essentially of, or still further consisting of the produced RNA.
Improving mRNA vaccine expression efficiency
To increase the expression efficiency of mRNA vaccines in mammalian cells, mRNA stability can be enhanced by partial chemical modification. To further increase translation efficiency, short-and double-stranded RNAs derived from aberrant RNA polymerase activity are removed. To increase the efficacy of mRNA vaccines, sequence optimization can be used, along with modified nucleosides such as pseudouridine (phi), 5-methylcytidine (5 mC) Cap-1 structures and optimized codons, to increase translation efficiency. During in vitro transcription of mRNA, immature mRNA can be produced as a contaminant that inhibits translation by stimulating innate immune activation. FPLC and HPLC purification can be used to remove these contaminants.
In the compositions presented herein, the template for in vitro transcription of mRNA contains five cis-acting structural elements, namely from 5 'to 3': (i) optimized cap structure, (ii) optimized 5 'untranslated region (UTR), (iii) codon optimized coding sequence, (iv) optimized 3' UTR and (v) a stretch of repeated adenine nucleotides (polyA tail) (fig. 12). These cis-acting structural elements are further optimized to obtain better mRNA characteristics. The 5' -UTRs provided herein include an initiation codon and some other elements, but do not encode a polypeptide (i.e., they are non-encoded). In some embodiments, the 5' -UTRs of the present disclosure comprise, consist essentially of, or still further consist of a cap structure having a 7-methylguanosine (7 mG) sequence. The 3'-UTR is located immediately downstream (3') of the stop codon (the codon representing the mRNA transcript of the stop signal) and does not encode a polypeptide (non-encoded). The polyA tail is a specific region of mRNA located downstream of the 3' -UTR and containing multiple consecutive adenosine monophosphates.
A typical mRNA production cassette comprises, consists essentially of, or still further of, a cap structure in its 5' -UTR region, followed by an in-frame mRNA sequence encoding the corresponding protein or peptide. In some embodiments, a 3' -UTR with a polyA tail is necessary for efficient mRNA production. In some embodiments, the expression cassette is used not only for the efficiency of mRNA production, but also for subsequent protein or peptide production (fig. 12).
In some embodiments, mRNA is produced by In Vitro Transcription (IVT) from a linear DNA template containing phage promoters, optimized UTRs, and codon optimized sequences using RNA polymerase (T7, T3, or SP 6) and a mixture of different nucleosides. In other embodiments, the linear DNA template may be cloned into plasmid DNA (pDNA) as a delivery vector. Plasmid vectors may be suitable for mRNA vaccine production. Commonly used plasmids include pSFV1, pcDNA3, and pTK126. One unique mRNA expression system is pEVL (see Grier et al, mol Ther Nucleic acids, 19; vol. 5: page e306, (2016), "pEVL: 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).
In some embodiments, the vaccine comprises, consists essentially of, or still further consists of an effective amount of RNA comprising, consisting essentially of, or still further of an open reading frame encoding one or more of the NY-ESO-1, NY-ESO-1 variants, and a pharmaceutically acceptable carrier. The effective amount is an amount effective to induce a neoantigen-specific (such as NY-ESO-1-specific) immune response in the subject. In one embodiment, the carrier comprises, consists essentially of, or still further consists of polymeric nanoparticles or liposomal nanoparticles. In some embodiments, the carrier is a histidine-lysine copolymer or a spermine-liposome conjugate. In some embodiments, the vector further comprises DOTAP or MC3 or both.
In some embodiments, the vaccine comprises, consists essentially of, or still further consists of an effective amount of an mRNA comprising, consisting essentially of, or still further consists of an open reading frame encoding a plurality of neoantigens separated by self-cleaving 2A peptide sites, a signal sequence that integrates the neoantigens into a membrane and/or is secreted using a different signal sequence (such as an albumin signal sequence).
In some embodiments, the vaccine comprises an effective amount of an mRNA comprising an open reading frame encoding one or more NY-ESO-1 based neoantigens and/or other neoantigens, and a pharmaceutically acceptable carrier. The effective amount is an amount effective to induce an NY-ESO-1 specific immune response in the subject. 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 vector further comprises DOTAP or MC3.
Histidine-lysine (HK) polypeptides as mRNA vaccine delivery systems
Despite significant progress in the past few years in the rational design of mRNA vaccines and in the elucidation of their mechanism of action, their widespread use has been limited by the presence of ubiquitous ribonucleases (RNases), and the need to facilitate the entry of vaccines into cells and subsequent escape from endosomes, and their targeting to lymphoid organs or specific cells. See, e.g., midoux and Pichon, experert Rev vaccines, 2015, volume 14, phase 2: pages 221-234. mRNA preparations with chemical vectors provide more specificity and internalization in Dendritic Cells (DCs) to achieve better immune responses and reduce dose.
Non-viral delivery systems are more advantageous than viral delivery systems. See, e.g., brito et al, adv genet.,2015, volume 89: pages 179-233. One non-limiting example is that non-viral methods are preferred over viral delivery systems because of their safety and cost effectiveness. See, e.g., juliano et al, nucleic Acids res.,2008, volume 36: pages 4158-4171. Non-viral methods for delivering vaccines include naked mRNA vaccines, gene guns, protamine condensation, adjuvant-based vaccines, and encapsulated mRNA vaccines. The sense RNA virus (alphavirus) can be used in a viral delivery system. The glycoproteins (E1 and E2) of the alphavirus are useful 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, mRNA is transfected into monocytes, macrophages, T cells, dendritic Cells (DCs) and Mesenchymal Stem Cells (MSCs) prior to administration, see, e.g., sahin et al, nat Rev Drug discovery, 2014, volume 13: pages 759-780. In contrast to bare mRNA vaccination, which only provides optimal expression, mRNA vaccination by ex vivo transfection can induce a strong immune response.
As described herein, a series of branched histidine-lysine (HKP) polypeptides (HKP) can be used to encapsulate mRNA by electrostatic interactions. As used herein, HKP is a group of linear and branched peptides consisting of histidine and lysine residues, and in most cases these peptides form spherical nanoparticles when mixed with nucleic acids. Such polypeptides are disclosed in U.S. Pat. No. 7,070,807 B2 issued 7/4/2006 and U.S. Pat. No. 7,163,695 B2 issued 16/1/2007. The disclosure of each of these patents is incorporated by reference herein in its entirety. Similar to other vectors, HKP vectors differ in their ability to carry various nucleic acids. For example, the four branched HK peptide (H2K 4 b) is a good vector for plasmids (see, e.g., chen et al, nucleic Acids Res.,2001, volume 29: pages 1334-1340; and Zhang et al Methods Mol biol.,2004, volume 245: pages 33-52), but is a poor vector for siRNA. In addition, H3K4b, H3K (+H) 4b and H3K8b are excellent vectors for siRNA (see, e.g., leng et al, J Gene Med.,2005, volume 7: pages 977-986), but only H3K (+H) 4b shows the effectiveness of carrying mRNA into target cells. In addition, H3K (+H) 4b is a more efficient mRNA vector than DOTAP liposomes. Furthermore, as described herein, a delivery vehicle combination of H3K (+h) 4b, MC3, and/or DOTAP may be used to enhance the therapeutic efficacy of mRNA delivery. The results described herein demonstrate that the H3k (+H) 4b, MC3 and/or DOTAP combinations are the most efficient mRNA vectors. This combination is synergistic in its ability to carry mRNA into cells.
Formulations and related methods
Thus, in one aspect, a composition (such as an immunogenic composition) is provided that comprises, consists essentially of, or still further of, an effective amount of an RNA disclosed herein, e.g., formulated in a pharmaceutically acceptable carrier. In some embodiments, the composition comprises, consists essentially of, or still further consists of RNA and a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutically acceptable carrier comprises, consists essentially of, or still further consists of nanoparticles. 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, consists essentially of, or still further consists of, a polymeric nanoparticle or a liposomal nanoparticle, or both.
In some embodiments, the polymeric nanoparticle carrier comprises, consists essentially of, or still further consists of a histidine-lysine copolymer (HKP). In further embodiments, the HKP comprises, consists essentially of, or still further consists of H3K (+h) 4 b. In yet further embodiments, the HKP comprises, consists essentially of, or still further consists of H3k (+h) 4 b. In some embodiments, HKP comprises a side chain selected from SEQ ID NO: 14-23.
In some embodiments, the mass ratio of HKP to RNA in the composition is about 10:1 to about 1:10, including any range or ratio therebetween, e.g., 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 1:1.5, 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 7:1, about 1:1, about 9:1, about 1:1.5, about 9:1, about 1:1, about 9:1. In one embodiment, the mass ratio of HKP to RNA in the composition is about 2.5:1. In another embodiment, the mass ratio of HKP to RNA in the composition is about 4:1.
In some embodiments, the polymeric nanoparticle carrier further comprises a lipid. In a further embodiment, the lipid is a cationic lipid. In yet a further embodiment, the cationic lipid is ionizable.
In some embodiments, the cationic lipid comprises, consists essentially of, or still further consists of Dlin-MC3-DMA (MC 3) or dioleoyloxy-3- (trimethylammonio) propane (DOTAP) or both.
In some embodiments, the lipid further comprises one or more of the following: helper lipids, cholesterol or pegylated lipids. In some embodiments, the lipid further comprises PLA or PLGA.
In some embodiments, HKP and mRNA self-assemble into nanoparticles when mixed.
In some embodiments, the liposome nanoparticle carrier comprises, consists essentially of, or still further consists of spermine-lipid cholesterol (SLiC). In a further embodiment, SLiC is selected from the group consisting of TM1-TM5, the structure of which is shown in FIG. 13.
In some embodiments, the pharmaceutically acceptable carrier is a Lipid Nanoparticle (LNP). In some embodiments, the lipid is a cationic lipid. In a further embodiment, the cationic lipid is ionizable. In some embodiments, the LNP comprises one or more of the following: 8- { (2-hydroxyethyl) [ 6-oxo-6- (undecyloxy) hexyl]Amino } 9-heptadecyl octanoate (SM-102), 2-diiodo-4-dimethylaminoethyl- [1,3 ]]Dioxolane (DLin-KC 2-DMA), diiodo-methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA), bis (-)Z) -non-2-en-1-yl) 9- ((4- (dimethylamino) butyryl) oxy) heptadecanedioate (L319) or an equivalent of each of them, or essentially consisting of, or still further consisting of. In some embodiments, the LNP further comprises one or more of the following: helper lipids, cholesterol or pegylated lipids.
In some embodiments, the mass ratio of LNP to RNA in the composition is about 10:1 to about 1:10, including any range or ratio therebetween, e.g., 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 1:1.5, 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 7:1, about 1:1:1, about 9:1, about 1:1.5, about 1:1, about 9:1. In one embodiment, the mass ratio of LNP to RNA in the composition is about 2.5:1. In another embodiment, the mass ratio of LNP to RNA in the composition is about 4:1.
In some embodiments, the helper lipid comprises one or more of the following: distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), (2)R) -3- (hexadecanoyloxy) -2- { [ (9)Z) -octadec-9-enoyl]Oxy } propyl 2- (trimethylammonium) ethyl phosphate (POPC) or dioleoyl phosphatidylethanolamine (DOPE), or consist essentially of, or still further of.
In some embodiments, the cholesterol comprises, consists essentially of, or still further consists of plant cholesterol or animal cholesterol, or both.
In some embodiments, the pegylated lipid comprises one or more of the following: PEG-c-DOMG (R-3- [ (-)ω-methoxy-poly (ethylene glycol) 2000) carbamoyl]-1, 2-dimyristoxypropyl-3-amine), PEG-DSG (1, 2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1, 2-dimyristoyl-sn-glycerol), optionally PEG2000-DMG ((1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000 ]]Or PEG-DPG (1, 2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol), or consist essentially of, or still further consist of.
In some embodiments, the mass ratio of cationic lipid to helper lipid is about 10:1 to about 1:10, including any range or ratio therebetween, e.g., 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 1:1.5, 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 7:1, about 1:1, about 9:1, about 1.5, about 1:1:1, about 9:1. In one embodiment, the mass ratio of cationic lipid to helper lipid is about 1:1.
In some embodiments, the mass ratio of cationic lipid to cholesterol is about 10:1 to about 1:10, including any range or ratio therebetween, e.g., 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 1:1.5, 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 7:1, about 1:1, about 9:1, about 1.5:1, about 1:1. In one embodiment, the mass ratio of cationic lipid to cholesterol is about 1:1.
In some embodiments, the mass ratio of cationic lipid to pegylated lipid is about 10:1 to about 1:10, including any range or ratio therebetween, e.g., 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 1:1.5, 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 7:1, about 1:1, about 9:1, about 1:1, about 5:1, about 9:1. In one embodiment, the mass ratio of cationic lipid to pegylated lipid is about 1:1.
The mass ratio of cationic lipid, helper lipid, cholesterol, and pegylated lipid can be calculated by one skilled in the art based on the ratio of cationic lipid and helper lipid, cationic lipid and cholesterol, and cationic lipid and pegylated lipid as disclosed herein.
In some embodiments, the LNP comprises, consists essentially of, or still further consists of SM-102, DSPC, cholesterol, and PEG 2000-DMG. In some embodiments, the mass ratio of SM-102, DSPC, cholesterol, and PEG200-DMG is about 1:1:1:1. In some embodiments, the molar ratio of SM-102, DSPC, cholesterol, and PEG2000-DMG is about 50:10:38.5:1.5.
In some embodiments, the mass ratios provided herein can be replaced with another parameter (such as a molar ratio, weight percent relative to total weight, component weight relative to total volume, or molar percent relative to total molar amount). Knowing the components and their molecular weights, one skilled in the art will not have difficulty converting the mass ratio to a molar ratio or other equivalent parameter.
In a further aspect, methods of producing the compositions disclosed herein are provided. The method comprises contacting the RNA disclosed herein with HKP, such that the RNA and HKP self-assemble into, consist essentially of, or still further consist of nanoparticles.
In some embodiments, the mass ratio of HKP to RNA in the contacting step is about 10:1 to about 1:10, including any range or ratio therebetween, e.g., 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 1:1.5, 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 7:1, about 1:1:1, about 9:1, about 1:1.5, about 1:1. In one embodiment, the mass ratio of HKP to RNA during the contacting step is about 2.5:1. In another embodiment, the mass ratio of HKP to RNA in the contacting step is about 4:1.
In some embodiments, the method further comprises contacting the HKP and RNA with a cationic lipid. In further embodiments, the cationic lipid comprises, consists essentially of, or still further consists of Dlin-MC3-DMA (MC 3) or DOTAP (dioleoyloxy-3- (trimethylammonio) propane) or both. In yet further embodiments, the mass ratio of cationic lipid to RNA in the contacting step is about 10:1 to about 1:10, including any range or ratio therebetween, e.g., 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 1:1.5, about 1:2.5, about 1:3.5, about 1:4.5, about 1:5, about 1:5.5, about 6:6, about 7:1, about 1:1, about 9:1, about 1:1.5, about 1:1, about 1:1.5, about 1:1:1, about 9:1. In one embodiment, the mass ratio of RNA to cationic lipid in the contacting step is about 1:1. Thus, the mass ratio of HKP, RNA and cationic lipid in the contacting step can be calculated based on the ratio between HKP and RNA and the ratio between RNA and cationic lipid. For example, if the ratio of HKP to RNA is about 4:1 and the ratio of RNA to cationic lipid is about 1:1, then the ratio of HKP to RNA to cationic lipid is about 4:1:1.
In yet another aspect, methods of producing the compositions disclosed herein are provided. The method comprises contacting the RNA disclosed herein with a lipid, such that the RNA and the lipid self-assemble into, consist essentially of, or still further consist of a Lipid Nanoparticle (LNP).
In some embodiments, the LNP comprises one or more of the following: 8- { (2-hydroxyethyl) [ 6-oxo-6- (undecyloxy) hexyl]Amino } 9-heptadecyl octanoate (SM-102), 2-diiodo-4-dimethylaminoethyl- [1,3 ]]Dioxolane (DLin-KC 2-DMA), diiodo-methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA), bis (-)Z) -non-2-en-1-yl) 9- ((4- (dimethylamino) butyryl) oxy) heptadecanedioate (L319) or an equivalent of each of them, or essentially consisting of, or still further consisting of.
In some embodiments, the LNP further comprises one or more of the following: helper lipids, cholesterol or pegylated lipids. In some embodiments, the helper lipid comprises one or more of the following: distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), (2) R) -3- (hexadecanoyloxy) -2- { [ (9)Z) -octadec-9-enoyl]Oxy } propyl 2- (trimethylammonium) ethyl phosphate (POPC) or dioleoyl phosphatidylethanolamine (DOPE), or consist essentially of, or still further of. In some embodiments, the cholesterol comprises, consists essentially of, or still further consists of plant cholesterol or animal cholesterol, or both. In some embodiments, the pegylated lipid comprises one or more of the following: PEG-c-DOMG (R-3- [ (-)ω-methoxy-poly (ethylene glycol) 2000) carbamoyl]-1, 2-dimyristoxypropyl-3-amine), PEG-DSG (1, 2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1, 2-dimyristoyl-sn-glycerol), optionally PEG2000-DMG ((1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000 ]]Or PEG-DPG (1, 2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol), or consist essentially of, or still further consist of.
In some embodiments, the LNP comprises, consists essentially of, or still further consists of SM-102, DSPC, cholesterol, and PEG 2000-DMG. In some embodiments, the mass ratio of SM-102, DSPC, cholesterol, and PEG200-DMG is about 1:1:1:1. Additionally or alternatively, the molar ratio of SM-102, DSPC, cholesterol, and PEG2000-DMG is about 50:10:38.5:1.5.
In some embodiments, the contacting step is performed in a microfluidic mixer. In further embodiments, the microfluidic mixer is a slit interdigital (slit interdigital) micromixer or an interlaced fish bone (staggered herringbone) micromixer (SHM).
Also provided are compositions prepared by the methods disclosed herein.
Compositions and carriers
Additional aspects of the disclosure relate to compositions comprising, or alternatively consisting essentially of, or still further consisting of, one or more of the vectors and products (e.g., RNA comprising an ORF encoding one or more NY-ESO-1 derived peptides). The carrier may be a pharmaceutically acceptable carrier. In one aspect, provided herein are compositions comprising, consisting essentially of, or still further of an RNA containing an ORF encoding one or more NY-ESO-1 derived peptides disclosed herein, and optionally a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle. In a further embodiment, the carrier comprises a histidine-lysine copolymer (HKP). In still further embodiments, the HKP may include H3K (+h) 4b. In other embodiments, the polymeric nanoparticle carrier may comprise PLA or PLGA.
In some embodiments, an mRNA or composition may comprise multiple mRNA molecules encoding the same or different polypeptides. In further embodiments, the plurality of mrnas are encapsulated in the same or different nanoparticles or liposomes. In some embodiments, the mRNA in the composition is encapsulated in 1, 2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP). In other embodiments, the liposome nanoparticle carrier comprises spermine-lipid cholesterol (SLiC). In a further embodiment, SLiC is selected from the structures TM1-TM5 shown in FIG. 13.
Briefly, pharmaceutical compositions of the present disclosure include, but are not limited to, any of the claimed compositions described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide) and preservatives. The 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 composition may be accomplished continuously or intermittently at a single dose throughout the course of treatment. Methods of determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for the treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations may be performed, with the dosage level and mode 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 compositions of the present disclosure may be administered in combination with other therapies. mRNA and compositions are administered to a host using methods known in the art.
In some embodiments, the carrier may be a polymeric nanoparticle or a liposomal nanoparticle. Non-limiting examples of polymer nanoparticles include H3K (+h) 4b, PLA or PLGA. A non-limiting example of a liposome nanoparticle is spermine-lipid cholesterol (SLiC).
Therapeutic method
Methods of treating a subject having, or at risk of having, or suspected of having cancer are also provided. In some embodiments, the cancer expresses NY-ESO-1. Methods of determining when this method is successful are known in the art and are briefly described herein.
Methods of inhibiting the growth of a tumor or cancer cell are also provided. The method comprises contacting the immune cells with any one or more of: an RNA disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a cell disclosed herein, or a composition disclosed herein, thereby activating an immune cell and contacting a tumor or cancer cell with, consisting essentially of, or still further consisting of the activated immune cell. In some embodiments, the cancer cell or tumor expresses NY-ESO-1. In one aspect, a method of treating a cancer subject having a tumor that expresses NY-ESO-1 comprises contacting a cancer cell with an RNA described in the present disclosure, optionally in combination with, or alternatively consisting essentially of, or still further consisting of a pharmaceutically acceptable carrier. The contacting may be in vitro or in vivo.
The pharmaceutical compositions of the present disclosure may be administered in a manner suitable for the disease to be treated or prevented. Although the appropriate dosage may be determined by clinical trials, the number and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, and the like. In one aspect, they are administered directly by direct injection or systemic injection, such as intravenous injection or infusion.
The subject to be treated may be an animal, such as a mammal or a human patient. The cancer to be treated may be a liquid tumor or cancer, or a solid tumor or cancer. In one aspect, the cancer expresses NY-ESO-1. The cancer may be stage I, stage II, stage III or stage IV. The treatment may be combined with other therapies described herein. In one aspect, the treatment is an adjunct treatment to the tumor resection. The treatment can be first line therapy, second line therapy, third line therapy, fourth line therapy, and fifth line therapy.
Additionally or alternatively, provided are screening steps of the screening methods or methods disclosed herein for personalizing or accurate methods, or alternatively for testing new combination therapies. The method comprises, consists essentially of, or still further consists of detecting expression of NY-ESO-1 as disclosed herein. In some embodiments, expression of NY-ESO-1 can be detected using sequencing, southern blotting, or northern blotting. In some embodiments, expression of the NY-ESO-1 protein may be detected using flow cytometry or Western blotting. The method can be practiced in an animal to produce an animal model for treatment or to treat the animal as determined by the veterinarian being treated. Methods of determining when this method is successful are known in the art and are briefly described herein.
In some embodiments, the cancer is an adenocarcinoma, adenoma, leukemia, lymphoma, carcinoma, melanoma, angiosarcoma, pancreatic cancer, colon cancer, colorectal cancer, rectal cancer, or seminoma. Cancers may be primary or metastatic. A subject in need thereof may have active cancer or be in remission, or be at risk of having primary or secondary cancer.
Additionally or alternatively, methods of inducing an immune response (e.g., an immune response expressing NY-ESO-1 as disclosed herein) in a subject in need thereof are provided. In some embodiments, the immune response includes any one or more of the following: th1 immune response, activation of CD8+ T cells or production of pro-inflammatory cytokines such as interleukin-2 (IL-2), interferon-gamma (IFN-gamma) or tumor necrosis factor-beta (TNF-beta), or consist essentially of, or still further of. Methods of determining when this method is successful are known in the art and are briefly described herein.
The methods comprise administering to the subject, for example, an effective amount (e.g., a pharmaceutically effective amount) of any one or more of: the DNA or RNA disclosed herein, the polynucleotide disclosed herein, the vector disclosed herein, the cell disclosed herein or the composition disclosed herein, or consist essentially of, or still further consist of, the DNA or RNA disclosed herein, the polynucleotide disclosed herein, the vector disclosed herein, the cell disclosed herein or the composition disclosed herein.
In some embodiments, the DNA encodes one or more of the following: 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 the following: SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7. In further embodiments, the RNA further comprises (e.g., comprises, or consists essentially of, or still further consists of) a 5'UTR (e.g., comprises, or consists essentially of, or still further consists of SEQ ID NO: 10) and a 3' UTR (e.g., comprises, or consists essentially of, or still further consists of, SEQ ID NO: 9). In some embodiments, the vector comprises, consists essentially of, or still further consists of SEQ ID NO. 11. In some embodiments, the composition comprises RNA formulated in a carrier (such as LNP or HKP nanoparticles disclosed herein).
In some embodiments, the administration is intratumoral, or intravenous, or intramuscular, or intradermal, or subcutaneous.
In some embodiments, the subject is a mammal or a human.
In some embodiments, the method further comprises administering to the subject an additional anti-cancer therapy. In some embodiments, the anti-cancer therapy is administered prior to, or concurrently with, or after, administration of any one or more of the following: RNA as disclosed herein, polynucleotides as disclosed herein, vectors as disclosed herein, cells as disclosed herein, or compositions as disclosed herein.
In some embodiments, the administration is repeated at least once, at least twice, 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 more.
In some embodiments, the method further comprises detecting expression of NY-ESO-1 disclosed herein in a biological sample of the subject (such as tumor biopsy or circulating tumor DNA) prior to administration.
In some embodiments, the method further comprises monitoring expression of NY-ESO-1 disclosed herein in a biological sample of the subject (such as tumor biopsy or circulating tumor DNA) after administration.
In some embodiments, the method further comprises detecting an antibody disclosed herein that recognizes and binds NY-ESO-1 in a biological sample (such as a blood sample) of the subject after administration.
As used herein, an effective dose of an RNA, or polynucleotide, or vector, or cell or composition disclosed herein is a dose required to generate a protective immune response in a subject to be administered. A protective immune response herein is an immune response that treats cancer in a subject. The RNA, or polynucleotide, or vector, or cell, or composition disclosed herein may be administered one or more times. Initial measurement of the vaccine immune response may be performed by measuring the production of antibodies in a subject receiving the RNA, or polynucleotide, or vector, or cell or composition. Methods for measuring antibody production in this manner are also well known in the art, and are dosages required for preventing, inhibiting the occurrence of cancer or for treatment (to some extent alleviating symptoms, preferably all symptoms). The pharmaceutically effective dosage will depend on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the particular mammal being considered, simultaneous administration, and other factors that will be recognized by those skilled in the medical arts. Typically, the amount of active ingredient applied will be from 0.1 mg/kg to 100 mg/kg body weight/day, depending on the potency of the formulated composition.
In some embodiments, the RNA composition may be administered at a dosage level sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05mg/kg, 0.005mg/kg to 0.05mg/kg, 0.001 mg/kg to 0.005mg/kg, 0.05mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30mg/kg, 0.01 mg/kg to 10mg/kg, 0.1 mg/kg to 10mg/kg, or 1 mg/kg to 25 mg/kg of subject body weight per day, one or more times per day, per week, month, etc., to obtain the desired therapeutic or prophylactic effect. In some embodiments, the RNA composition is administered at a dose of about 10 μg/kg to about 500 μg/kg body weight, or any dose or subrange therein, such as a dose of about 28.5 μg/kg to 285 μg/kg body weight. The desired dose may be delivered three times per day, twice per day, once every other day, once every three days, once per week, once every two weeks, once every three weeks, once every four weeks, once every 2 months, once every three months, once every 6 months, etc. In certain embodiments, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, twelve, thirteen, fourteen or more administrations) may be used to deliver the desired dose. When multiple administrations are employed, separate dosing regimens such as those described herein may be employed. In some embodiments, the RNA composition may be administered at a dosage level sufficient to deliver 0.0005mg/kg to 0.01 mg/kg, e.g., about 0.0005mg/kg to about 0.0075 mg/kg, e.g., about 0.0005mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004mg/kg, or about 0.005 mg/kg. In some embodiments, the RNA composition may be administered once or twice (or more) at a dosage level sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.
In some embodiments, the RNA composition can be administered at a total dose of or sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675mg, 0.700 mg, 0.725 mg, 0.775mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875mg, 0.925 mg, 0.37, 0.950 mg, 0.37, 0.900 mg, or two times (e.971), day 0 and 7, day 0 and 14, day 0 and 21, day 0 and 28, day 0 and 60, day 0 and 90, day 0 and 120, day 0 and 150, day 0 and 180, day 0 and 3 months, day 0 and 6 months, day 0 and 9 months, day 0 and 12 months, day 0 and 18 months, day 0 and 2 years, day 0 and 5 years or day 0 and 10 years later. The present disclosure encompasses higher and lower doses and frequency of administration. For example, the RNA composition can be administered three or four times.
In certain embodiments, the RNA or composition is further combined with another therapy (such as a purified protein drug). Non-limiting examples include therapeutic mabs (e.g., avastin ™, herceptin ™, yervoy ™, keytruda ™, opdivo ™, tecantriq ™, etc.) or therapeutic proteins such as GM-CSF, interleukins, interferons, thymosin a, sea sarrelin (hexarelin), or adiponectin. In one embodiment, the combination therapy comprises simultaneous, sequential and/or separate administration of an mRNA or composition disclosed herein and a combination therapy to a subject in need thereof. In one embodiment, the mRNA or composition and the combination therapy are administered to the subject via the same route of administration. In another embodiment, a different route of administration is used. In certain embodiments, there is an interval of about 0.5 hours, 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 month in either of the two administrations.
In certain embodiments, any combination therapy as described herein achieves a synergistic effect in the treatment of a disease (such as cancer).
Also provided are methods of treating cancer comprising administering to a subject in need thereof an effective amount of one or more of the RNAs or compositions described herein. In one embodiment, the method comprises administering an effective amount of an RNA or composition described herein. In a further embodiment, the method further comprises administering an effective amount of a combination therapy, thereby achieving a synergistic effect in the treatment of cancer.
Additionally, methods for delivering an RNA or composition described herein to a subject in need thereof are provided.
In one embodiment, the subject is suspected of having and/or is diagnosed with a cancer that expresses NY-ESO-1.
In one embodiment, the cancer is a solid tumor (e.g., carcinoma or sarcoma) or a non-solid cancer (e.g., leukemia). Additionally or alternatively, the cancer or tumor is a primary or metastatic cancer or tumor. In further embodiments, the cancer is selected from the group consisting of gastric cancer, colorectal cancer, prostate cancer, breast cancer, triple negative breast cancer, ovarian cancer, renal cell carcinoma, ewing's sarcoma, melanoma, mesothelioma, lung cancer, non-small cell lung cancer, stage IV lung cancer, brain cancer, glioblastoma, lymphoma, leukemia, or Multiple Myeloma (MM). In another embodiment, the cancer affects blood and/or bone marrow.
In one aspect, the method further comprises identifying the treatment of the subject by determining the expression of NY-ESO-1 from a cancer sample from the patient. The therapy is then administered to subjects expressing NY-ESO-1.
Any of the methods disclosed herein further comprise monitoring efficacy prior to, concurrent with, and/or after any of the steps described in the method or the method are performed. In one embodiment, the efficacy is assessed and/or quantified as treatment of a disease (e.g., cancer) in a subject, inhibition of cancer cell growth, and/or biomarker change.
In another embodiment, both the efficacy outcome and the biomarker can be determined simultaneously in the cancer or tumor of the subject. In a non-limiting example, rodents bearing human tumor xenografts are used. Human tumor cells (preferably engineered to express a bioluminescent gene, such as luciferase) are transplanted into a rodent (such as a mouse or rat) and visualization is allowed by injection of an activatable fluorescent optical imaging agent in the rodent.
Kit for detecting a substance in a sample
As described herein, the present disclosure provides methods for generating and administering an RNA comprising an ORF encoding NY-ESO-1 or a variant thereof. In a particular aspect, the present disclosure provides kits for performing these methods and instructions for performing the methods of the disclosure, such as administering an effective amount of an mRNA of the present disclosure to a subject.
In one aspect, the kit comprises, or alternatively consists essentially of, or still further consists of, any of the RNA molecules disclosed herein. In another aspect, disclosed herein are kits comprising, or alternatively consisting essentially of, or still further consisting of, the compositions disclosed herein, and optionally instructions for use. Such a kit may also comprise, or alternatively consist essentially of, or still further consist of, a culture medium or other agent suitable for administration of an RNA molecule, such as the agents disclosed herein.
The kits of the present disclosure may further comprise, for example, buffers, preservatives, or protein stabilizers. The kit may also comprise components necessary for detecting the detectable label, such as enzymes or substrates. The kit may also comprise a control sample or a series of control samples that can be analyzed and compared to the test sample. Each component of the kit may be packaged in a separate container, and all of the various containers may be in a single package along with instructions for interpreting the results of the assays performed using the kit. The kits of the present disclosure may comprise written products on or in the kit containers. The written product describes how to use the reagents contained in the kit.
If necessary, these suggested kit components may be packaged in a conventional manner used by those skilled in the art. For example, these suggested kit components may be provided as solutions or as liquid dispersions or the like.
The following examples illustrate methods that may be used in various circumstances to validate the present disclosure.
In some embodiments, the kit comprises instructions for use and one or more of the following: the RNA disclosed herein, the polynucleotide disclosed herein, the vector disclosed herein, the cell disclosed herein or the composition disclosed herein, or alternatively, consists essentially of, or still further consists of, the RNA disclosed herein, the polynucleotide disclosed herein, the vector disclosed herein, the cell disclosed herein or the composition disclosed herein. In further embodiments, the kits are suitable for use in the methods of treatment disclosed herein. In some embodiments, the kit further comprises an anti-cancer therapy.
In some embodiments, the kit comprises instructions for use and one or more of the following: RNA as disclosed herein, polynucleotides as disclosed herein, vectors as disclosed herein, cells as disclosed herein, compositions as disclosed herein, HKP or lipids, optionally a cationic lipid, or alternatively consists essentially of, or still further consists of, a cationic lipid. In further embodiments, the kit is suitable for use in a method of producing an RNA or composition disclosed herein.
In some embodiments, the kit comprises, or alternatively consists essentially of, or still further consists of, instructions for use, a polynucleotide or vector disclosed herein, an RNA polymerase, ATP, CTP, GTP, and UTP or chemically modified UTP. In a further embodiment, the kit is suitable for use in an in vitro method of producing an RNA or composition disclosed herein.
Experimental method
The following examples illustrate methods that may be used in various circumstances to validate the present disclosure.
Example 1: in Vitro Transcription (IVT) assay
In vitro transcription was performed according to SOP of crown-reaching organisms (RNAimmune) in vitro transcription. Plasmids producing NY-ESO-1 mRNA were linearized using XhoI (NEB) at 37℃for 4 hours and the linearized plasmids were purified using a QIAquick PCR purification kit according to the instructions of the manufacturer (Qiagen). After purification, an in vitro transcription reaction was performed in 20. Mu.L reaction, and all components were added in the following order: 1. mu.g of linearized DNA, 1. Mu.L of ATP, CTP, GTP and N1-methyl-pseudouridine triphosphate, 0.8. Mu.L of CleanCap AG (3' Ome), 2. Mu.L of 10 XIVT reaction buffer, 0.5. Mu.L of murine RNase inhibitor, 0.4. Mu.L of E.coli inorganic pyrophosphatase and 3.2. Mu.LT 7 RNA polymerase. The IVT reaction was incubated for 4 hours at 37 ℃. The 10 x transcription buffer components 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 the in vitro transcription reaction, mRNA was purified using RNeasy Mini kit according to the manufacturer's instructions (Qiagen).
The IVT reaction was adjusted to a volume of 100. Mu.L with RNase-free water. The mRNA was then mixed with 350. Mu.L of buffer RLT. Then 250. Mu.L of 100% ethanol was added to the mixture, the mixture was blown and mixed well, and 700. Mu.L of the sample was transferred to an RNeasy Mini spin column (spincolumn) and centrifuged at > 8000 Xg (. Gtoreq.10000 rpm) for 15 seconds. After removal of the flow-through, the spin column was washed twice with 500. Mu.L of buffer RPE. Spin columns were then transferred to 1.5 mL collection tubes (kit supplied) and mRNA was eluted with 30. Mu.L of RNase-free water. If the expected RNA yield is > 30. Mu.g, the elution step is repeated using an additional 30. Mu.L of RNase-free water.
mRNA quality was determined by RNA gel electrophoresis. (FIGS. 1A to 1B) the quality of purified mRNA was evaluated using a 1% agarose Gel northern Gold (1:20,000) containing northern Gold ™ -Gly Gel Prep solution. Running buffer was prepared by diluting a 10 Xstock of northern Ax ™ -Gly Gel Prep into DEPC treated ultrapure water. RNA samples were prepared by adding a volume of RNA (up to 30. Mu.g total RNA) to a volume of northern Ax-Gly sample loading buffer and incubated at 65℃for 30 minutes to prevent the formation of secondary structures. Gel markers were similarly treated. RNA samples and markers were electrophoresed on a 1% agarose gel at 5V/cm. When the bromophenol blue dye front had migrated about the gel length of the microsphere, the electrophoresis was stopped and the mRNA was visualized with blue light.
Example 2: lipid nanoparticle formulations
To prepare a 4 x stock of each component in ethanol, each component was prepared as follows: 100 mg SM-102 was added to 5.63 mL 100% ethanol; 39.5 mg DSPC was added to 10 mL 100% ethanol; adding 74.4 mg cholesterol to 100% ethanol; and 18.8mg of DMG-PEG2000 was added to 100% ethanol. The final concentrations of the 4 Xstock were 25 mM SM-102, 5mM DSPC, 19.5mM cholesterol and 0.75 mM DMG-PEG2000. The lipid components were then added to a 5 mL tube in a 1:1:1:1 ratio to prepare a 1 x working solution. The final working concentrations were 6.25 mM SM-102, 1.25 mM DSPC, 4.815mM cholesterol, and 0.1875 mM DMG-PEG2000. To prepare the mRNA working concentration, the mRNA stock solution was diluted to 25 mM sodium acetate pH 5.0 to a final concentration of 0.13 mg/mL.
The formulation of lipid nanoparticles was performed using NanoAssmblr Ignite (PNI) according to the manufacturer's instructions, with an mRNA: lipid ratio of 3:1. After formulation, the formulated LNP was transferred to a pre-soaked dialysis cartridge and dialyzed in 20mM Tris-HCl, 8% sucrose dialysis buffer at 4 ℃ for at least 18 hours. After dialysis, LNP was removed from the dialysis cartridge using a 3 mL to 5 mL syringe, filter sterilized using an acrodisc filter (0.2 μm pore size) and poured into the top compartment of an Amicon Ultra-15 centrifugal filter tube. The tube was rotated at 2000 Xg at 4℃until the solution was re-concentrated to the desired volume. LNP was transferred to new tubes and characterized by dynamic light scattering using ZetasizerUltra (Malven Panalytical) to measure particle size, polydispersity and zeta potential. Encapsulation efficiency was measured using the Ribogreen assay (Invitrogen). mRNA concentrations were measured using a Ribogreen assay and Nanodrop. The final LNP was stored at-80℃for future use.
Example 3: western blot analysis
To select the NY-ESO-1 vaccine candidates, the in vitro expression levels of the vaccine candidates were examined. (FIG. 2) 293T cells were transfected with various forms of NY-ESO-1 mRNA using Lipofectamin MessengerMax transfection reagents. Forty-eight hours after transfection, cells were collected, cell lysates were prepared, and BCA assays were used to determine protein concentration. Using Western blot analysis, the expression levels of each construct were compared with anti-NY-ESO-1 antibodies (anti-CTAG 1B, abcam).
To test the quality of the formulated NY-ESO-1 vaccine, various amounts of mRNA-LNP (0.5. Mu.g, 1. Mu.g, and 2.5. Mu.g) were transfected into 293T cells. (FIG. 4) forty-eight hours after transfection, cells were collected, cell lysates were prepared, and protein concentrations were determined using the BCA assay. Using Western blot analysis, the expression levels of each construct were compared with anti-NY-ESO-1 antibodies (anti-CTAG 1B, abcam).
Example 4: ELISA assay
Balb/c female mice were immunized with 1. Mu.g, 5. Mu.g, and 10. Mu.g of NY-ESO-1 vaccine at weeks 0 and 3. (FIG. 3) serum was collected 2 weeks after immunization. The ratio of total IgG to IgG2a/IgG1 was measured. Briefly, 96-well plates were coated with 0.1. Mu.g of NY-ESO-1 protein (RayBiotech) per well and incubated overnight at 4 ℃. To measure total IgG, each serum was diluted 3-fold to 1:437400 dilution starting at 1:200 in 1 x ELISA dilution buffer (Biolegn) and incubated for 2 hours at room temperature. Subsequently, 100 μl of horseradish peroxidase (HRP) -conjugated goat anti-mouse IgG (Jackson ImmunoResearch) diluted 1:5000 was added, followed by incubation for 1 hour at room temperature. The chromogenic reaction was allowed to proceed with TMB substrate (Biolgend) for 15 minutes at room temperature and then stopped with 1 NHCl. Absorbance was detected at 450 nm. (FIGS. 5A to 5B).
To measure the IgG1 and IgG2a subtypes, each serum of immunization 1 was diluted 1:200 in a 1 x ELISA dilution buffer, while the serum of immunization 2 was diluted 3-fold to 1:48600 starting at 1:1800. 100. Mu.L of diluted serum was added to NY-ESO-1 coated 96-well plates and incubated for 2 hours at room temperature. After 2 hours of incubation, 100 μl of horseradish peroxidase (HRP) -conjugated goat anti-mouse IgG1 and IgG2a subtype (Abcam) diluted 1:5000 were added, followed by incubation for 1 hour at room temperature. The chromogenic reaction was allowed to proceed with TMB substrate (Biolgend) for 15 minutes at room temperature and then quenched with 1N HCl. Absorbance was detected at 450 nm. The IgG2a/IgG1 ratio was calculated based on the o.d. readings. (FIGS. 7A to 7B and FIGS. 8A to 8C).
For IgG quantification, serum collected 35 days after the second immunization (fig. 6A to 6B) was used to quantify the amount of IgG. anti-CTAG 1B antibody (Abcam) was used as a standard and diluted 3-fold at 200 ng/. Mu.L as initial concentration. The absorbance of the standard was measured at 450 nm according to the determination method of total IgG. Standard curves were plotted using sigmoid, 4PL, X functions in GraphPad. The amount of IgG per immunization dose was calculated by means of an interpolated standard curve.
Example 5: ELISA spot assay
Eight weeks after the last boost, the spleens of the mice were collected, mechanically dissociated into single cell suspensions, passed through a 70- μm cell filter (Miltenyi Biotec), and lysed by ACK lysis buffer (KD Medical). ThenCells were resuspended in RPMI supplemented with 10% FBS, 1 XPen/Strep and 1X 2-mercaptoethanol, counted and adjusted to 2X 10 6 Individual cells/mL. Thereafter, 100. Mu.L of spleen cells were added to the wells of IFN-. Gamma.coated 96-well enzyme-linked immunosorbent spot plate (Mabtech). Spleen cells were then incubated in a cell incubator for 16 hours with or without stimulation using a full set of overlapping peptide libraries of NY-ESO-1 proteins or single NY-ESO-1 157-165 peptide (iba) prepared according to the manufacturer's instructions (JPT peptides). After incubation, biotinylated anti-IFN-. Gamma.R 4-A2-biotin monoclonal antibody was used as detection Ab according to the manufacturer's instructions; and using streptavidin-ALP complex and BCIP/NBT-plus substrate (Mabtech) to reveal the presence of spots. Spots formed by IFN-gamma secreting cells were counted using Cystation 7 (Biotek) and the results were expressed as every 2X 10 5 Spot forming cells of individual spleen cells. (FIGS. 9 and 10).
Example 6: intracellular staining (ICS) assay
Induction of antigen-specific T cells was determined using ICS. Spleen cells were prepared as described above. In the presence of protein transport inhibitors (brefeldin A, bioLegend) 1X 10 in the presence of stimulation with or without a full set of overlapping peptide libraries of NY-ESO-1 proteins prepared according to the manufacturer's instructions (JPT peptides) or a single NY-ESO-1 157-165 peptide (iba) 6 Individual spleen cells were added to 12 x 75 mm plastic tubes. After 16 hours incubation, FACS analysis was performed to determine cytokine spleen cells. Cell surface staining was performed using antibodies against CD3 (FITC), CD4 (BV 421) and CD8 (BV 650). Intracellular staining was performed using antibodies against IL-4 (APC) and IFN-gamma (PE). eFluor450 was used to differentiate between live and dead cells (Invitrogen). Cells were collected using a BD Celesta flow cytometer (BD Biosciences) and flow cytometry data was analyzed using FlowJo software. (FIGS. 11A to 11D).
Sequence listing
SEQ ID NO: 1(WT NY-ESO-1 RNA)
AUGCAGGCCGAAGGCCGGGGCACAGGGGGUUCGACGGGCGAUGCUGAUGGCCCAGGAGGCCCUGGCAUUCCUGAUGGCCCAGGGGGCAAUGCUGGCGGCCCAGGAGAGGCGGGUGCCACGGGCGGCAGAGGUCCCCGGGGCGCAGGGGCAGCAAGGGCCUCGGGGCCGGGAGGAGGCGCCCCGCGGGGUCCGCAUGGCGGCGCGGCUUCAGGGCUGAAUGGAUGCUGCAGAUGCGGGGCCAGGGGGCCGGAGAGCCGCCUGCUUGAGUUCUACCUCGCCAUGCCUUUCGCGACACCCAUGGAAGCAGAGCUGGCCCGCAGGAGCCUGGCCCAGGAUGCCCCACCGCUUCCCGUGCCAGGGGUGCUUCUGAAGGAGUUCACUGUGUCCGGCAACAUACUGACUAUCCGACUGACUGCUGCAGACCACCGCCAACUGCAGCUCUCCAUCAGCUCCUGUCUCCAGCAGCUUUCCCUGUUGAUGUGGAUCACGCAGUGCUUUCUGCCCGUGUUUUUGGCUCAGCCUCCCUCAGGGCAGAGGCGCUAA
SEQ ID NO. 2 (WT NY-ESO-1 peptide)
MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR
SEQ ID NO. 3 (codon optimized NY-ESO-1 RNA)
AUGGCACAGGCCGAGGGCAGAGGCACCGGCGGCAGCACCGGCGACGCCGAUGGCCCUGGAGGCCCUGGCAUCCCCGACGGCCCUGGCGGCAACGCCGGAGGCCCCGGCGAGGCCGGCGCUACAGGAGGAAGAGGCCCCAGAGGCGCCGGCGCCGCCCGGGCCAGCGGCCCAGGCGGCGGCGCCCCUAGAGGUCCCCACGGCGGAGCCGCUUCCGGCCUGAACGGCUGCUGCAGGUGUGGCGCUAGAGGACCUGAGAGCAGACUGCUGGAAUUCUACCUGGCCAUGCCUUUCGCCACACCUAUGGAAGCCGAGCUGGCUAGACGGAGCCUGGCCCAGGACGCCCCUCCACUGCCUGUCCCCGGAGUGCUGCUGAAGGAAUUUACCGUGUCUGGCAAUAUCCUGACCAUCCGCCUGACAGCCGCUGAUCACCGGCAGCUGCAGCUCUCCAUCAGCAGCUGCCUGCAGCAGCUGUCUCUGCUGAUGUGGAUCACCCAGUGCUUCCUGCCCGUGUUCCUGGCCCAACCUCCUAGCGGCCAGCGGAGAAUAAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAA
SEQ ID NO. 4 (codon optimized NY-ESO-1 peptide)
MAQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRRIKLAFLLSNFY
SEQ ID NO. 5 (WT NY-ESO-1 with COVID Signal RNA)
ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTCAGCAGCCAGCAGGCCGAAGGCCGGGGCACAGGGGGTTCGACGGGCGATGCTGATGGCCCAGGAGGCCCTGGCATTCCTGATGGCCCAGGGGGCAATGCTGGCGGCCCAGGAGAGGCGGGTGCCACGGGCGGCAGAGGTCCCCGGGGCGCAGGGGCAGCAAGGGCCTCGGGGCCGGGAGGAGGCGCCCCGCGGGGTCCGCATGGCGGCGCGGCTTCAGGGCTGAATGGATGCTGCAGATGCGGGGCCAGGGGGCCGGAGAGCCGCCTGCTTGAGTTCTACCTCGCCATGCCTTTCGCGACACCCATGGAAGCAGAGCTGGCCCGCAGGAGCCTGGCCCAGGATGCCCCACCGCTTCCCGTGCCAGGGGTGCTTCTGAAGGAGTTCACTGTGTCCGGCAACATACTGACTATCCGACTGACTGCTGCAGACCACCGCCAACTGCAGCTCTCCATCAGCTCCTGTCTCCAGCAGCTTTCCCTGTTGATGTGGATCACGCAGTGCTTTCTGCCCGTGTTTTTGGCTCAGCCTCCCTCAGGGCAGAGGCGCTAA
SEQ ID NO. 6 (WT NY-ESO-1 with a COVID Signal peptide)
MFVFLVLLPLVSSQQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR
SEQ ID NO. 7 (codon optimized NY-ESO-1 with COVID Signal RNA)
AUGGCAUUCGUGUUUCUGGUCCUGCUGCCUCUGGUGUCCAGCCAGCAGGCCGAGGGCAGAGGUACAGGCGGCAGCACCGGCGAUGCCGAUGGCCCCGGAGGCCCCGGCAUCCCCGACGGCCCAGGCGGCAACGCCGGCGGCCCUGGAGAGGCCGGAGCCACCGGCGGAAGAGGCCCAAGAGGAGCCGGCGCCGCUCGGGCCUCUGGCCCUGGAGGCGGAGCUCCUAGAGGCCCCCACGGCGGCGCUGCUUCUGGCCUGAACGGCUGCUGCAGAUGCGGCGCCAGGGGCCCUGAGAGCAGACUGCUGGAGUUCUACCUGGCCAUGCCUUUCGCCACACCUAUGGAAGCCGAACUGGCCCGCCGGAGCCUGGCUCAGGACGCCCCUCCUCUGCCUGUGCCCGGCGUGCUGCUGAAGGAAUUCACCGUGUCUGGCAAUAUCCUGACCAUCCGGCUGACAGCCGCCGACCACAGACAGCUGCAGCUGAGCAUCAGCAGCUGCCUGCAACAGCUUUCCCUGCUGAUGUGGAUCACCCAGUGUUUUCUGCCCGUGUUCCUGGCCCAACCUCCAAGCGGCCAGCGGAGAAUAAAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAA
SEQ ID NO. 8 (codon optimized NY-ESO-1 with a COVID Signal peptide)
MAFVFLVLLPLVSSQQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRRIKLAFLLSNFY
SEQ ID NO: 9(3' UTR)
AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAAGCGAUCGCAAGCUUCUCGAGC
SEQ ID NO: 10(5' UTR)
UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACC
SEQ ID NO. 11 (pUC 57 plasmid)
/>
SEQ ID NO. 12 (PolyA tail)
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO. 13 (PolyA tail)
CGGCAAUAAAAAGACAGAAUAAAACGCACGGUGUUGGGUCGUUUGUUC
SEQ ID NO. 14 (HK peptide branched chain)
KHKHHKHHKHHKHHKHHKHK
SEQ ID NO. 15 (HK peptide branched chain)
KHHHKHHHKHHHKHHHK
SEQ ID NO. 16 (HK peptide branched chain)
KHHHKHHHKHHHHKHHHK
SEQ ID NO. 17 (HK peptide branched chain)
kHHHkHHHkHHHHkHHHk
SEQ ID NO. 18 (HK peptide branched chain)
HKHHHKHHHKHHHHKHHHK
SEQ ID NO. 19 (HK peptide branched chain)
HHKHHHKHHHKHHHHKHHHK
SEQ ID NO. 20 (HK peptide branched chain)
KHHHHKHHHHKHHHHKHHHHK
SEQ ID NO. 21 (HK peptide branched chain)
KHHHKHHHKHHHKHHHHK
SEQ ID NO. 22 (HK peptide branched chain)
KHHHKHHHHKHHHKHHHK
SEQ ID NO. 23 (HK peptide branched chain)
KHHHKHHHHKHHHKHHHHK。
Reference to the literature
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Claims (66)
1. An isolated deoxyribonucleic acid (DNA) comprising an Open Reading Frame (ORF) encoding at least one of:
1) Ribonucleic acid (RNA) sequences as shown in SEQ ID NO. 1 or peptides as shown in SEQ ID NO. 2;
2) Ribonucleic acid (RNA) sequences as shown in SEQ ID NO. 3 or peptides as shown in SEQ ID NO. 4;
3) A ribonucleic acid (RNA) sequence as shown in SEQ ID NO. 5 or a peptide as shown in SEQ ID NO. 6; or (b)
4) Ribonucleic acid (RNA) sequences as shown in SEQ ID No. 7 or peptides as shown in SEQ ID No. 8.
2. An isolated ribonucleic acid (RNA) comprising at least one of:
1) A ribonucleic acid (RNA) sequence as shown in SEQ ID NO. 1;
2) A ribonucleic acid (RNA) sequence as shown in SEQ ID NO. 3;
3) A ribonucleic acid (RNA) sequence as shown in SEQ ID NO. 5; or (b)
4) A ribonucleic acid (RNA) sequence as shown in SEQ ID NO. 7.
3. The DNA of claim 1, further encoding one or more of the following: 3'-UTR, 5' -UTR, cap structure and poly A tail (polyA tail).
4. A DNA according to claim 3, wherein the 5'-UTR comprises a cap structure and an initiation codon, optionally wherein the 5' -UTR encodes UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACC (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 AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAAGCGAUCGCAAGCUUCUCGAGC (SEQ ID NO: 9) or an equivalent thereof.
6. The RNA of claim 2, further comprising one or more of the following: 3'-UTR, 5' -UTR, cap structure and poly A tail (polyA tail).
7. The RNA of claim 6, wherein the 5'-UTR comprises a cap structure and an initiation codon, optionally wherein the 5' -UTR comprises UAAUACGACUCACUAUAAGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACC (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 AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCCAAUAGGCCGAAAUCGGCAAGACGCGUAAAGCGAUCGCAAGCUUCUCGAGC (SEQ ID NO: 9) or an equivalent thereof.
9. The RNA according to any one of claims 2 or 6 to 8, which is prepared by transcription of a polynucleotide encoding the RNA in an In Vitro Transcription (IVT) system.
10. The RNA according to any one of claims 2 or 6 to 9, which is prepared by transcription of a plasmid DNA (pDNA) vector encoding the RNA, optionally the vector is a pUC57 plasmid, optionally wherein the plasmid comprises SEQ ID No. 11 or an equivalent thereof.
11. The RNA according to any one of claims 2 or 6 to 10, wherein the GC content of the full-length RNA is from about 35% to about 70% of the total RNA content.
12. The RNA according to any one of claims 2 or 6 to 11, wherein the RNA is chemically modified, optionally wherein the chemical modification comprises one or both of incorporation of an N1-methyl-pseudouridine residue or incorporation of a pseudouridine residue, further optionally wherein at least about 50% to about 100% of 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, the polynucleotide encoding the RNA of any one of claims 2 or 6 to 12, or a polynucleotide complementary to the polynucleotide, or any combination thereof.
14. The polynucleotide of claim 13, wherein the polynucleotide is selected from the group consisting of: deoxyribonucleic acid (DNA), RNA, hybrids of DNA and RNA, or analogs of each of them.
15. A vector comprising the polynucleotide of claim 13 or 14.
16. The vector of claim 15, further comprising a regulatory sequence operably linked to the polynucleotide to direct its transcription.
17. The vector of claim 16, wherein the regulatory sequence comprises a promoter.
18. The vector of claim 17, wherein the promoter comprises: phage RNA polymerase promoter, T7 promoter, SP6 promoter or 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 polyA tail is SEQ ID No. 12, and the cap structure is a 7-methylguanosine (7 mG) cap.
21. The vector according to any one of claims 15 to 18, wherein the vector is a non-viral vector, optionally a plasmid, or a liposome or 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 according to any one of claims 15 to 18, wherein the vector is a viral vector, optionally an adenovirus vector, or an adeno-associated viral vector, or a retrovirus vector, or a lentivirus 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 mammalian cell, an insect cell, or a yeast cell.
26. A composition, the composition comprising: a carrier, optionally a pharmaceutically acceptable carrier, and one or more of the following: 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 RNA, the method comprising culturing the cell of any one of claims 24 or 25 under conditions suitable for transcription of DNA encoding the RNA into the RNA.
28. A method of producing RNA, the method comprising contacting the polynucleotide or the vector of any one of claims 13 or 14 with an RNA polymerase, adenosine Triphosphate (ATP), cytidine Triphosphate (CTP), guanosine 5' -triphosphate (GTP) and Uridine Triphosphate (UTP) or chemically modified UTP under conditions suitable for transcribing the polynucleotide or the vector of any one of claims 15 to 18 or 21 to 23 into 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 DNA according to any one of claims 1 or 3 to 5 or RNA according to any one of claims 2 or 6 to 12 formulated in a pharmaceutically acceptable carrier.
32. The composition of 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 copolymer (HKP).
34. The composition of claim 33, wherein the HKP comprises 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 (MC 3) 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 the following: helper lipids, cholesterol or pegylated lipids.
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 34-39, wherein the HKP and mRNA self-assemble into nanoparticles upon mixing.
41. The composition of any one of claims 33 to 40, wherein the liposomal nanoparticle carrier comprises spermine-lipid cholesterol (SLiC).
42. The composition of claim 41, wherein said SLIC is selected from the group consisting of TM1-TM5.
43. The composition of claim 31, wherein the pharmaceutically 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: 8- { (2-hydroxyethyl) [ 6-oxo-6- (undecyloxy) hexyl]Amino } 9-heptadecyl octanoate (SM-102), 2-diiodo-4-dimethylaminoethyl- [1,3 ] ]Dioxolane (DLin-KC 2-DMA), diiodo-methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA), bis (-)Z) -non-2-en-1-yl) 9- ((4- (dimethylamino) butyryl) oxy) heptadecanedioate (L319) or an equivalent of each of them.
45. The composition of claim 44, wherein the LNP further comprises one or more of the following: helper lipids, cholesterol or pegylated lipids.
46. The composition of claim 38 or 45, wherein the helper lipid comprises one or more of: distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), (2)R) -3- (hexadecanoyloxy) -2- { [ (9)Z) -octadec-9-enoyl]Oxy } propyl 2- (trimethylammonium) ethyl phosphate (POPC) or dioleoyl phosphatidylethanolamine (DOPE).
47. The composition of any one of claims 38, 45 or 46, wherein the cholesterol comprises plant cholesterol or animal cholesterol or both.
48. The composition of any one of claims 38 or 45-47, wherein the pegylated lipid comprises one or more of: PEG-c-DOMG (R-3- [ (-) ω-methoxy-poly (ethylene glycol) 2000) carbamoyl]-1, 2-dimyristoxypropyl-3-amine), PEG-DSG (1, 2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1, 2-dimyristoyl-sn-glycerol), optionally PEG2000-DMG ((1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000 ]]Or PEG-DPG (1, 2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
49. The composition of any one of claims 43-48, wherein said LNP comprises SM-102, DSPC, cholesterol, and PEG2000-DMG.
50. The composition of claim 49, wherein the mass ratio of SM-102, DSPC, cholesterol, and PEG200-DMG is about 1:1:1:1, and/or wherein the molar ratio of SM-102, DSPC, cholesterol, and PEG2000-DMG is about 50:10:38.5:1.5.
51. A method of producing a composition according to any one of claims 31 to 40 or 46 to 49, the method comprising contacting the DNA according to any one of claims 1 or 3 to 5 or the RNA according to any one of claims 2 or 6 to 12 with HKP, thereby self-assembling the DNA or RNA and the HKP 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 the DNA or RNA with a cationic lipid, optionally wherein the cationic lipid comprises Dlin-MC3-DMA (MC 3) or DOTAP (dioleoyloxy-3- (trimethylammonium) propane) or both.
54. The method of claim 53, wherein the mass ratio of the cationic lipid to 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 HKP, the DNA or RNA, and the cationic lipid in the contacting step is about 4:1:1.
56. A method of producing a composition according to any one of claims 31 or 43 to 48, the method comprising contacting the DNA according to any one of claims 1 or 3 to 5 or the RNA according to any one of claims 2 or 6 to 12 with a lipid, thereby causing self-assembly of the DNA or RNA and the lipid into a Lipid Nanoparticle (LNP).
57. The method of claim 56, wherein the LNP comprises one or more of the following: 8- { (2-hydroxyethyl) [ 6-oxo-6- (undecyloxy) hexyl]Amino } 9-heptadecyl octanoate (SM-102), 2-diiodo-4-dimethylaminoethyl- [1,3 ]]Dioxolane (DLin-KC 2-DMA), diiodo-methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA), bis (-)Z) -non-2-en-1-yl) 9- ((4- (dimethylamino) butyryl) oxy) heptadecanedioate (L319) or an equivalent of each of them.
58. The method of claim 57 wherein the LNP further comprises one or more of: a helper lipid, cholesterol, or pegylated lipid, optionally wherein the helper lipid comprises one or more of: distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), (2)R) -3- (hexadecanoyloxy) -2- { [ (9)Z) -octadec-9-enoyl]Oxy } propyl 2- (trimethylammonium) ethyl phosphate (POPC)Or dioleoyl phosphatidylethanolamine (DOPE), optionally wherein the cholesterol comprises plant cholesterol or animal cholesterol or both, and optionally wherein the pegylated lipid comprises one or more of: PEG-c-DOMG (R-3- [ (-) ω-methoxy-poly (ethylene glycol) 2000) carbamoyl]-1, 2-dimyristoxypropyl-3-amine), PEG-DSG (1, 2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1, 2-dimyristoyl-sn-glycerol), optionally PEG2000-DMG ((1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000 ]]Or PEG-DPG (1, 2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
59. The method of any one of claims 56-58, wherein said LNP comprises SM-102, DSPC, cholesterol, and PEG2000-DMG, optionally wherein the mass ratio of SM-102, DSPC, cholesterol, and PEG200-DMG is about 1:1:1:1, or optionally wherein the molar ratio of 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 an interlaced fish bone micromixer (SHM).
61. Use of a DNA according to any one of claims 1 or 3 to 5, an RNA according to any one of claims 2 or 6 to 12, a polynucleotide according to claim 13 or 14, a vector according to any one of claims 15 to 18 or 21 to 23, a cell according to any one of claims 24 or 25 or a composition according to any one of claims 26 or 31 to 39 in the manufacture of a medicament for treating cancer in a subject.
62. The use of claim 61, wherein the medicament is formulated for intratumoral administration, or intravenous administration, or intramuscular administration, or intradermal administration or subcutaneous administration.
63. The use of claim 61 or 62, wherein the subject is a mammal or a human.
64. The use of any one of claims 61-63, wherein the cancer comprises a 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 cancer.
65. The use of any one of claims 61 to 64, the medicament being formulated for administration with an additional anti-cancer therapy.
66. A kit comprising instructions for use and one or more of the following: 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 of any one of claims 26 or 31 to 50, or an anti-cancer therapy.
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