VACCINE, NUCLEIC ACID MOLECULE, AND AMINO ACID MOLECULE

专利摘要:
vaccine, nucleic acid molecule, and, method of preventing or treating cancer, vaccine compositions comprising one or more nucleic acids encoding one or more amino acid sequence(s) selected from the group consisting of tyrosinase (tyr) amino acid sequences are described , tyrosinase-related protein 1 (tyrp1), tyrosinase-related protein 2 (tyr2), melanoma-associated antigen protein 4 (magea4), growth hormone releasing hormone (ghrh), melan-a/mart- antigen 1, testis cancer antigen ny-eso-1, testis cancer antigen ny-eso-2, preferably antigen expressed in melanoma (prame), wilms tumor 1 (wt1) and telo-reverse transcriptase human merase (htert). vaccine compositions can further comprise a nucleic acid encoding one or more other antigens including prostate specific antigen (psa). Further described are methods of preventing or treating cancer in a subject in need comprising administering to the subject a vaccine composition comprising a particular number of cancer antigens to treat or prevent a particular cancer.
公开号:BR112015022367B1
申请号:R112015022367-2
申请日:2014-03-14
公开日:2021-06-22
发明作者:David Weiner；Jian Yan；Karuppiah Muthumani；Jewell Walters
申请人:The Trustees Of The University Of Pennsylvania；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[001] This application claims priority to U.S. Provisional Patent Application No. 61/799,952, filed March 15, 2013, which is incorporated herein by reference. TECHNICAL FIELD
[002] Disclosed in this document are compositions and methods for treating cancer and, in particular, vaccines that treat and offer protection against tumor growth. FUNDAMENTALS
[003] Cancer is one of the leading causes of death in the world, and in the United States, it is the second most common cause of death, accounting for about 1 in 4 deaths. Cancer arises from a single cell that has changed from a normal cell into a tumor cell. This transformation is often a multistep process, progressing from a precancerous lesion to malignant tumors. Several factors contribute to this progression, including aging, genetic contributions, and exposure to external agents such as physical carcinogens (eg, ultraviolet and ionizing radiation), chemical carcinogens (eg, asbestos, components of tobacco smoke, etc. .) and biological (eg certain viruses, bacteria and parasites).
[004] Prevention, diagnosis and treatment of cancer can take many different forms. Prevention may include screening for predisposing factors (eg, specific genetic variants), behavioral change (eg, smoking, diet and amount of physical activity), and virus vaccination (eg, human papilloma virus, hepatitis B virus). Treatment may include chemotherapy, radiation therapy, and surgical removal of a tumor or cancerous tissue. Despite the availability of various prevention and treatment methods, such methods often have limited success in effectively preventing and/or rapidly treating cancer.
[005] Therefore, there is a need to identify and develop compositions and methods for the prevention and/or treatment of cancer to facilitate clinical management of disease protection and progression. In addition, more effective treatments are needed to slow disease progression and/or decrease mortality in cancer patients. SUMMARY OF THE INVENTION
[006] The present invention is directed to a vaccine comprising one or more nucleic acid or amino acid sequences of cancer antigens that are no longer self-antigens and stimulate an immune response to a particular cancer or tumor associated with a particular cancer. The vaccine may additionally comprise immune checkpoint inhibitors such as anti-PD-1 anti-PDL-1 antibodies that prevent suppression of any component in the immune system, such as MHC presentation class, T cell presentation and/or differentiation, presentation and/or differentiation of B cells, any cytokine, chemokine or signaling for proliferation and/or differentiation of immune cells. The one or more cancer antigens of the vaccine may be a nucleic acid encoding or more amino acid sequence(s) or amino acid sequence that is selected from the group consisting of: amino acid sequence that is 95% identical to or greater than the sequence tyrosinase amino acid (Tyr); tyrosinase-related protein 1 amino acid sequence (TYRP1); amino acid sequence that is 95% identical to or greater than the amino acid sequence of tyrosinase-related protein 2 (TYRP2); amino acid sequence that is 95% identical to or greater than the melanoma-associated antigen protein 4 amino acid sequence (MAGEA4); amino acid sequence that is 95% identical to or greater than the growth hormone-releasing hormone (GHRH) amino acid sequence; amino acid sequence that is 95% identical to or greater than the amino acid sequence of the MART-1/melan-A antigen (MART-1/Melan-A); amino acid sequence that is 95% identical to or greater than the testicular cancer antigen amino acid sequence (NY-ESO-1); amino acid sequence that is 95% identical to or greater than the testicular cancer antigen II amino acid sequence (NY-ESO-2); amino acid sequence that is 95% identical to or longer than the PRAME amino acid sequence; amino acid sequence that is 95% identical to or longer than the WT1 amino acid sequence; amino acid sequence that is 95% identical to or longer than the hTERT amino acid sequence; or a combination of them. The vaccine may further comprise a nucleic acid encoding one or more antigens selected from the group consisting of: PSA, PSMA, STEAP, PSCA, MAGE A1, gp100, a viral antigen, and combinations thereof.
[007] The present invention is further directed to a method for the prevention or treatment of cancer in a patient in need thereof, the method comprising administering to a subject in need of a vaccine comprising a number of cancer antigens for treatment or prevention of a particular cancer. The method may include administering to a patient in need of a vaccine comprising a CMV cancer antigen to treat or prevent glioblastoma, or administering to a patient in need of a vaccine comprising a CMV cancer antigen in combination with one or more of the cancer antigens, hTERT, NY-ESO-1, MAGE-A1 or WT1 to treat or prevent glioblastoma; administering to a patient in need of a vaccine comprising one or more PSA, PSMA or STEAP cancer antigens to treat or prevent prostate cancer, or administering to a subject in need thereof a vaccine comprising PSA, PSMA or STEAP in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1 or WT1 to treat or prevent prostate cancer; administration to a patient in need of a vaccine composed of one or more cancer antigens, tyrosinase, PRAME or GP-100 to treat or prevent melanoma, administration to a patient in need of a vaccine comprising tyrosinase, PRAME or GP- 100 in combination with one or more of the cancer antigens, hTERT, NY-ESO-1, MAGE-A1 or WT1 to treat or prevent melanoma; administration to a patient in need of a vaccine comprising one or more HPV 16 E6 or HPV 16 E7 cancer antigens to treat or prevent head and neck cancer, administration to a patient in need of a vaccine composed of HPV 16 E6 or HPV 16 E7 in combination with any one or more of the cancer antigens, hTERT, NY-ESO-1, MAGE-A1 or WT1 to treat or prevent head and neck cancer; administering to a patient in need of a vaccine comprising one or more tyrosinase, PRAME or GP-100 cancer antigens to treat or prevent melanoma, or administering to a patient in need of a vaccine comprising tyrosinase, PRAME or GP-100 in combination with any one or more of the hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens to treat or prevent melanoma; administering to a patient in need of a vaccine comprising one or more HPV6, HPV11 or HPV 16 cancer antigens to treat or prevent anal cancer, or administering to a patient in need of a vaccine comprising HPV6, HPV 11 or HPV 16 in one combination with any of the hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens to treat or prevent anal cancer; administration to a patient in need of a vaccine comprising one or more of the central HBV cancer antigens, superficial HBV antigens, HCV NS34A, HCV NS5A, HCV NS5B or HCV NS4B in combination with one or more of the cancer antigens hTERT, NY-ESO -1, MAGE-A1 or WT1 to treat or prevent liver cancer; or administering to a patient in need of a vaccine comprising core HBV, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B or HCV NS4B in combination with any or more of a hTERT, NY-ESO-1, MAGE- A1 or WT1 to treat or prevent liver cancer; administering to a patient in need of a vaccine comprising one or more HPV 16 E6/E7 or HPV 18 E6/E7 cancer antigens to treat or prevent cervical cancer, or administering to a patient in need of a vaccine comprising HPV 16 E6/E7 or HPV 18 E6/E7 in combination with any one or more of the hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens to treat or prevent cervical cancer; or administering to a patient in need of a vaccine comprising one or more of the PRAME, WT-1 or hTERT cancer antigens to treat or prevent blood cancers, or administering to a patient in need of a vaccine comprising PRAME, WT- 1 or hTERT in combination with any one or more of the NY-ESO-1 or MAGE-A1 cancer antigens to treat or prevent blood cancers, where the method may further comprise the combination of steps of (a)-(i ) with a checkpoint inhibitor selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody and a combination thereof. BRIEF DESCRIPTION OF THE FIGURES
[008] Figures 1A-E show the construction of pTyr.
[009] Figures 2A and 2B show an immunization strategy and induction of cellular immune responses mediated by Tyr DNA vaccination, respectively.
[0010] Figure 3 shows the flux of fluorescence-activated cell sorting (FACS) from controlled and immunized mice.
[0011] Figures 4A and 4B show the induction of tyrosinase-specific antibodies in immunized mice.
[0012] Figures 5A and 5B show Kaplan-Meier survival curves and tumor volume curves, respectively, after tumor challenge in controlled and immunized mice.
[0013] Figures 6A and 6B show MDSC cell populations in immunized and unimmunized mice.
[0014] Figure 7 shows staining for MDSCs in mice immunized with pVax1 and pTyr.
[0015] Figures 8A and 8B show secretion of MCP-1 by MDSCs.
[0016] Figure 9 shows the phylogenetic relationship of Tyr nucleotide sequences among the indicated organisms.
[0017] Figure 10 shows (A) a schematic illustrating a plasmid map of pPRAME (also known herein as pGX1411); (B) staining of RD and 293T cells for DAPI nuclei and for the PRAME antigen consensus; and (C), and Western blot technique for consensus PRAME antigen in lysates from non-transfected cells ("control"), cells transfected with pVAX ("pVAX"), and cells transfected with pPRAME ("PRAME-pVAX") .
[0018] Figure 11 shows in (A) and (B) graphs representing mouse group vs. spot forming unit (SFU) / 106 splenocytes for interferon gamma (IFN-y).
[0019] Figure 12 shows (A) a schematic illustrating a plasmid map of pNY-ESO-1 (also known herein as pGX1409); (B) cell staining for nuclei with DAPI and for the consensus NY-ESO-1 antigen; and (C), and Western blot technique for consensus NY-ESO-1 antigen in RD and 293T lysates from non-transfected cells ("control"), pVAX-transfected cells ("pVAX"), and cells transfected with pNY-ESO-1 ("pNY-ESO-1").
[0020] Figure 13 shows a graph representing the group of mice vs. spot-forming units (SFU) / 106 splenocytes for interferon gamma (IFN-y).
[0021] Figure 14 shows a graph representing the group of mice vs. spot-forming units (SFU)/106 splenocytes for interferon gamma (IFN-Y).
[0022] Figure 15 shows a schematic diagram illustrating various cancers with some of their associated cancer antigens. DETAILED DESCRIPTION
[0023] The present invention is directed to a vaccine that can be customized for certain cancers and tumors. Antigen consensus sequences have been designed for certain cancer-related antigens, such as tyrosinase (Tyr), melanoma preferentially expressed antigen (PRAME), tyrosinase-related protein 1 (Tyrp1), testicular cancer antigen (NY-ESO- 1), hepatitis B virus antigen and Wilms' tumor antigen 1 (WT-1) to be used in the vaccine to enable personalized prevention of and treatment of certain cancers. For example, the tyrosinase antigen can be used in a vaccine for the prevention or treatment of melanomas. The vaccine of the invention can provide any combination of cancer antigens specific to a particular cancer prevention or treatment of a patient in need of treatment.
One way of designing the nucleic acid and its encoded amino acid sequence of the recombinant cancer antigen is by introducing mutations that alter certain amino acids in the overall amino acid sequence of the native cancer antigen. The introduction of mutations does not alter the cancer antigen to the point that it cannot be universally applied to a mammal, preferably a human subject or a dog, but alters it sufficiently so that the resulting amino acid sequence breaks tolerance or is considered an antigen. foreign in order to generate an immune response. Another way might be to create a recombinant consensus cancer antigen that has at least 85% and up to 99% amino acid sequence identity to its corresponding native cancer antigen; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In some cases, the recombinant cancer antigen is 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. Native cancer antigen is the antigen normally associated with a particular cancer or tumor. Depending on the cancer antigen, the cancer antigen consensus sequence may be about mammalian species or within subtypes of a species, or about viral strains or serotypes. Some cancer antigens do not vary much from the wild-type amino acid sequence of the cancer antigen. Some cancer antigens have nucleic acid/amino acid sequences so divergent across species that a consensus sequence cannot be generated. In these cases, a recombinant cancer antigen that will break tolerance and generate an immune response is generated having at least 85% and up to 99% amino acid sequence identity to its corresponding native cancer antigen; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In some cases, the recombinant cancer antigen is 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. The above mentioned approaches can be combined so that the final recombinant cancer antigen has a percent similarity with the native cancer antigen amino acid sequence as discussed above.
The recombinant cancer antigen can induce antigen-specific T-cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (IFN--Y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule.
The vaccine can be further combined with antibodies to checkpoint inhibitors such as DP-1 and PDL-1 to enhance stimulation of both cellular and humoral immune responses. Using anti-PDL-1 or anti-DP-1 antibodies prevents DP-1 or PDL-1 from suppressing T-cell and/or B-cell responses. In general, designing cancer antigens to be recognized by the immune system helps to overcome other forms of immune suppression by tumor cells, and these vaccines can be used in combination with suppression or inhibition therapies (such as anti-DP antibody therapies). -1 and anti-PDL-1) to further enhance T-cell and/or B-cell responses. 1. Definitions
[0027] Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, this document, including definitions, will prevail. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention. All publications, patent applications, patents, and other references mentioned in this document are incorporated by reference in their entirety. The materials, methods and examples disclosed in this document are illustrative only and are not intended to be a limiting factor. The terminology used in this document is for the purpose of describing specific modalities only and is not intended to be limiting.
[0028] The terms "comprises", "includes", "have", "has", "may", "contains", and variants thereof, as used in this document, are intended themselves to be open-ended transitional sentences, terms or words that do not exclude the possibility of additional acts or structures. The singular forms "an", "an" and "a/o" include plural references, unless the context clearly dictates otherwise. The present disclosure also contemplates other modalities "comprising", "consisting of" and "consisting essentially of" modalities or elements presented herein, whether explicitly stated or not.
[0029] For the recitation of numerical intervals here, each intermediate number with the same degree of precision is explicitly contemplated. For example, for the 6-9 scale, the numbers 7 and 8 are included in addition to 6 and 9, and for the 6.0-7.0 scale, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly covered.
"Adjuvant", as used herein, means any molecule added to the plasmid DNA vaccines described herein to enhance the immunogenicity of the antigens encoded by the DNA plasmids and the encoding nucleic acid sequences described below.
"Antibody", as used herein, means an antibody of the IgG, IgM, IgA, IgD or IgE classes, or fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and their derivatives. The antibody may be an antibody isolated from the mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or mixtures thereof, which exhibits sufficient binding specificity to a desired epitope or sequence derived therefrom.
"Coding sequence" or "coding nucleic acid", as used herein, means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence that encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.
"Complementary" or "complementary" as used herein means that a nucleic acid may mean a Watson-Crick (eg AT/U and CG) or a Hoogsteen base pairing between the nucleotides or nucleotide analogues of the nucleic acid molecules.
"Consensus" or "consensus sequence" as used herein means a polypeptide sequence based on an alignment analysis of multiple sequences for the same gene from different organisms. Nucleic acid sequences that encode a polypeptide consensus sequence can be prepared. Vaccines comprising proteins comprising the consensus sequences and/or nucleic acid molecules encoding such proteins can be used to induce broad immunity against an antigen.
[0035] "Electroporation", "electropermeabilization," or "electrokinetic enhancement" ("EP"), as used interchangeably herein, means the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; its presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water to pass from one side of the cell membrane to the other.
"Fragment", as used herein with respect to nucleic acid sequences, means a nucleic acid sequence or a portion thereof, which encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. document. The fragments can be DNA fragments selected from at least one of several nucleotide sequences that encode the protein fragments defined below. Fragments may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences shown below. In some embodiments, fragments can span at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides, or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 300 nucleotides or more, at least at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more, at least at least 700 nucleotides or more, at least 750 nucleotides or more, at least 800 nucleotides or more, at least 850 nucleotides or more, at least 900 nucleotides or more, at least 950 nucleotides or more more, or at least 1000 nucleotides, or more of at least one of the nucleic acid sequences shown below.
[0037] "Fragment" or "immunogenic fragment" with respect to polypeptide sequences means a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. Fragments can be fragments of polypeptides selected from at least one of the various amino acid sequences below. Consensus protein fragments may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90 % or at least 95% of a consensus protein. In some embodiments, consensus protein fragments can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more , at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids, or more than at least 110 amino acids, or more, at least 120 amino acids, or more, at least 130 amino acids or more, at least 140 amino acids, or more, at least 150 amino acids, or more, at least 160 or more amino acids, at least 170 amino acids, or more at least 180 or more amino acids of a sequence of disclosed protein disclosed in this document.
As used herein, the term "genetic construct" refers to those DNA or RNA molecules that comprise a sequence of nucleotides that encode a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of directing expression in the cells of the individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to those gene constructs that contain the necessary operable regulatory elements linked to a coding sequence that encodes a protein such that when present in the individual's cell, the coding sequence will be express.
[0039] The term "homologous", as used in this document, refers to the degree of complementarity. There can be a partial homology or a complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits a fully complementary sequence from hybridizing to a target nucleic acid is called by the functional term "substantially homologous." When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the The term "substantially homologous" as used herein refers to a probe that can hybridize to a strand of a double-stranded nucleic acid sequence under conditions of low stringency. When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous", as used herein, refers to a probe that can hybridize to (ie, is the complement of) a template nucleic acid sequence. tape under low stringency conditions.
"Identical" or "identity", as used herein in the context of two or more nucleic acid or polypeptide sequences, means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions where the identical residue occurs in both sequences to produce the number of corresponding positions, dividing the number of corresponding positions by the total number of positions in the specified region, and multiplying the result by 100 to produce the percent sequence identity. In cases where the two sequences are of different length or the alignment produces one or more staggered ends and the specified region of comparison includes only a unique sequence, the residues of the unique sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.
[0041] "Immune response", as used herein, means the activation of a host's immune system, for example that of a mammal, in response to the introduction of antigen. The immune response can be in the form of a cellular or a humoral response, or both.
"Nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein means at least two nucleotides covalently linked together. The representation of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a single strand depicted. Many variants of a nucleic acid can be used for the same purpose as a particular nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereto. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
Nucleic acids may be single-stranded or double-stranded, or may contain portions of the double-stranded or single-stranded sequences. The nucleic acid can be DNA, genomic and cDNA, RNA or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides and base combinations including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine , isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.
[0044] "Operably linked", as used herein, means that the expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, the variation of this distance can be adjusted without loss of promoter function.
A "peptide", a "protein" or a "polypeptide" as used herein may mean a sequence of linked amino acids and may be natural, synthetic or a modification or combination of natural and synthetic.
"Promoter", as used herein, means a synthetic or naturally derived molecule that is capable of conferring, activating or enhancing the expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial and/or temporal expression thereof. A promoter can also comprise distal enhancer or repressor elements, which can be localized as far as thousands of base pairs from the transcription start site. A promoter can be derived from sources including viral, bacterial, fungal, plant, insect and animal. A promoter can regulate the expression of a gene component constitutively or differentially with respect to the cell, tissue or organ in which expression occurs, with respect to the stage of development at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions or inducing agents. Representative examples of the promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or the SV40 late promoter and the CMV IE promoter.
"Signal peptide" and "main sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein set forth herein. Signal peptides/major sequences normally direct the location of a protein. Signal peptides/major sequences used herein preferentially facilitate the secretion of the protein from the cell in which they are produced. Signal peptides/major sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides/main sequences are linked at the amino terminus (i.e., N-terminus) of the protein.
"Strict hybridization conditions", as used herein, means the conditions under which a first nucleic acid sequence (eg probe) will hybridize to a second nucleic acid sequence (eg target) such as in a complex mixture of nucleic acids. Restrictive conditions are sequence dependent and will be different under different circumstances. The stringent conditions can be selected to be about 5-10°C below the sequence-specific thermal melting point (Tm) at a pH of defined ionic strength. The Tm can be the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the target-complementary probes hybridize to the target sequence in equilibrium (as the target sequences are present in excess, in the Tm, 50% of the probes are occupied in equilibrium). Restrictive conditions can be those where the salt concentration is less than about 1.0 M sodium ion, such as a concentration of approximately 0.01-1.0 M sodium ions (or other salts) at a pH of 7.0 to 8.3, and the temperature is at least about 30°C for short probes (eg, approximately 1050 nucleotides) and at least about 60°C for long probes (eg, greater than about 50 nucleotides). The restrictive conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be background hybridization at least 2 to 10 times. Examples of stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS incubating at 42°C or 5x SSC, 1% SDS incubating at 65°C, with washing in 0.2x SSC and 0.1 % SDS at 65°C.
"Object" as used herein may mean a mammal that desires or is in need of being immunized with the vaccine described in this document. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse or rat.
[0050] "Substantially complementary" as used in this document may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%.94%, 95%, 96%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.
[0051] "Substantially identical", as used herein, means that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to complement of the second sequence.
[0052] "Treatment" or "treat", as used herein, may mean to protect an animal from a disease by means of prevention, suppression, suppression or total elimination of the disease. Preventing the disease involves administering a vaccine of the present invention to an animal before the onset of the disease. Suppressing the disease involves administering a vaccine of the present invention to an animal after disease induction, but before its clinical onset. Repressing the disease involves administering a vaccine of the present invention to an animal after the clinical onset of the disease.
"Variant", as used herein with respect to a nucleic acid, means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or part thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement or a substantially identical sequence thereto.
"Variant" with respect to a peptide or polypeptide that differs in amino acid sequence by conservative amino acid insertion, deletion or substitution, but retains at least one biological activity. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, that is, the substitution of an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a small change. These small changes can be identified, in part, by considering the hydropathic amino acid index, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indices can be substituted and still maintain protein function. In one aspect, amino acids having hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide allows the calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 incorporated in its entirety herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, e.g. immunogenicity, as is known in the art. Substitutions can be performed with amino acids that have hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to be dependent on the relative similarity of the amino acids, and particularly the side chains of such amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size and other properties.
A variant may be a nucleic acid sequence that is substantially identical over the full length of the entire gene sequence or a fragment thereof. The nucleic acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or a fragment thereof. The nucleic acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the amino acid sequence or a fragment thereof.
"Vector", as used herein, means a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector can be a DNA or an RNA vector. A vector can be a self-replicating extrachromosomal vector and is preferably a DNA plasmid. The vector can contain or include one or more heterologous nucleic acid sequences. The. Vaccine
[0057] The present invention is directed to an anti-cancer vaccine. The vaccine may comprise one or more cancer antigens. The vaccine can stop the tumor from growing. The vaccine can reduce tumor growth. The vaccine can prevent tumor cell metastasis. Depending on the cancer antigen, the vaccine may be targeted to treat liver cancer, prostate cancer, melanomas, blood cancers, head and neck cancer, glioblastoma, recurrent respiratory papillomatosis, anal cancer, cervical cancer, and brain cancer.
[0058] The first step in vaccine development is to identify a cancer antigen that is not recognized by the immune system and is a self-antigen. The identified cancer antigen was changed from an auto-antigen to a foreign antigen in order to be recognized by the immune system. Redesigning the recombinant cancer antigen amino acid nucleic acid sequence from the foreign antigen itself breaks the tolerance of the antigen by the immune system. In order to break tolerance, various reconception measures can be applied to the cancer antigen as described below.
[0059] The vaccine's recombinant cancer antigen is not recognized as self, therefore, it breaks tolerance. Tolerance breakdown can induce T-cell specific antigen/and or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response against the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (IFN--Y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule.
[0060] In a given modality, the vaccine can mediate clearance or prevent the growth of tumor cells by inducing (1) humoral immunity through B cell responses to generate antibodies that block the production of monocyte chemotactic protein-1 (MCP- 1), thereby slowing myeloid-derived suppressor cells (MDSC) and suppressing tumor growth; (2) increase in cytotoxic T lymphocytes like CD8+ (CTL) to attack and kill tumor cells; (3) increase T-cell helper responses; (4) and enhance inflammatory responses through IFN-y and TNF-α or, preferably, all of the above. The vaccine can increase tumor-free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43 %, 44%, and 45%. The vaccine can reduce tumor mass by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% , 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% after immunization. The vaccine can prevent and block increases in monocyte chemotactic protein-1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. The vaccine can increase tumor survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% , 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%.
[0061] The vaccine can enhance a cellular immune response in a patient who has had the vaccine administered about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to about 6000 times, compared to a cellular immune response in a patient who has not had the vaccine administered. In some modalities the vaccine can increase the cellular immune response in the patient who had the vaccine administered about 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times , 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 fold, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times , 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times compared to the cellular immune response in the patient who did not have the vaccine administered.
[0062] The vaccine can increase interferon gamma (IFN-Y) levels in a patient who has had the vaccine administered about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to about 6000 times, compared to IFN-Y levels in a patient who did not have the vaccine administered. In some modalities the vaccine can increase IFN-Y levels in the patient who has had the vaccine administered about 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times , 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times compared to the IFN-Y levels in the patient who does not have you see the vaccine administered.
[0063] The vaccine may be a DNA vaccine. DNA vaccines are disclosed in US Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are incorporated in this document in its entirety by reference. The DNA vaccine can additionally comprise elements or reagents that inhibit it from integrating into the chromosome.
[0064] The vaccine may be an RNA of one or more cancer antigens. The RNA vaccine can be introduced into the cell.
The vaccine may be a live attenuated vaccine, a vaccine using recombinant vectors to deliver antigen, subunit vaccines, and glycoprotein vaccines, for example, but not limited to the vaccines described in US Patent Nos.: 4,510,245 ; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 64 5.453.3; 5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529, which are incorporated herein by reference.
[0066] The vaccine of the present invention may have characteristics required of effective vaccines, such as being safe so that the vaccine itself does not cause illness or death; being protective against disease; inducing the neutralizing antibody; inducing T cell protection responses; and providing ease of administration, few side effects, biological stability and low cost per dose. The vaccine may achieve one or all of these characteristics by containing the cancer antigen, as discussed below.
[0067] As described in more detail below, the vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., an immune checkpoint inhibitor). Immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component of the immune system, such as MHC presentation class, T cell presentation and/or differentiation, B cell presentation and/or differentiation, any cytokine, chemokine or signaling and/or differentiation for immune cell proliferation. As also described in detail below, the vaccine can be further combined with antibodies to checkpoint inhibitors such as DP-1 and PDL-1 to enhance stimulation of both cellular and humoral immune responses. Using anti-PDL-1 or anti-DP-1 antibodies prevents DP-1 or PDL-1 from suppressing T-cell and/or B-cell responses. 1. Cancer Antigen
[0068] The vaccine may comprise one or more cancer antigens. The cancer antigen can be a nucleic acid sequence, an amino acid sequence, or a combination of these. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also include additional sequences that encode the linker or signal sequences that are linked to the antigens by a peptide bond. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The cancer antigen may be a recombinant cancer antigen.
One way of designing the nucleic acid and its encoded amino acid sequence of the recombinant cancer antigen is by introducing mutations that alter certain amino acids in the overall amino acid sequence of the native cancer antigen. The introduction of mutations does not alter the cancer antigen to the point that it cannot be universally applied to a mammal, preferably a human subject or a dog, but alters it sufficiently so that the resulting amino acid sequence breaks tolerance or is considered an antigen. foreign in order to generate an immune response. Another way might be to create a recombinant consensus cancer antigen that has at least 85% and up to 99% amino acid sequence identity to its corresponding native cancer antigen; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In some cases, the recombinant cancer antigen is 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. Native cancer antigen is the antigen normally associated with a particular cancer or tumor. Depending on the cancer antigen, the cancer antigen consensus sequence may be about mammalian species or within subtypes of a species, or about viral strains or serotypes. Some cancer antigens do not vary much from the wild-type amino acid sequence of the cancer antigen. Some cancer antigens have nucleic acid/amino acid sequences so divergent across species that a consensus sequence cannot be generated. In these cases, a recombinant cancer antigen that will break tolerance and generate an immune response is generated having at least 85% and up to 99% amino acid sequence identity to its corresponding native cancer antigen; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In some cases, the recombinant cancer antigen is 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. The above mentioned approaches can be combined so that the final recombinant cancer antigen has a percent similarity with the native cancer antigen amino acid sequence as discussed above.
[0070] The cancer antigen may be one or more of the following antigens: tyrosinase (Tyr), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein 2 (TYRP2), melanoma-associated antigen 4 protein (MAGEA4 ), growth hormone releasing hormone (GHRH) amino acid sequence, MART-1/melan-A antigen amino acid sequence (MART-1 / Melan-A), testicular cancer antigen (NY-ESO-1) , testicular cancer antigen II (NY-ESO 1), and PRAME. The vaccine may be a DNA vaccine comprising polynucleotide sequences encoding tyrosinase (Tyr), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein 2 (TYRP2), melanoma-associated antigen 4 protein (MAGEA4), growth hormone releasing hormone amino acid sequence (GHRH), amino acid sequence of MART-1/melan-A antigen (MART-1 / Melan-A), testicular cancer antigen (NY-ESO-1), antigen testicular cancer II (NY-ESO-1), PRAME, a viral antigen, or combinations thereof. The viral antigen may be one or more of the antigens from the following viruses: Hepatitis B virus (eg, core protein and surface proteins), Hepatitis C virus (eg, non-structural protein (NS) 34A (NS34A), NS5A, NS5B, NS4B), and Human Papilloma Virus (HPV) 6, HPV 11, HPV 16 and HPV 18. (1) Tyrosinase (Tyr)
[0071] The vaccine of the present invention may comprise the cancer antigen tyrosinase (Tyr), a fragment thereof, or a variant thereof. Tyrosinase is a copper-containing enzyme having tyrosine hydroxylase and catalytic dopa oxidase activities that can be found in microorganisms and plant and animal tissues. Specifically, tyrosinase catalyzes the production of melanin and other pigments by oxidizing phenols such as tyrosine. Mutations in the TYR gene result in oculocutaneous albinism in mammals and non-pathological polymorphisms in the TYR gene contribute to variation in skin pigmentation.
[0072] Additionally, in cancers or tumors such as melanoma, tyrosinase can become irregular, resulting in increased melanin synthesis. Thus, tyrosinase may be a cancer antigen associated with melanoma. In patients suffering from melanoma, tyrosinase may be a target for cytotoxic T-cell recognition. In some cases, however, the immune response to cancer or tumor (including melanoma) can be suppressed, which leads to a microenvironment that supports tumor formation and/or growth and thus disease progression.
[0073] Immunosuppression can be facilitated by myeloid-derived suppressor cells (MDSC), which are a mixed population of immature macrophages, granulocytes, dendritic cells and myeloid cells. Myeloid cells can be a heterogeneous population of myeloid progenitor cells and immature myeloid cells (IMCs). MDSC markers can include the expression of Gr-1 and CD11b (ie, Gr-1+ and CD11b+ cells).
[0074] Circulation of MDSCs may increase due to chronic infection and expansion of MDSC populations may be associated with autoimmunity and inflammation. Particularly, expanding MDSCs (or presence in tumor or cancer tissue) can facilitate tumor growth and escape detection and/or immune regulation, and thus, MDSCs can affect immune responses to cancer vaccines.
[0075] MDSCs can be regulated by regulators of G protein signaling2 (Rgs2) and Rgs2 can be highly expressed in tumor-derived MDSCs. Rgs2 can also be widely expressed in a variety of cells, eg myeloid cells. MDSCs derived from tumor-bearing mice may function differently from MDSCs derived from non-tumor-bearing mice. One of the differences may be the increased regulation of the production of the chemokine MCP-1, which is secreted by MDSCs. MCP-1 can promote cell migration through signaling by CCR2, a G protein-coupled receptor (GPCR) found in monocytes, endothelial cells, and T cells. vascularization. Blocking MCP-1 through neutralizing antibodies can inhibit angiogenesis and, therefore, can lead to a decrease in tumor metastases and increased survival. As such, MCP-1 can be considered an angiogenic factor. In addition to secreting MCP-1, MDSCs can secrete growth factors, further contributing to tumor growth.
The Tyr antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (IFN--Y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[0077] As demonstrated in this document, the Tyr antigen induces T-cell specific antigens and high titer antibody responses against cancer or tumor cells (eg melanoma cells). Specifically, the Tyr antigen is an important target for immunological clearance mediated by inducing (1) humoral immunity through B cell responses to produce antibodies that block monocyte chemotactic protein-1 (MCP-1), thereby retarding suppressor cell-derived of myeloids (MDSCs) and suppressing tumor growth; (2) increase in cytotoxic T lymphocytes, such as CD8+ (CTL) to attack and destroy tumor cells; (3) increase helper T cell responses; and (4) enhance inflammatory responses through IFN-y and TNF-α or preferably all of the above. As such, a protective immune response is provided against tumor formation and tumor growth by vaccines comprising the Tyr antigen (for example, the Tyr consensus antigen, which is described in more detail below), because these vaccines prevent immune suppression by decrease in the population of MDSCs found within the cancerous or tumor-bearing tissue, blocking the vascularization of the cancerous or tumor-bearing tissue, reducing the production or secretion of MCP-1. Thus, any user can design a vaccine of the present invention to include a Tyr antigen to provide broad immunity against tumor formation, tumor metastasis, and tumor growth.
The Tyr antigen may comprise protein epitopes which make them particularly effective as immunogens against which anti-Tyr immune responses can be induced. The Tyr antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. Tyr antigen can comprise a consensus protein.
The nucleic acid sequence encoding the Tyr consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. Nucleic acid encoding the Tyr consensus antigen may be an optimized codon and RNA for expression. In some embodiments, the nucleic acid sequence encoding the Tyr consensus antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the Try consensus antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the Tyr consensus antigen may also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the Tyr consensus antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the Tyr consensus antigen amino acid sequence by a peptide bond. Nucleic encoding the Tyr consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the Tyr consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
The Tyr consensus antigen can be the nucleic acid sequence SEQ ID NO: 1, which encodes the amino acid sequence SEQ ID NO: 2. SEQ ID NO: 1 encodes the Tyr consensus protein linked to an IgE core sequence. The Tyr consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the Tyr consensus protein may be free or unbound to an IgE core sequence and/or an HA tag.
[0082] In some embodiments, the Tyr consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the full length of the nucleic acid sequence defined in the SEQ ID NO:1. In some embodiments, the Tyr consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO :two. The Tyr consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:2.
[0083] Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to Tyr consensus protein, immunogenic fragment of Tyr consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[0085] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of Tyr consensus protein, immunogenic fragment of Tyr consensus protein and immunogenic fragments of proteins that have identity to the consensus protein Tyr. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity with a full-length Tyr consensus sequence, up to 85% identity with a full-length Tyr consensus sequence, up to 90% identity with a consensus sequence of full length Tyr, up to 91% identity with a full length Tyr consensus sequence, up to 92% identity with a full length Tyr consensus sequence, up to 93% identity with a full length Tyr consensus sequence , up to 94% identity with a full length Tyr consensus sequence, up to 95% identity with a full length Tyr consensus sequence, up to 96% identity with a full length Tyr consensus sequence, up to 97% of identity with a full-length Tyr consensus sequence, up to 98% identity with a full-length Tyr consensus sequence, and up to 99% identity with a full-length Tyr consensus sequence can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those noted above for the Tyr proteins set forth herein are also provided.
[0086] In some embodiments, the nucleic acid sequence is free of coding sequence encoding a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[0087] Some embodiments refer to fragments of SEQ ID NO: 1. Fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35 %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO: 1. Fragments may be at least 95%, at least minus 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO:1. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:1. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
[0088] Additionally, a consensus Tyr protein amino acid sequence is SEQ ID NO:2. The amino acid sequence of the Tyr consensus protein linked to a major IgE is SEQ ID NO:2. The amino acid sequence of the Tyr consensus protein linked to the major IgE can be linked to the HA tag.
[0089] Some modalities refer to proteins that are homologous to SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences , as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have 98% homology to the consensus protein sequences, as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO:2.
[0090] Some modalities refer to proteins that are identical to SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown. in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full length consensus amino acid sequences as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO: 2. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the amino acid sequences full-length consensus as shown in SEQ ID NO: 2. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO: 2 Some modalities refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have a sequence of amino acid which is 96% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:2. modalities refer to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have an amino acid sequence which is 98% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO: 2. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences. , as shown in SEQ ID NO: 2.
[0091] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of major IgE. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of a consensus protein. Immunogenic fragments of SEQ ID NO:2 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, in minus 97%, at least 98%, or at least 99% of SEQ ID NO: 2. In some embodiments, fragments include a major sequence such as a major immunoglobulin such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[0092] Immunogenic fragments of proteins with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO:2 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of proteins that are 95% or more homologous to SEQ ID NO:2. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[0093] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 2 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequences shown in SEQ ID NO:2. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[0094] As noted herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (2) Tyrosinase 1 Related Protein (TYRP1)
[0095] The vaccine of the present invention may comprise the tyrosinase 1-related protein cancer antigen (TYRP1), a fragment thereof, or a variant thereof. TYRP1, encoded by the TYRP1 gene, is a 75kDa transmembrane glycoprotein and is expressed in melanoma cells and normal and malignant melanocytes. Like tyrosinase, TYRP1 contains a certain modified M-box that can bind to microphthalmia transcription factor (MITF), which plays a central role within the melanocyte in the activation of pigmentation, proliferation and cell differentiation. TYRP1 can help stabilize tyrosinase and can form a heterodimer, which can prevent premature death of melanocytes by attenuating tyrosinase-mediated cytotoxicity.
[0096] As described above for tyrosinase, tyrosinase-related protein 1 (TYRP-1) may also be involved in the melanin synthesis and pigmented mechanism of the melanocyte, and may be recognized by the immune system in individuals suffering from melanoma. Therefore, TYRP-1 may be the melanoma-associated antigen.
The TRYP-1 antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (JFN--y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
The TYRP-1 antigen may comprise protein epitopes that make them particularly effective as immunogens against which anti-TYRP-1 immune responses can be induced. The TYRP-1 antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The TYRP-1 antigen can comprise a consensus protein.
The nucleic acid sequence encoding the TYRP-1 consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the TYRP-1 consensus antigen can be an optimized codon and RNA for expression. In some embodiments, the nucleic acid sequence encoding the TYRP-1 consensus antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the TYRP-1 consensus antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the TYRP-1 consensus antigen can also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the TYRP-1 consensus antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the TYRP-1 consensus antigen amino acid sequence by a peptide bond. Nucleic encoding the TYRP-1 consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the TYRP-1 consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
The TYRP-1 consensus antigen may be the nucleic acid sequence SEQ ID NO: 3, which encodes the amino acid sequence SEQ ID NO: 4. SEQ ID NO: 3 encodes the TYRP-1 consensus protein linked to a sequence main IgE. The TYRP-1 consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the TYRP-1 consensus protein may be free or unbound to an IgE main sequence and/or an HA tag.
[00102] In some embodiments, the TYRP-1 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:3. In some embodiments, the TYRP-1 consensus antigen can be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:4 . The TYRP-1 consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:4.
[00103] Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to the TYRP-1 consensus protein, immunogenic fragment of the TYRP-1 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[00105] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of the TYRP-1 consensus protein, immunogenic fragment of the TYRP-1 consensus protein and immunogenic fragments of proteins that have identity in relation to the TYRP-1 consensus protein. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity to a full length TYRP-1 consensus sequence, up to 85% identity to a full length TYRP-1 consensus sequence, up to 90% identity to a TYRP-1 full length consensus sequence, up to 91% identity with a TYRP-1 full length consensus sequence, up to 92% identity with a TYRP-1 full length consensus sequence, up to 93% identity with a sequence TYRP-1 full length consensus sequence, up to 94% identity with a TYRP-1 full length consensus sequence, up to 95% identity with a TYRP-1 full length consensus sequence, up to 96% identity with a TYRP-1 full length consensus sequence full length TYRP-1, up to 97% identity with a full length TYRP-1 consensus sequence, up to 98% identity with a full length TYRP-1 consensus sequence, and up to 99% identity with a full length consensus sequencefull TYRP-1 can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those indicated above for the TYRP-1 proteins presented herein are also provided.
[00106] In some embodiments, the nucleic acid sequence is free of coding sequence encoding a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[00107] Some modalities refer to fragments of SEQ ID NO: 3. Fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35 %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO: 3. Fragments may be at least 95%, at least minus 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO:3. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:3. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
[00108] Additionally, an amino acid sequence of the TYRP-1 consensus protein is SEQ ID NO:4. The amino acid sequence of the consensus protein TYRP-1 linked to a major IgE is SEQ ID NO:4 The amino acid sequence of the consensus protein TYRP-1 linked to the major IgE can be linked to the HA tag.
[00109] Some modalities refer to proteins that are homologous to SEQ ID NO: 4. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO: 4. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO: 4. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences , as shown in SEQ ID NO: 4. Some modalities refer to immunogenic proteins that have 98% homology to the consensus protein sequences, as shown in SEQ ID NO: 4. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO: 4.
[00110] Some modalities refer to proteins that are identical to SEQ ID NO: 4. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown. in SEQ ID NO:4 Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO: 4. Some modalities refer to immunogenic proteins that have a sequence of amino acid that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:4.
[00111] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of major IgE. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of a TYRP-1 consensus protein. Immunogenic fragments of SEQ ID NO:4 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98%, or at least 99% of SEQ ID NO: 4. In some embodiments, fragments include a major sequence such as a major immunoglobulin such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00112] Immunogenic fragments of proteins with homologous amino acid sequences to the immunogenic fragments of SEQ ID NO:4 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of proteins that are 95% or more homologous to SEQ ID NO:4. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00113] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 4 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences shown in SEQ ID NO:4. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00114] As stated herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (3) Tyrosinase-related protein 2 (TYRP2)
[00115] The vaccine of the present invention may comprise the cancer antigen Tyrosinase-related Protein 2 (TYRP2; also known as dopachrome tautomerase (DCT)), a fragment thereof, or a variant thereof. TYRP2/DCT, encoded by the TYRP2/DCT gene, is a protein made up of 519 amino acids and is expressed in both normal and malignant cells and melanoma and melanocytes. TYRP2/DCT is a well-characterized melanocyte-specific enzyme that, together with tyrosinase and TYRP1, functions in the conversion of L-tyrosine to melanin in melanocytes. DCT specifically catalyzes the tautomerization of L-dopachrome melanin precursors to 5,6-dihydroxy-indole-2-carboxylic acid (DHICA), which is subsequently oxidized by TYRP1 (as discussed above) to form eumelanin. Studies have shown that TYRP2/DCT may be a mediator of drug resistance in melanoma cells, with specificity for agents that damage DNA. Since it has often been reported that TYRP2/DCT is highly expressed in melanomas, this melanocyte-specific enzyme plays an important role in contributing to the intrinsic resistance phenotype of melanomas to various drugs that damage anticancer DNA.
[00116] As described above for tyrosinase, tyrosinase-related protein 2 (TYRP-2) may also be involved in melanin synthesis and recognized by the immune system in patients suffering from melanoma. Furthermore, TYRP-2 can mediate drug resistance in melanoma cells. Therefore, TYRP-2 may be a melanoma-associated antigen.
[00117] The TRYP-2 antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (JFN--y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00118] The TYRP2 antigen may comprise protein epitopes that make them particularly effective as immunogens against which anti-TYRP2 immune responses can be induced. The TYRP2 antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The TYRP2 antigen can comprise a consensus protein.
[00119] The nucleic acid sequence encoding the TYRP2 consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the TYRP2 consensus antigen can be a codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the TYRP2 consensus antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the TYRP2 consensus antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
The nucleic acid encoding the TYRP2 consensus antigen may also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the TYRP2 consensus antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the TYRP2 consensus antigen amino acid sequence by a peptide bond. Nucleic encoding the TYRP2 consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the TYRP2 consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
The TYRP2 consensus antigen can be the nucleic acid sequence SEQ ID NO: 5, which encodes the amino acid sequence SEQ ID NO: 6. SEQ ID NO: 5 encodes the TYRP2 consensus protein linked to an IgE core sequence. The TYRP2 consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the TYRP2 consensus protein may be free or unbound to an IgE main sequence and/or an HA tag.
[00122] In some embodiments, the TYRP2 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the full length of the nucleic acid sequence defined in the SEQ ID NO:5. In some embodiments, the TYRP2 consensus antigen can be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:6 . The TYRP2 consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:6.
[00123] Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to the TYRP2 consensus protein, immunogenic fragment of the TYRP2 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[00125] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of the TYRP2 consensus protein, immunogenic fragment of the TYRP2 consensus protein and immunogenic fragments of proteins that have identity to the consensus protein TYRP2. Such nucleic acid molecules encoding immunogenic proteins have up to 80% identity with a full length TYRP2 consensus sequence, up to 85% identity with a full length TYRP2 consensus sequence, up to 90% identity with a full length TYRP2 consensus sequence of full length, up to 91% identity with a full length TYRP2 consensus sequence, up to 92% identity with a full length TYRP2 consensus sequence, up to 93% identity with a full length TYRP2 consensus sequence, up to 94% identity with a full length TYRP2 consensus sequence, up to 95% identity with a full length TYRP2 consensus sequence, up to 96% identity with a full length TYRP2 consensus sequence, up to 97% identity with a full length TYRP2 consensus sequence , up to 98% identity with a full-length TYRP2 consensus sequence, and up to 99% identity with a full-length TYRP2 consensus sequence can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those indicated above for the TYRP2 proteins presented in this document are also provided.
[00126] In some embodiments, the nucleic acid sequence is free of coding sequence that encodes a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[00127] Some modalities refer to fragments of SEQ ID NO: 5. Fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35 %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO: 5. Fragments can be at least 95%, at least minus 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO:5. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:5. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
[00128] Additionally, a consensus TYRP2 protein amino acid sequence is SEQ ID NO:6. The amino acid sequence of the consensus TYRP2 protein linked to a major IgE is SEQ ID NO:6. The amino acid sequence of the consensus TYRP2 protein linked to the major IgE can be linked to the HA tag.
[00129] Some modalities refer to proteins that are homologous to SEQ ID NO: 2. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO: 6. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO: 6. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences , as shown in SEQ ID NO: 6. Some modalities refer to immunogenic proteins that have 98% homology to the consensus protein sequences, as shown in SEQ ID NO: 6. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO:6.
[00130] Some embodiments refer to proteins that are identical to SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:6.
[00131] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of major IgE. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of a consensus protein. Immunogenic fragments of SEQ ID NO:6 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, in minus 97%, at least 98%, or at least 99% of SEQ ID NO: 6. In some embodiments, fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00132] Immunogenic fragments of proteins with homologous amino acid sequences to the immunogenic fragments of SEQ ID NO:6 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of proteins that are 95% or more homologous to SEQ ID NO:6. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00133] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 6 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences shown in SEQ ID NO:6. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00134] As noted herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (4) . Melanoma-associated antigen 4 (MAGEA4)
[00135] The vaccine of the present invention may comprise the cancer antigen, the melanoma-associated antigen 4 (MAGEA4), a fragment thereof or a variant thereof. MAGE-A4, encoded by the MAGE-A4 gene, is a protein formed by 317 amino acids and is expressed in male germ cells and tumor cells of various histological types, such as gastrointestinal, esophageal and lung cancers. MAGE-A4 binds the oncoprotein, Gankyrin. This specific MAGE-A4 binding is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 can partially inhibit the adhesion-independent growth of overexpressing Gankyrin cells in vitro and suppress the formation of tumors that migrated from these cells in nude mice. This inhibition is dependent on the binding between MAGE-A4 and Gankyrin, suggesting that interactions between Gankyrin and MAGE-A4 inhibit Gankyrin-mediated carcinogenesis. It is likely that MAGE expression in tumor tissue is not a cause but a result of tumor genesis, and MAGE genes take part in the immunization process by targeting the destruction of early tumor cells.
[00136] Melanoma-associated antigen protein 4 (MAGEA4) may be involved in embryonic development and tumor transformation and/or progression. MAGEA4 is normally expressed in testes and placenta. MAGEA4, however, can be expressed in many different types of tumors, eg melanoma, head and neck squamous cell cancer, lung carcinoma and breast carcinoma. Therefore, MAGEA4 may be an antigen associated with a variety of tumors.
[00137] The MAGEA4 antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (JFN--y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00138] The MAGEA4 antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-MAGEA4 immune responses can be induced. The MAGEA4 antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The MAGEA4 antigen can comprise a consensus protein.
[00139] The nucleic acid sequence encoding the MAGEA4 consensus antigen can be optimized with regard to the use of the codon and the corresponding RNA transcripts. The nucleic acid encoding the MAGEA4 consensus antigen can be an optimized codon and RNA for expression. In some embodiments, the nucleic acid sequence encoding the MAGEA4 consensus antigen may include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the MAGEA4 consensus antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
The nucleic acid encoding the MAGEA4 consensus antigen may also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the Tyr consensus antigen may further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the MAGEA4 consensus antigen amino acid sequence by a peptide bond. Nucleic encoding the MAGEA4 consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the MAGEA4 consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
The MAGEA4 consensus antigen can be the nucleic acid sequence SEQ ID NO: 7, which encodes the amino acid sequence SEQ ID NO: 8. SEQ ID NO: 7 encodes the MAGEA4 consensus protein linked to an IgE core sequence. The MAGEA4 consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the MAGEA4 consensus protein may be free or unbound to an IgE core sequence and/or an HA tag.
[00142] In some embodiments, the MAGEA4 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO: 7. In some embodiments, the MAGEA4 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:8 . The MAGEA4 consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:8.
[00143] Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to the MAGEA4 consensus protein, immunogenic fragment of the MAGEA4 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[00145] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of the MAGEA4 consensus protein, immunogenic fragment of the MAGEA4 consensus protein and immunogenic fragments of proteins that have identity to the consensus protein MAGEA4. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity to a full length MAGEA4 consensus sequence, up to 85% identity to a full length MAGEA4 consensus sequence, up to 90% identity to a full length MAGEA4 consensus sequence of full length MAGEA4, up to 91% identity with a full length MAGEA4 consensus sequence, up to 92% identity with a full length MAGEA4 consensus sequence, up to 93% identity with a full length MAGEA4 consensus sequence , up to 94% identity with a full length MAGEA4 consensus sequence, up to 95% identity with a full length MAGEA4 consensus sequence, up to 96% identity with a full length MAGEA4 consensus sequence, up to 97% of identity with a full-length MAGEA4 consensus sequence, up to 98% identity with a full-length MAGEA4 consensus sequence, and up to 99% identity with a full length MAGEA4 consensus sequence can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those indicated above for the MAGEA4 proteins presented in this document are also provided.
In some embodiments, the nucleic acid sequence is free of coding sequence that encodes a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[00147] Some embodiments refer to fragments of SEQ ID NO: 7. Fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35 %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO: 7. Fragments can be at least 95%, at least minus 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO:7. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:7. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
[00148] Additionally, a consensus MAGEA4 protein amino acid sequence is SEQ ID NO:8. The amino acid sequence of the consensus MAGEA4 protein linked to a major IgE is SEQ ID NO:8. The amino acid sequence of the MAGEA4 consensus protein linked to the major IgE can be linked to the HA tag.
[00149] Some modalities refer to proteins that are homologous to SEQ ID NO: 8. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO:8. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO: 8. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences , as shown in SEQ ID NO: 8. Some embodiments refer to immunogenic proteins that have 98% homology to the consensus protein sequences as shown in SEQ ID NO:8. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO: 8.
[00150] Some embodiments refer to proteins that are identical to SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:8.
[00151] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of major IgE. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of a consensus protein. Immunogenic fragments of SEQ ID NO:8 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, in minus 97%, at least 98%, or at least 99% of SEQ ID NO: 8. In some embodiments, fragments include a major sequence such as a major immunoglobulin such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00152] Immunogenic fragments of proteins with homologous amino acid sequences to the immunogenic fragments of SEQ ID NO:8 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of proteins that are 95% or more homologous to SEQ ID NO:8. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00153] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO:8 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences shown in SEQ ID NO:8. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
As noted herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (5) . Growth Hormone Releasing Hormone (GHRH)
[00155] The vaccine of the present invention may comprise the cancer antigen growth hormone releasing hormone (GHRH, also known as growth hormone releasing factor (GRF or GHRF) or somatocrinin), a fragment thereof, or a variant thereof your. GHRH is a 44-amino acid peptide hormone produced in the arcuate nucleus of the hypothalamus. GHRH is secreted by the hypothalamus and stimulates the release of growth hormone, a regulator of growth, metabolism, and body structure, from the pituitary gland. GHRH also stimulates growth hormone product. GHRH antagonists can inhibit the growth of a variety of cancers, for example, osteosarcomas, glioblastomas, prostate cancer, kidney cancer, pancreatic cancer, colorectal cancer, and breast cancer. Therefore, GHRH may be an antigen associated with a variety of tumors.
[00156] The GHRH antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (JFN--y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00157] The GHRH antigen may comprise protein epitopes that make them particularly effective as immunogens against which anti-GHRH immune responses can be induced. The GHRH antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The GHRH antigen can comprise a consensus protein.
[00158] The nucleic acid sequence encoding the GHRH consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. Nucleic acid encoding the GHRH consensus antigen can be an optimized codon and RNA for expression. In some embodiments, the nucleic acid sequence encoding the consensus GHRH antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the consensus GHRH antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
The nucleic acid encoding the consensus GHRH antigen may also encode an immunoglobulin E (IgE) main sequence. Nucleic acid encoding the GHRH consensus antigen may further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the GHRH consensus antigen amino acid sequence by a peptide bond. Nucleic encoding the consensus GHRH antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the GHRH consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
[00160] The GHRH consensus antigen may be the nucleic acid sequence SEQ ID NO:9, which encodes the amino acid sequence SEQ ID NO:10. SEQ ID NO:9 encodes the GHRH consensus protein linked to an IgE core sequence. The GHRH consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the GHRH consensus protein may be free or unbound to an IgE core sequence and/or an HA tag.
[00161] In some embodiments, the GHRH consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:9. In some embodiments, the GHRH consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:10 . The GHRH consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:10.
Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to the GHRH consensus protein, immunogenic fragment of the GHRH consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[00164] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of the GHRH consensus protein, immunogenic fragment of the GHRH consensus protein and immunogenic fragments of proteins that have identity to the consensus protein GHRH. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity with a full length GHRH consensus sequence, up to 85% identity with a full length consensus sequence, up to 90% identity with a full length GHRH consensus sequence full length, up to 91% identity with a full length GHRH consensus sequence, up to 92% identity with a full length GHRH consensus sequence, up to 93% identity with a full length GHRH consensus sequence, up to 94% identity with a full length GHRH consensus sequence, up to 95% identity with a full length GHRH consensus sequence, up to 96% identity with a full length GHRH consensus sequence, up to 97% identity with a full length GHRH consensus sequence, up to 98% identity with a full-length GHRH consensus sequence, and up to 99% identity with a full-length GHRH consensus sequence can be provided. mentioned. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those noted above for the GHRH proteins set forth herein are also provided.
In some embodiments, the nucleic acid sequence is free of a coding sequence that encodes a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[00166] Some modalities refer to fragments of SEQ ID NO: 9. Fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35 %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 9. Fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO:9. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:9. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
[00167] Additionally, an amino acid sequence of the GHRH consensus protein is SEQ ID NO:10. The amino acid sequence of the consensus GHRH protein linked to a major IgE is SEQ ID NO:10. The amino acid sequence of the consensus GHRH protein linked to the major IgE can be linked to the HA tag.
Some embodiments refer to proteins that are homologous to SEQ ID NO:10. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO:10. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO:10. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences as shown in SEQ ID NO:10. Some modalities refer to immunogenic proteins that have 98% homology to the consensus protein sequences as shown in SEQ ID NO:10. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO:10.
Some embodiments refer to proteins that are identical to SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:10.
[00170] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of major IgE. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of a consensus protein. Immunogenic fragments of SEQ ID NO:10 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, in at least 97%, at least 98%, or at least 99% of SEQ ID NO: 10. In some embodiments, fragments include a major sequence, such as a major immunoglobulin, such as a major IgE. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00171] Immunogenic fragments of proteins with homologous amino acid sequences to the immunogenic fragments of SEQ ID NO:10 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of proteins that are 95% or more homologous to SEQ ID NO:10. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00172] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO:10 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences shown in SEQ ID NO:10. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00173] As noted herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (6) MART-1/Melan-A
The vaccine of the present invention may comprise the cancer antigen MART-1 (also known as Melan-A), a fragment thereof, or a variant thereof. MART-1, encoded by the MLANA gene, is a 118 amino acid protein containing a single transmembrane domain and is expressed in most melanoma cells. MART-1 forms a complex with a structural protein and affects its expression, stability, diapedesis (trafficking) and processing that are necessary for melanosome structure and maturation. Therefore, MART-1 is indispensable for the regulation of mammalian pigmentation. Defects in melanosome maturation have been associated with susceptibility to cancer. MART-1 can be expressed in numerous cancers, including, but not limited to, melanomas.
[00175] Melan-A, also known as T-cell recognized melanoma antigen (MART-1,) is a melanocyte differentiation antigen and can be found on normal skin, retina, and melanocytes. Melan-A can be associated with the endoplasmic reticulum and melanosomes. Melan-A can be recognized by cytotoxic T cells as an antigen on melanoma cells, but it can also be associated with other tumors with melanocytic origin or differentiation (ie, cells have melanosomes), eg, clear cell sarcoma and melanotic neurofibroma. Therefore, Melan-A may be an antigen associated with a variety of tumors derived from cells bearing melanosomes.
The Melan-A antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (JFN--y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
The Melan-A antigen may comprise protein epitopes which make them particularly effective as immunogens against which anti-Melan-A immune responses can be induced. The Melan-A antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The Melan-A antigen can comprise a consensus protein.
The nucleic acid sequence encoding the Melan-A consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. Nucleic acid encoding the Melan-A consensus antigen can be a codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the Melan-A consensus antigen may include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the Melan-A consensus antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
The nucleic acid encoding the Melan-A consensus antigen may also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the Melan-A consensus antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the Melan-A consensus antigen amino acid sequence by a peptide bond. Nucleic encoding the Melan-A consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the Melan-A consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
The Melan-A consensus antigen may be the nucleic acid sequence SEQ ID NO:11, which encodes the amino acid sequence SEQ ID NO:12. SEQ ID NO: 11 encodes the consensus MELAN-A protein linked to an IgE leader sequence. The Melan-A consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the Melan-A consensus protein may be free or unbound to an IgE main sequence and/or an HA tag.
[00181] In some embodiments, the Melan-A consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in the SEQ ID NO:11. In some embodiments, the Melan-A consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO :12. The Melan-A consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:12.
[00182] Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to the Melan-A consensus protein, immunogenic fragment of the Melan-A consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[00184] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of the Melan-A consensus protein, immunogenic fragment of the Melan-A consensus protein and immunogenic fragments of proteins that have identity in relation to Melan-A consensus protein. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity to a full-length Melan-A consensus sequence, up to 85% identity to a full-length Melan-A consensus sequence, up to 90% identity to a full-length Melan-A consensus sequence, up to 91% identity with a full-length Melan-A consensus sequence, up to 92% identity with a full-length Melan-A consensus sequence, up to 93% identity with a full length Melan-A consensus sequence, up to 94% identity with a full length Melan-A consensus sequence, up to 95% identity a full length Melan-A consensus sequence, up to 96% identity with a full-length Melan-A consensus sequence, up to 97% identity to a full-length Melan-A consensus sequence, up to 98% identity to a full-length Melan-A consensus sequence, and up to 99% of identity a Melan-A consensus sequence of ex full voltage can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those indicated above for the Melan-A proteins presented in this document are also provided.
In some embodiments, the nucleic acid sequence is free of coding sequence encoding a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[00186] Some embodiments refer to fragments of SEQ ID NO:11. Fragments can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 11. Fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO:11. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:11. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
[00187] In addition, the amino acid sequence of the Melan-A consensus protein is SEQ ID NO:12. The amino acid sequence of the Melan-A consensus protein linked to a major IgE is SEQ ID NO:12. The amino acid sequence of the consensus Melan-A protein linked to the major IgE can be linked to the HA tag.
[00188] Some embodiments refer to proteins that are homologous to SEQ ID NO:12. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO:12. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO:12. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences as shown in SEQ ID NO:12. Some modalities refer to immunogenic proteins that have 98% homology to the consensus protein sequences as shown in SEQ ID NO:12. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO:12.
[00189] Some embodiments refer to proteins that are identical to SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:12.
[00190] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of major IgE. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of a consensus protein. Immunogenic fragments of SEQ ID NO:12 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, in at least 97%, at least 98%, or at least 99% of SEQ ID NO: 12. In some embodiments, fragments include a major sequence, such as a major immunoglobulin, such as a major IgE. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00191] Immunogenic fragments of proteins with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO:12 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of proteins that are 95% or more homologous to SEQ ID NO:12. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00192] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO:12 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences shown in SEQ ID NO:12. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00193] As noted herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (7) NY-ESO-1
[00194] The vaccine of the present invention may comprise the esophageal cancer NY-ESO-1 cancer antigen (NY-ESO-1; also called CTAG1), a fragment thereof, or a variant thereof. NY-ESO-1, encoded by the CTAG1B gene, is a protein 180 amino acids long, with a glycine-rich N-terminal region and an extremely hydrophobic C-terminal region. NY-ESO-1 has restricted expression in normal tissues but is frequent in cancer. NY-ESO-1 can be expressed in numerous types of cancer, including, but not limited to, bladder, colorectal, esophagus, stomach, hepatocarcinoma, head and neck, melanoma, non-small cell lung, ovary, pancreas, carcinoma synovial and prostate cancers.
[00195] Testicular cancer antigen (NY-ESO-1) can be expressed in the testis and ovaries. NY-ESO-1 may be associated with a variety of cancers and may induce humoral immune responses. Patients suffering from cancer or tumors may develop immunogenicity for NY-ESO-1. Therefore, NY-ESO-1 may be an antigen associated with a variety of tumors.
The NY-ESO-1 antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (JFN--y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00197] The NY-ESO-1 antigen can enhance a cellular immune response in a patient who has had NY-ESO-1 administered about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to about 6000 times, compared to a cellular immune response in a patient who did not have NY-ESO-1 administered. In some modalities the NY-ESO-1 antigen can enhance the cellular immune response in the patient who has had NY-ESO-1 administered about 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times , 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times , 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times if compared to cell immune response air in the patient who did not have NY-ESO-1 administered.
[00198] The NY-ESO-1 antigen can increase interferon gamma (IFN-y) levels in a patient who has had the NY-ESO-1 antigen administered about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to about 6000 times, compared to IFN-Y levels in a patient who did not have NY-ESO-1 administered. In some modalities the NY-ESO-1 antigen can increase IFN-y levels in the patient who has had the NY-ESO-1 antigen administered about 50 times, 100 times, 150 times, 200 times, 250 times, 300 times , 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times , 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times compared to IFN- Y in the patient who did not have NY-ESO-1 antigen administered.
The NY-ESO-1 antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-NY-ESO-1 immune responses can be induced. The NY-ESO-1 antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The NY-ESO-1 antigen can comprise a consensus protein.
The nucleic acid sequence encoding the NY-ESO-1 consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the NY-ESO-1 consensus antigen can be an optimized codon and RNA for expression. In some embodiments, the nucleic acid sequence encoding the NY-ESO-1 consensus antigen may include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the NY-ESO-1 consensus antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the NY-ESO-1 consensus antigen can also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the NY-ESO-1 consensus antigen may further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the NY-ESO-1 consensus antigen amino acid sequence by a bond peptide. Nucleic encoding the NY-ESO-1 consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the NY-ESO-1 consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
The NY-ESO-1 consensus antigen may be the nucleic acid sequence SEQ ID NO:13, which encodes the amino acid sequence SEQ ID NO:14. SEQ ID NO:13 encodes the NY-ESO-1 consensus protein linked to an IgE core sequence. The NY-ESO-1 consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the NY-ESO-1 consensus protein may be free or unbound to an IgE main sequence and/or an HA tag.
In some embodiments, the NY-ESO-1 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:13. In some embodiments, the NY-ESO-1 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in the SEQ ID NO:14. The NY-ESO-1 consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:14 .
[00204] Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to the consensus protein NY-ESO-1, immunogenic fragment of the consensus protein NY-ESO-1, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[00206] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of the consensus protein NY-ESO-1, immunogenic fragment of the consensus protein NY-ESO-1 and immunogenic fragments of proteins that have identity to the NY-ESO-1 consensus protein. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity with a full-length NY-ESO-1 consensus sequence, up to 85% identity with a full-length NY-ESO-1 consensus sequence, up to 90% identity with a full-length NY-ESO-1 consensus sequence, up to 91% identity with a full-length NY-ESO-1 consensus sequence, up to 92% identity with a NY-ESO consensus sequence -1 full length, up to 93% identity with a full length NY-ESO-1 consensus sequence, up to 94% identity with a full length NY-ESO-1 consensus sequence, up to 95% identity with a full-length NY-ESO-1 consensus sequence, up to 96% identity with a full-length NY-ESO-1 consensus sequence, up to 97% identity with a full-length NY-ESO-1 consensus sequence , up to 98% identity with a full-length NY-ESO-1 consensus sequence, and up to 99% identity with a sequence full-length NY-ESO-1 consensus can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those indicated above for the NY-ESO-1 proteins presented herein are also provided.
In some embodiments, the nucleic acid sequence is free of coding sequence encoding a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
Some embodiments refer to fragments of SEQ ID NO:13. Fragments can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 13. Fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % homologous to the fragments of SEQ ID NO:13. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:13. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
Additionally, an amino acid sequence of the consensus protein NY-ESO-1 is SEQ ID NO:14. The amino acid sequence of the consensus protein NY-ESO-1 linked to a major IgE is SEQ ID NO:14. The amino acid sequence of the consensus protein NY-ESO-1 linked to the major IgE can be linked to the HA tag.
Some embodiments refer to proteins that are homologous to SEQ ID NO:14. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO:14. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO:14. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences as shown in SEQ ID NO:14. Some modalities refer to immunogenic proteins that have 98% homology to the consensus protein sequences as shown in SEQ ID NO:14. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO:14.
Some embodiments refer to proteins that are identical to SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:14.
[00212] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of major IgE. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of a consensus protein. The immunogenic fragments of SEQ ID NO:14 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, in at least 97%, at least 98%, or at least 99% of SEQ ID NO: 14. In some embodiments, the fragments include a major sequence, such as, for example, a major immunoglobulin, such as a major IgE. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00213] Immunogenic fragments of proteins with homologous amino acid sequences to the immunogenic fragments of SEQ ID NO:14 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of proteins that are 95% or more homologous to SEQ ID NO:14. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00214] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO:14 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% of proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences shown in SEQ ID NO:14. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00215] As noted herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (8) NY-ESO-2
[00216] The vaccine of the present invention may comprise esophageal cancer NY-ESO-2 cancer antigen (NY-ESO-2, also known as cancer antigen/testis antigen 2, ESO2, and LAGE1), a fragment yours, or a variant of it. NY-ESO-2 is an auto-immunogenic tumor antigen that belongs to the ESO/LAGE family of testicular cancer antigens. NY-ESO-2 can be expressed in a variety of cancers including melanoma, breast cancer, bladder cancer and prostate cancer and is normally expressed in testicular tissue. In addition, NY-ESO-2 can be seen in 25-50% of tumor samples from melanomas, non-small cell lung carcinomas, bladder, prostate, and head and neck cancers. The gene encoding NY-ESO-2 also contains an alternative open reading frame that encodes the protein called CAMEL, a tumor antigen that is recognized by melanoma-specific cytotoxic T lymphocytes.
[00217] Similar to NY-ESO-1, NY-ESO-2 can be expressed in the testis and ovary. NY-ESO-2 can also be associated with a variety of cancers and immunogenic in individuals suffering from cancer or tumors. Therefore, NY-ESO-2 may be an antigen associated with numerous tumors.
The NY-ESO-2 antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (JFN--y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00219] The NY-ESO-2 antigen can enhance a cellular immune response in a patient who has had NY-ESO-2 administered about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to about 6000 times, compared to a cellular immune response in a patient who did not have NY-ESO-2 administered. In some modalities the NY-ESO-2 antigen can increase the cellular immune response in the patient who has had NY-ESO-2 administered about 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times , 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 5900 times or 6000 times if compared to the answer cellular immune system in the patient who did not have NY-ESO-2 administered.
[00220] The NY-ESO-2 antigen can increase interferon gamma (IFN-Y) levels in a patient who has had the NY-ESO-2 antigen administered about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to about 6000 times, compared to IFN-y levels in a patient who did not have onY-ESO-2 administered. In some modalities the NY-ESO-2 antigen can increase IFN-Y levels in the patient who has had the NY-ESO-2 antigen administered about 50 times, 100 times, 150 times, 200 times, 250 times, 300 times , 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times , 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times compared to IFN- y in the patient who did not have NY-ESO-2 antigen administered.
The NY-ESO-2 antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-NY-ESO-2 immune responses can be induced. The NY-ESO-2 antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The NY-ESO-2 antigen can comprise a consensus protein.
The nucleic acid sequence encoding the NY-ESO-2 consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the NY-ESO-2 consensus antigen can be an optimized codon and RNA for expression. In some embodiments, the nucleic acid sequence encoding the NY-ESO-2 consensus antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the NY-ESO-2 consensus antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the NY-ESO-2 consensus antigen can also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the NY-ESO-2 consensus antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the NY-ESO-2 consensus antigen amino acid sequence by a bond peptide. Nucleic encoding the NY-ESO-2 consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the NY-ESO-2 consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
The NY-ESO-2 consensus antigen can be the nucleic acid sequence SEQ ID NO:15, which encodes the amino acid sequence SEQ ID NO:16. SEQ ID NO:1 encodes the NY-ESO-2 consensus protein linked to an IgE core sequence. The NY-ESO-2 consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the NY-ESO-2 consensus protein may be free or unbound to an IgE main sequence and/or an HA tag.
In some embodiments, the NY-ESO-2 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:15. In some embodiments, the NY-ESO-2 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in the SEQ ID NO:16. The consensus antigen NY-ESO-2 can be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:16 .
Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to the consensus protein NY-ESO-2, immunogenic fragment of the consensus protein NY-ESO-2, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[00228] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of the consensus protein NY-ESO-2, immunogenic fragment of the consensus protein NY-ESO-2 and immunogenic fragments of proteins that have identity to the NY-ESO-2 consensus protein. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity to a full-length NY-ESO-2 consensus sequence, up to 85% a full-length NY-ESO-2 consensus sequence, up to 90% of identity with a full-length NY-ESO-2 consensus sequence, up to 91% identity with a full-length NY-ESO-2 consensus sequence, up to 92% identity with a NY-ESO-2 consensus sequence of full length, up to 93% identity with a full length NY-ESO-2 consensus sequence, up to 94% identity with a full length NY-ESO-2 consensus sequence, up to 95% identity with a consensus sequence of full-length NY-ESO-2, up to 96% identity with a full-length NY-ESO-2 consensus sequence, up to 97% identity with a full-length NY-ESO-2 consensus sequence, up to 98 % identity with a full-length NY-ESO-2 consensus sequence, and up to 99% identity with a NY-ES consensus sequence Full extension O-2 can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those indicated above for the NY-ESO-2 proteins presented herein are also provided.
[00229] In some embodiments, the nucleic acid sequence is free of coding sequence that encodes a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[00230] Some embodiments refer to fragments of SEQ ID NO:15. Fragments can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 15. The fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO:15. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:15. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
[00231] Additionally, an amino acid sequence of the NY-ESO-2 consensus protein is SEQ ID NO:16. The amino acid sequence of the consensus protein NY-ESO-2 linked to a major IgE is SEQ ID NO:16. The amino acid sequence of the consensus protein NY-ESO-2 linked to the major IgE can be linked to the HA tag.
Some embodiments refer to proteins that are homologous to SEQ ID NO:16. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO:16. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO:16. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences as shown in SEQ ID NO:16. Some modalities refer to immunogenic proteins that have 98% homology to the consensus protein sequences as shown in SEQ ID NO:16. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO:16.
Some embodiments refer to proteins that are identical to SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:16.
In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of major IgE. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of a consensus protein. Immunogenic fragments of SEQ ID NO:16 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, in minus 97%, at least 98%, or at least 99% of SEQ ID NO:16. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00235] Immunogenic fragments of proteins with homologous amino acid sequences to the immunogenic fragments of SEQ ID NO:16 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of proteins that are 95% or more homologous to SEQ ID NO:16. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00236] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO:16 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% of proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences shown in SEQ ID NO:16. In some embodiments, the fragments include a major sequence, such as a major immunoglobulin, such as an IgE major. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a core sequence, such as an IgE core.
[00237] As stated herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (9) PRAME
[00238] The vaccine of the present invention may comprise the cancer antigen PRAME, a fragment thereof, or a variant thereof. PRAME, encoded by the PRAME gene, is a protein made up of 509 amino acids and is expressed in testes, placenta, endometrium, ovary, adrenal glands, and in tissues derived from melanoma, lung, kidney, and head and neck carcinomas. PRAME is also expressed in adult and pediatric acute leukemias, and in multiple myeloma. PRAME contains an immunogenic nonapeptide capable of inducing a cytotoxic response when presented by HLA-A24. Studies show that overexpression of PRAME in cultured cells induces a casspace-independent cell death responsible for a slower proliferation rate. Other studies demonstrate that overexpression of PRAME also confers growth or survival advantages by antagonizing retinoic acid receptor (RAR) signaling, and is causally involved in the tumorigenic process. Interference of RAR signaling leads to a loss in the regulation of cell proliferation, development and differentiation.
[00239] PRAME may have an expression pattern similar to the testis cancer antigens MAGE, BAGE, and GAGE. PRAME, however, can be expressed in human melanomas and acute leukemias. PRAME can be recognized by cytolytic T lymphocytes. Therefore, PRAME may be an antigen associated with melanoma and leukemia.
The PRAME antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon gamma (JFN--y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00241] The PRAME antigen can enhance a cellular immune response in a patient who has had PRAME administered about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times , about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times , or about 300 times to about 6000 times, compared to a cellular immune response in a patient who did not have PRAME administered. In some modalities the PRAME antigen can increase the cellular immune response in the patient who has had PRAME administered approximately 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times , 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times compared to the cellular immune response in the patient who did not have PRAME administered.
[00242] The PRAME antigen can increase interferon gamma (IFN-Y) levels in a patient who has had the PRAME antigen administered about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to about 6000 times, compared to IFN-y levels in a patient who did not have PRAME administered. In some modalities the PRAME antigen may increase IFN-Y levels in the patient who has had the PRAME antigen administered approximately 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times , 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times compared to IFN-Yno pac levels patient who did not have PRAME antigen administered.
The PRAME antigen may comprise protein epitopes that make them particularly effective as immunogens against which anti-PRAME immune responses can be induced. The PRAME antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PRAME antigen can comprise a consensus protein.
[00244] The nucleic acid sequence encoding the consensus PRAME antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the PRAME consensus antigen can be an optimized codon and RNA for expression. In some embodiments, the nucleic acid sequence encoding the PRAME consensus antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the PRAME consensus antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the PRAME consensus antigen may also encode an immunoglobulin E (IgE) major sequence. The nucleic acid encoding the PRAME consensus antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the PRAME consensus antigen amino acid sequence by a peptide bond. Nucleic encoding the PRAME consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the PRAME consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
The PRAME consensus antigen can be the nucleic acid sequence SEQ ID NO:17, which encodes the amino acid sequence SEQ ID NO:18. SEQ ID NO:17 encodes the PRAME consensus protein linked to an IgE core sequence. The PRAME consensus protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the PRAME consensus protein may be free or unbound to an IgE core sequence and/or an HA tag.
[00247] In some embodiments, the PRAME consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:17. In some embodiments, the PRAME consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence defined in SEQ ID NO:18 . The PRAME consensus antigen can be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:18.
[00248] Some modalities are related to nucleic acid sequences encoding proteins identical or homologous to the PRAME consensus protein, immunogenic fragment of the PRAME consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence and up to 99% of a homology with a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
[00249] Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a coding sequence of a consensus protein disclosed herein include sequences that encode an IgE core sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed in this document.
[00250] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full extent of the PRAME consensus protein, immunogenic fragment of the PRAME consensus protein and immunogenic fragments of proteins that have identity to the consensus protein TO ME. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity to a full-length PRAME consensus sequence, up to 85% identity to a full-length PRAME consensus sequence, up to 90% identity to a full-length PRAME consensus sequence, up to 91% identity with a full-length PRAME consensus sequence, up to 92% identity with a full-length PRAME consensus sequence, up to 93% identity with a full-length PRAME consensus sequence of full length, up to 94% identity with a full length PRAME consensus sequence, up to 95% identity with a full length PRAME consensus sequence, up to 96% identity with a full length PRAME consensus sequence, up to 97 % identity with a full-length PRAME consensus sequence, up to 98% identity with a full-length PRAME consensus sequence, and up to 99% identity with a PRAME consensus sequence of full extension can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those noted above for the PRAME proteins set forth herein are also provided.
In some embodiments, the nucleic acid sequence is free of coding sequence encoding a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[00252] Some embodiments refer to fragments of SEQ ID NO:15. Fragments can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 17. Fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % homologous to the fragments of SEQ ID NO:17. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:17. In some embodiments, fragments include a sequence that encodes a major sequence, such as a major immunoglobulin, such as major IgE. In some embodiments, the fragments are free of coding sequences that encode a core sequence. In some embodiments, fragments are free of coding sequences that encode a core sequence, such as, for example, core IgE.
[00253] Additionally, an amino acid sequence of the PRAME consensus protein is SEQ ID NO:18. The amino acid sequence of the consensus PRAME protein linked to a major IgE is SEQ ID NO:18. The amino acid sequence of the consensus PRAME protein linked to major IgE can be linked to the HA tag.
Some embodiments refer to proteins that are homologous to SEQ ID NO:18. Some modalities refer to immunogenic proteins that have 95% homology to the consensus protein sequences as shown in SEQ ID NO:18. Some modalities refer to immunogenic proteins that have 96% homology to the consensus protein sequences as shown in SEQ ID NO:18. Some modalities refer to immunogenic proteins that have 97% homology to the consensus protein sequences as shown in SEQ ID NO:18. Some modalities refer to immunogenic proteins that have 98% homology to the consensus protein sequences as shown in SEQ ID NO:18. Some modalities refer to immunogenic proteins that have 99% homology to the consensus protein sequences as shown in SEQ ID NO:18.
[00255] Some embodiments refer to proteins that are identical to SEQ ID NO:18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences as shown in SEQ ID NO:18. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 96% identical to the consensus full length amino acid sequences as shown in SEQ ID NO: 18. Some modalities refer to immunogenic proteins that have a sequence of amino acid that is 97% identical to the consensus full length amino acid sequences as shown in SEQ ID NO: 18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the amino acid sequences of full length consensus as shown in SEQ ID NO: 18. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full length consensus amino acid sequences as shown in SEQ ID NO: 18.
[00256] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of the IgE leader. Consensus protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a consensus protein. The immunogenic fragments of SEQ ID NO: 18 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 18. In some embodiments, fragments include a core sequence, such as an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a major sequence, such as an IgE leader.
[00257] Immunogenic fragments of proteins with amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 18 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% proteins that are 95% or more homologous to SEQ ID NO:18. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the consensus protein sequences in this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences of this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the consensus protein sequences of this document. In some embodiments, the fragments include a core sequence, such as an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a major sequence, such as an IgE leader.
[00258] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 18 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequences shown in SEQ ID NO:18. In some embodiments, the fragments include a core sequence, such as an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a major sequence, such as an IgE leader.
[00259] As stated herein, with respect to binding a signal peptide or a main sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. (10) PSA
[00260] The vaccine of the present invention may comprise the cancer antigen, the prostate specific antigen (PSA; also known as gamma semiprotein or kallikrein-3 (KLK3)), a fragment thereof or a variant thereof. PSA is an androgen-regulated serine protease produced by prostate epithelial cells and prostate cancer cells and encoded by the KLK3 gene. PSA is commonly used as a marker serum for prostate cancer. PSA is a member of the kallikrein tissue family and cleaves semenogelins in the seminal clot after cleavage of the proenzyme to release the old enzyme, thus liquefying the semen to allow the sperm to move freely. Furthermore, the enzymatic activity of PSA is regulated by the concentration of zinc, that is, high concentrations of zinc inhibit the enzymatic activity of PSA.
The PSA antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon-gamma (JFN--y) and/or tumor necrosis of alpha factor (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00262] The PSA antigen may include protein epitopes that make them particularly effective as immunogens against which anti-PSA immune responses can be induced. The PSMA antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PSA antigen can comprise a consensus protein.
The nucleic acid sequence encoding the PSA consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the PSA consensus antigen can be an optimized codon or RNA for expression. In some embodiments, the nucleic acid sequence encoding the PSA consensus antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the PSA antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the PSA consensus antigen may also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the PSA consensus PSA antigen can further encode the IgE main sequence such that the IgE main sequence amino acid sequence is linked to the PSA consensus P antigen P amino acid sequence by a peptide bond. Nucleic encoding the PSA antigen may also include a nucleotide sequence encoding the main sequence of IgE. In some embodiments, the nucleic acid encoding the PSA consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
In some embodiments, the nucleic acid encoding the PSA consensus antigen may be a heterologous nucleic acid sequence and/or contain one or more heterologous nucleic acid sequences. (11) . PSMA
[00266] The vaccine of the present invention may comprise the cancer antigen, the prostate specific membrane antigen (PSMA; also known as carboxypeptidase II glutamate (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) and NAAG peptidase), a fragment thereof or a variant thereof. PSMA is encoded by the folate hydrolase 1 (FOLH1) gene. PSMA is a zinc metalloenzyme residing in membranes and in the extracellular space. PSMA is highly expressed in the human prostate and is up-regulated in prostate cancer. PSMA is also overexpressed in other cancers, such as solid tumors of the kidney, breast and colon.
[00267] The PSMA antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon-gamma (JFN--y) and/or tumor necrosis of alpha factor (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00268] The PSMA antigen may comprise protein epitopes that make them particularly effective as immunogens against which anti-PSMA immune responses can be induced. The PSMA antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PSMA antigen can comprise a consensus protein.
[00269] The nucleic acid sequence encoding the PSMA consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the PSMA consensus antigen can be a codon or RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the PSMA consensus antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the PSMA antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the PSMA consensus antigen may also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the PSMA consensus antigen may further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the PSMA consensus PSMA P antigen amino acid sequence by a peptide bond. Nucleic encoding the PSMA consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the PSMA consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
In some embodiments, the nucleic acid encoding the PSMA consensus antigen may be a heterologous nucleic acid sequence and/or contain one or more heterologous nucleic acid sequences. (12)STEAP
[00272] The vaccine of the present invention may comprise the cancer antigen, the six-transmembrane epithelial antigen of the prostate antigen (STEAP), a fragment thereof or a variant thereof. STEAP is a metalloreductase encoded by the STEAP1 gene. STEAP is widely expressed in prostate tissues and is up-regulated in cancer cells. STEAP is predicted to be a protein with six transmembranes and a cell surface antigen found at cell-cell junctions.
[00273] The STEAP antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN--Y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
The STEAP antigen can comprise protein epitopes that make them particularly effective as immunogens against which anti-STEAP immune responses can be induced. The STEAP antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The STEAP antigen can comprise a consensus protein.
The nucleic acid sequence encoding the consensus STEAP antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the consensus STEAP antigen can be a codon or RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus STEAP antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the STEAP antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the consensus STEAP antigen may also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the consensus STEAP antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the consensus STEAP antigen amino acid sequence by a peptide bond. Nucleic encoding the consensus STEAP antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the consensus STEAP antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
In some embodiments, the nucleic acid encoding the consensus STEAP antigen can be a heterologous nucleic acid sequence and/or contain one or more heterologous nucleic acid sequences. (13) . PSCA
[00278] The vaccine of the present invention may comprise the cancer antigen, the prostate specific stem cell antigen (PSCA), a fragment thereof or a variant thereof. PSCA is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein and is encoded by an androgen-responsive gene. PSCA is a member of the Thy-1/Ly-6 family of GPI-anchored cell surface antigens. PSCA is up-regulated in many cancers, including prostate, bladder and pancreatic cancers.
[00279] The PSCA antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon-gamma (JFN--y) and/or tumor necrosis of alpha factor (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
The PSCA antigen may comprise protein epitopes which make them particularly effective as immunogens against which anti-PSCA immune responses can be induced. The PSCA antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PSCA antigen can comprise a consensus protein.
[00281] The nucleic acid sequence encoding the PSCA consensus antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the PSCA consensus antigen can be an optimized codon or RNA for expression. In some embodiments, the nucleic acid sequence encoding the PSCA consensus antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the PSCA antigen can include multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the PSCA consensus antigen may also encode an immunoglobulin E (IgE) major sequence. Nucleic acid encoding the PSCA consensus antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the PSCA consensus antigen amino acid sequence by a peptide bond. Nucleic encoding the PSCA consensus antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the PSCA consensus antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
In some embodiments, the nucleic acid encoding the PSCA consensus antigen may be a heterologous nucleic acid sequence and/or contain one or more heterologous nucleic acid sequences. (14) . hTERT
[00284] The vaccine of the present invention may comprise the hTERT cancer antigen, a fragment thereof or a variant thereof. hTERT is a human telomerase reverse transcriptase that synthesizes a TTAGGG tag at the telomere end to prevent cell death due to chromosome shortening. Hyperproliferative cells may have abnormally high expression of hTERT. Abnormal expression of hTERT can also occur in hyperproliferative cells infected with HCV and HPV. Thus, immunotherapy for both HPV and HCV can be enhanced, targeting cells that express hTERT at abnormal levels. HPV and HCV antigens are discussed in more detail below. The hTERT cancer antigen may further be defined by US Patent Application No. 14/139,660, filed December 23, 2013, which is incorporated herein by reference in its entirety.
[00285] Furthermore, the expression of hTERT in dendritic cells transfected with hTERT genes can induce cytotoxic CD8+ T cells and elicit CD4+ T cells in an antigen-specific manner. Therefore, the use of hTERT expression in antigen presenting cells (APCs) to delay senescence and maintain their ability to present the antigen of choice can be used in immunotherapeutic methods such as the methods described here.
[00286] The hTERT antigen can be associated with or expressed by any number of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, recurrent papillary respiratory (RRP), anal cancer, breast cancer. head and neck and blood cancers. Therefore, the vaccine, when it includes the hTERT antigen described here, can be used to treat individuals suffering from a variety of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, respiratory papilloates recurrent (RRP), anal cancer, head and neck cancer, and blood cancers.
The nTERT antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon-gamma (JFN--y) and/or tumor necrosis of alpha factor (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00288] The hTERT antigen may comprise protein epitopes that make them particularly effective as immunogens against which anti-hTERT immune responses can be induced. The hTERT antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The hTERT antigen may comprise a consensus protein.
The nucleic acid sequence encoding the hTERT antigen or the consensus hTERT antigen can be optimized with regard to codon usage and the corresponding RNA transcripts. The nucleic acid encoding the hTERT antigen or the consensus hTERT antigen can be an optimized codon or RNA for expression. In some embodiments, the nucleic acid sequence encoding the hTERT antigen or the consensus consensus hTERT antigen can include a Kozak sequence (e.g., GCC ACC) to increase translation efficiency. Nucleic acid encoding the hTERT antigen or the consensus hTERT antigen can include multiple stop codons (e.g., TGA TGA) to increase translation termination efficiency.
Nucleic acid encoding the hTERT antigen or the consensus hTERT antigen may also encode an immunoglobulin E (IgE) main sequence. Nucleic acid encoding the hTERT antigen or the consensus hTERT antigen can further encode the IgE core sequence such that the IgE core sequence amino acid sequence is linked to the hTERT antigen or the antigen amino acid sequence. consensus hTERT by a peptide bond, respectively. Nucleic encoding the hTERT antigen or the consensus hTERT antigen may also include a nucleotide sequence encoding the IgE core sequence. In some embodiments, the nucleic acid encoding the hTERT antigen or the consensus HTERT antigen is free or does not contain a nucleotide sequence encoding the IgE core sequence.
In some embodiments, the nucleic acid encoding the hTERT antigen or the consensus hTERT antigen may be a heterologous nucleic acid sequence and/or contain one or more heterologous nucleic acid sequences. Nucleic acid encoding the hTERT antigen or consensus hTERT antigen can be mutated from the wild-type hTERT antigen such that one or more amino acids or residues in the amino acid sequence of the hTERT antigen or the hTERT antigen Consensus hTERT, respectively, is replaced by another amino acid or residue. The nucleic acid encoding the hTERT antigen or the consensus hTERT antigen can be mutated relative to the wild-type hTERT antigen, as that residue or residues in the amino acid sequence of the hTERT antigen or consensus hTERT antigen, respectively , is replaced by another residue, thus causing the immune system to cease to be tolerant to hTERT in the mammal that has received the nucleic acid encoding the hTERT antigen or the consensus hTERT antigen, or combinations thereof. Nucleic acid encoding the hTERT antigen or consensus hTERT antigen can be mutated from the wild-type hTERT antigen such that arginine 589, aspartate 1005 or arginine 589 and aspartate 1005 in the amino acid sequence of the hTERT antigen or consensus hTERT antigen are replaced by a tyrosine residue.
The hTERT antigen can be the nucleic acid sequence of SEQ ID NO:23, which encodes the amino acid sequence of SEQ ID NO:24. SEQ ID NO:23 encodes the hTERT protein linked to a sequence main IgE. The hTERT protein can be linked to the IgE main sequence and an HA tag. In other embodiments, the hTERT protein may be free or unbound to an IgE core sequence and/or an HA tag.
[00293] In some embodiments, the hTERT antigen may be the nucleic acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over a full length of the nucleic acid sequence defined in SEQ ID NO:23. In some embodiments, the hTERT consensus antigen can be the nucleic acid sequence encoding the amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the sequence of amino acids shown in SEQ ID NO:24. The hTERT antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a full length of the amino acid sequence shown in SEQ ID NO:24.
[00294] Some modalities are related to nucleic acid sequences encoding proteins homologous to hTERT protein, immunogenic fragment of hTERT protein and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding immunogenic proteins that have up to 95% homology to a sequence, up to 96% homology to a sequence, up to 97% homology to a sequence, up to 98% homology to a sequence, and up to 99% of a homology to a sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments shown here and the immunogenic fragments of proteins homologous to the proteins shown here are also provided.
[00295] Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments refer to nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences disclosed herein that are homologous to a consensus protein coding sequence disclosed herein include sequences that encode an IgE leader sequence linked to the 5' end of the coding sequence encoding the sequences of homologous proteins disclosed herein.
[00296] Some modalities are related to nucleic acid sequences encoding proteins with a specific percentage of identity in relation to the full length of the hTERT protein, immunogenic fragment of the hTERT protein and immunogenic fragments of proteins that have identity to the protein of hTERT. Such nucleic acid molecules encoding immunogenic proteins that have up to 80% identity to a full length hTERT sequence, up to 85% identity to a full length hTERT sequence, up to 90% identity to a full length hTERT sequence , up to 91% identity to a full length hTERT sequence, up to 92% identity to a full length hTERT sequence, up to 93% identity to a full length hTERT sequence, up to 94% identity to a full length hTERT sequence, up to 95% identity to a full length hTERT sequence, up to 96% identity to a full length hTERT sequence, up to 97% identity to a full length hTERT sequence, up to 98% identity to a full-length hTERT sequence and up to 99% identity to a full-length hTERT sequence can be provided. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins with percentage identities similar to those indicated above for the hTERT proteins presented herein are also provided.
[00297] In some embodiments, the nucleic acid sequence is free of coding sequence encoding a core sequence. In some embodiments, the nucleic acid sequence is free of coding sequence that encodes major IgE.
[00298] Some embodiments refer to fragments of SEQ ID NO: 23. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35 %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO: 23. Fragments can be at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO:23. Fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to the fragments of SEQ NO:23. In some embodiments, fragments include a sequence that encodes a leader sequence, such as, for example, an immunoglobulin leader, such as the IgE leader. In some embodiments, the fragments are free of coding sequences that encode a leader sequence. In some embodiments, fragments are free of coding sequences that encode a leader sequence, such as the IgE core.
[00299] Furthermore, the amino acid sequence of the hTERT protein is SEQ ID NO: 24. The amino acid sequence of the hTERT protein linked to a major IgE is SEQ ID NO: 24. The amino acid sequence of the protein of hTERT hTERT linked to the main IgE can be linked to the HA tag.
Some modalities refer to proteins that are homologous to SEQ ID NO: 24. Some modalities refer to immunogenic proteins that have 95% homology to the protein sequences as shown in SEQ ID NO: 24. Some modalities refer to immunogenic proteins that have 96% homology to the protein sequences as shown in SEQ ID NO: 24. Some modalities refer to immunogenic proteins that have 97% homology to the protein sequences as shown in SEQ ID NO: 24. Some modalities refer to immunogenic proteins that have 98% homology to the protein sequences as shown in SEQ ID NO: 24. Some modalities refer to immunogenic proteins that have 99% homology with the protein sequences as shown in SEQ ID NO: 24.
[00301] Some modalities refer to proteins that are identical to SEQ ID NO: 24. Some modalities refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the consensus full length amino acid sequences, as per shown in SEQ ID NO: 24. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 85% identical to the full length amino acid sequences as shown in SEQ ID NO: 24. Some embodiments relate to Immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length amino acid sequences as shown in SEQ ID NO: 24. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length amino acid sequences as shown in SEQ ID NO: 24. Some modalities refer to immunogenic proteins that have a sequence of a. amino acids that are 92% identical to the full length amino acid sequences as shown in SEQ ID NO: 24. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 93% identical to the full length amino acid sequences, as shown in SEQ ID NO: 24. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 94% identical to the full length amino acid sequences as shown in SEQ ID NO: 24. Some embodiments relate to to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length amino acid sequences as shown in SEQ ID NO: 24. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 96% identical to full-length amino acid sequences as shown in SEQ ID NO: 24. Some modalities refer to immunogenic proteins having m an amino acid sequence that is 97% identical to the full-length amino acid sequences as shown in SEQ ID NO: 24. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 98% identical to the amino acid sequences full length amino acid sequences as shown in SEQ ID NO: 24. Some embodiments refer to immunogenic proteins that have an amino acid sequence that is 99% identical to the full length amino acid sequences as shown in SEQ ID NO: 24.
[00302] In some embodiments, the protein is free of a major sequence. In some embodiments, the protein is free of the IgE leader. Protein fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% , at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% of a protein. Immunogenic fragments of SEQ ID NO:24 can be provided. Immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 24. In some embodiments, fragments include a core sequence, such as an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a major sequence, such as an IgE leader.
[00303] Immunogenic fragments of proteins with homologous amino acid sequences to the immunogenic fragments of SEQ ID NO:24 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% proteins that are 95% or more homologous to SEQ ID NO:18. Some modalities refer to immunogenic fragments that have 96% homology to the immunogenic fragments of the protein sequences in this document. Some modalities refer to immunogenic fragments that have 97% homology to the immunogenic fragments of the protein sequences in this document. Some modalities refer to immunogenic fragments that have 98% homology to the immunogenic fragments of the protein sequences in this document. Some modalities refer to immunogenic fragments that have 99% homology to the immunogenic fragments of the protein sequences in this document. In some embodiments, the fragments include a core sequence, such as an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a major sequence, such as an IgE leader.
[00304] Immunogenic fragments of proteins with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 24 can be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least minus 97%, at least 98% or at least 99% proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequences shown in SEQ ID NO:24. In some embodiments, the fragments include a core sequence, such as an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments are free from a main sequence. In some embodiments, the fragments are free of a major sequence, such as an IgE leader.
[00305] As noted herein, with respect to binding a signal peptide or a core sequence to the N-terminus of a protein, the signal peptide/main sequence replaces the N-terminal methionine of a protein that is encoded by the start codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences.
The fragments of SEQ ID NO: 23 may comprise 30 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 45 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 60 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 75 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 90 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 120 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 150 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 180 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 210 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 240 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 270 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 300 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 360 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 420 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 480 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 540 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 600 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 300 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 660 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 720 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 780 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 840 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:23 can comprise 900 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 960 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1020 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1080 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1140 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1200 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1260 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1320 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1380 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1440 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1500 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1560 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1620 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1680 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1740 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1800 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1860 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 1920 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 34 can comprise 1980 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2040 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2100 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2160 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2220 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2280 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2340 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2400 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2460 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2520 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2580 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2640 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2700 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2760 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2820 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2880 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 2940 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3000 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3060 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3120 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3180 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3240 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3300 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3360 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3420 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise 3480 or more nucleotides, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 can comprise coding sequences for the IgE leader sequences. In some embodiments, the fragments of SEQ ID NO: 23 do not comprise coding sequences for the IgE leader sequences.
[00307] Fragments may comprise less than 60 nucleotides, in some embodiments less than 75 nucleotides, in some embodiments less than 90 nucleotides, in some embodiments less than 120 nucleotides, in some embodiments less than 150 nucleotides, in some embodiments less than 180 nucleotides, in some embodiments less than 210 nucleotides, in some embodiments less than 240 nucleotides, in some embodiments less than 270 nucleotides, in some embodiments less than 300 nucleotides, in some embodiments less than 360 nucleotides, in some embodiments less than 360 nucleotides in some embodiments some embodiments less than 420 nucleotides, in some embodiments less than 480 nucleotides, in some embodiments less than 540 nucleotides, in some embodiments less than 600 nucleotides, in some embodiments less than 660 nucleotides, in some embodiments less than 720 nucleotides, in some embodiments less than 720 nucleotides in some modalities less than 780 nucleotides, in some modalities less than 840 nucleotides, in al some embodiments less than 900 nucleotides, in some embodiments less than 960 nucleotides, in some embodiments less than 1020 nucleotides, in some embodiments less than 1080 nucleotides, in some embodiments less than 1140 nucleotides, in some embodiments less than 1200 nucleotides , in some embodiments less than 1260 nucleotides, in some embodiments less than 1320 nucleotides, in some embodiments less than 1380 nucleotides, in some embodiments less than 1440 nucleotides, in some embodiments less than 1500 nucleotides, in some embodiments less than 1,500 nucleotides in some embodiments than 1560 nucleotides, in some embodiments less than 1620 nucleotides, in some embodiments less than 1680 nucleotides, in some embodiments less than 1740 nucleotides, in some embodiments less than 1800 nucleotides, in some embodiments less than 1860 nucleotides, in some modalities less than 1920 nucleotides, in some modalities me nos than 2040 nucleotides, in some embodiments less than 2100 nucleotides, in some embodiments less than 2160 nucleotides, in some embodiments less than 2220 nucleotides, in some embodiments less than 2280 nucleotides, in some embodiments less than 2340 nucleotides , in some embodiments less than 2400 nucleotides, in some embodiments less than 2460 nucleotides, in some embodiments less than 2520 nucleotides, in some embodiments less than 2580 nucleotides, in some embodiments less than 2640 nucleotides, in some embodiments less than 2580 nucleotides than 2700 nucleotides, in some embodiments less than 2760 nucleotides, in some embodiments less than 2820 nucleotides, in some embodiments less than 2860 nucleotides, in some embodiments less than 2940 nucleotides, in some embodiments less than 3000 nucleotides, in some modalities less than 3060 nucleotides, in some modalities less than 3120 nucleotides, in some embodiments less than 3180 nucleotides, in some embodiments less than 3240 nucleotides, in some embodiments less than 3300 nucleotides, in some embodiments less than 3420 nucleotides, in some embodiments less than 3480 nucleotides and in some embodiments modalities less than 35 10 nucleotides.
The fragments of SEQ ID NO: 24 may comprise 15 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 can comprise 18 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 21 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 24 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 30 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 36 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 42 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 48 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 54 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 60 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 66 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 724 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 90 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 120 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 150 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 180 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 210 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 240 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 270 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 300 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 330 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 360 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 390 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 420 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 450 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 480 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 510 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 540 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 570 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 600 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 630 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 660 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 690 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 720 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 750 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 780 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 810 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 840 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 870 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 900 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 930 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 960 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 990 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1020 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1050 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:24 can comprise 1080 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1110 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1140 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1170 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1200 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1230 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1260 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1290 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1320 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1350 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1380 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1410 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1440 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1470 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise 1500 or more amino acids, preferably including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO:24 can comprise coding sequences for the IgE core sequences. In some embodiments, the fragments of SEQ ID NO: 24 do not comprise coding sequences for the core IgE sequences.
Fragments may include less than 24 amino acids, in some embodiments less than 30 amino acids, in some embodiments less than 36 amino acids, in some embodiments less than 42 amino acids, in some embodiments less than 48 amino acids, in some embodiments less than 54 amino acids , in some embodiments less than 60 amino acids, in some embodiments less than 72 amino acids, in some embodiments less than 90 amino acids, in some embodiments less than 120 amino acids, in some embodiments less than 150 amino acids, in some embodiments less than 180 amino acids, in some embodiments some embodiments less than 210 amino acids, in some embodiments less than 240 amino acids, in some embodiments less than 260 amino acids, in some embodiments less than 290 amino acids, in some embodiments less than 320 amino acids, in some embodiments less than 350 amino acids, in some embodiments less than 380 amino acids, in some embodiments less than 410 amino acids, in some modality less than 440 amino acids, in some embodiments less than 470 amino acids, in some embodiments less than 500 amino acids, in some embodiments less than 530 amino acids, in some embodiments less than 560 amino acids, in some embodiments less than 590 amino acids, in some embodiments less than 590 amino acids of 620 amino acids, in some embodiments less than 650 amino acids, in some embodiments less than 680 amino acids, in some embodiments less than 710 amino acids, in some embodiments less than 740 amino acids, in some embodiments less than 770 amino acids, in some embodiments less than 800 amino acids, in some embodiments less than 830 amino acids, in some embodiments less than 860 amino acids, in some embodiments less than 890 amino acids, in some embodiments less than 920 amino acids, in some embodiments less than 950 amino acids, in some embodiments less than 980 amino acids, in some embodiments less than 980 amino acids, in some embodiments less than 1010 amino acids, in some embodiments less than 1040 to mino acids, in some embodiments less than 1070 amino acids, in some embodiments less than 1200 amino acids, in some embodiments less than 1230 amino acids, in some embodiments less than 1260 amino acids, in some embodiments less than 1320 amino acids, in some embodiments less than 1350 amino acids, in some embodiments less than 1380 amino acids, in some embodiments less than 1410 amino acids, in some embodiments less than 1440 amino acids, in some embodiments less than 1470 amino acids, and in some embodiments less than 1500 amino acids. (15) . MAGE A1
[00310] The vaccine of the present invention may comprise the cancer antigen, the melanoma associated antigen (MAGE A1), a fragment thereof or a variant thereof. MAGE A1, encoded by the MAGEA1 gene, is a 280 amino acid protein, and found to be expressed only by tumor cells or germ cells. MAGE A1 relies on DNA methylation for its repression in normal somatic tissues. These genes become activated in many types of tumors in the course of the genome-wide demethylation process that often accompanies tumorigenesis. Specifically, during neoplastic transformation, these genes are activated, expressed, and can become antigenic targets that are recognized and attacked by the immune system. Thus, MAGE genes participate in the immune process, targeting some early tumor cells for immune destruction. MAGE A1 can be expressed in numerous types of cancer, including, but not limited to, melanomas, lung carcinomas, and esophageal squamous cell carcinomas.
The MAGE A1 antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon-gamma (JFN--y) and/or tumor necrosis of alpha factor (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below. (16) . WT1
[00312] The vaccine of the present invention may comprise Wilm's tumor 1 (WT1) cancer antigen, a fragment thereof, or a variant thereof. WT1 is a transcription factor containing at the N-terminus, a proline/glutamine-rich DNA-binding domain and at the C-terminus, four zinc finger motifs. WT1 plays a role in the normal development of the urogenital system and interacts with a number of factors, for example, p53, a known tumor suppressor, and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with a cytotoxic drug.
[00313] Mutation of WT1 can lead to tumor or cancer formation, eg Wilm's tumor or tumors expressing WT1. Wilm's tumor usually forms in one or both kidneys before metastasizing to other tissues, for example, but not limited to, liver tissue, urinary tract system tissue, lymph tissue, or lung tissue. Consequently, Wilm's tumor can be considered a metastatic tumor. Wilm's tumor usually occurs in younger children (eg, less than 5 years of age) in both sporadic and hereditary forms. The WT1 cancer antigen may further be defined by PCT/US13/75141, filed December 23, 2013, which are incorporated herein by reference in their entirety.
The WT-1 antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon-gamma (JFN--y) and/or tumor necrosis of alpha factor (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below.
[00315] Indeed, the vaccine can be used to treat objects suffering from Wilm's tumor. The vaccine can be used to treat individuals suffering from a range of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papilloate (RRP), anal cancer, head cancer, and neck and blood cancers. The vaccine can also be used to treat a subject with cancer types or tumors that express WT1 to prevent the development of such tumors in the objects. The WT1 antigen may differ from the native type, "normal" TW1 gene, and therefore provide therapy or prophylaxis against a WT1 antigen-expressing tumor. Indeed, WT1 antigen sequences that differ from the native WT1 gene (e.g. mutant WT1 genes or sequences) are presented in this document.
The native WT1 gene transcripts are processed into a variety of mRNAs and the resulting proteins are not all of equal value for inducing an immune response. The mutant WT1 genes described in this document prevent alternative splicing, producing a full-length transcript and resulting in a stronger induction of effector T and B cell responses. The first mutant WT1 sequence is referred to as CON WT1 with modified Zinc Fingers or ConWT1-L. SED ID NO: 19 is a nucleic acid sequence encoding the WT1 CON WT1 antigen with modified Zinc Fingers. SEQ ID NO:20 is the amino acid sequence of the WT1 CON WT1 antigen with modified Zinc Fingers. The second mutant WT1 sequence is referred to as CON WT1 without Zinc Fingers or ConWT1-S. SED ID NO:21 is a nucleic acid sequence encoding the WTI CON WT1 antigen without Zinc Fingers. SEQ ID NO:22 is the amino acid sequence of the WT1 CON WT1 antigen without modified Zinc Fingers.
[00317] The WT1 antigen may be a consensus antigen (or immunogen) sequence derived from two or more species. The WT1 antigen may comprise a consensus sequence and/or modifications for enhanced expression. Modification can include codon optimization, RNA optimization, additional of a kozak sequence (eg, GCC ACC) for increased translation initiation and/or the addition of an immunoglobulin leader sequence to increase the immunogenicity of the WT1 antigen. The WT1 antigen may comprise a signal peptide such as an immunoglobulin signal peptide, for example, not limited to, an immunoglobulin E (IgE) or an immunoglobulin G (IcG) signal peptide. In some embodiments, the WT1 consensus antigen may comprise a hemagglutinin (HA) marker. The WT1 consensus antigen can be designed to elicit stronger and broader cellular and/or humoral immune responses than a codon-optimized WT1 antigen.
The WT1 consensus antigen may comprise one or more mutations in one or more zinc fingers, thereby triggering stronger and broader cellular and/or humoral immune responses than a codon-optimized WT1 antigen. One or more mutations can be a substitution of one or more amino acids that coordinate the zinc ion in one or more zinc fingers. One or more amino acids that coordinate the zinc ion can be a CCHH motif. Indeed, in some embodiments, one or more mutations can replace 1, 2, 3 or all 4 amino acids of the CCHH motif.
[00319] In another embodiment, the one or more mutations are such that residues 312, 317, 342 and 347 of SEQ ID NO:20 are any residues except cysteine (C) and residues 330, 334, 360 and 364 of SED ID NO:20 are any residues except histidine (H). In particular, one or more mutations are of a form in which residues 312, 317, 330, 334, 342, 360 and 364 of SEQ ID NO:20 are glycine (G).
[00320] In other modalities, one or more zinc fingers may be removed from the WT1 consensus antigen. One, two, three or all four of the zinc fingers can be removed from the WT1 consensus antigen.
The WT1 consensus antigen may be SEQ ID NO:19 of the nucleic acid encoding SEQ ID NO:20. In some embodiments, the WT1 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity over a full length nucleic acid sequence defined in SEQ ID NO:19 . In some embodiments, the WT1 consensus antigen may be the nucleic acid sequence encoding the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a full length defined amino acid sequence in SEQ ID NO:20.
In still other embodiments, the WT1 consensus antigen may be the nucleic acid sequence encoding the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity relative to a full length sequence defined in SEQ ID NO:20, provided that residues 312, 317, 342, and 347 of SEQ ID NO:20 are any residues except cysteine (C) and residues 330, 334, 360, and 364 of SEQ ID NO:20 are any residues except histidine (H). In other modalities, the WT1 consensus antigen may be the nucleic acid sequence encoding the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% identity relative to a full length amino acid sequence defined in SEQ ID NO:20, provided that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:20 are glycine (G).
The WT1 consensus antigen may be the amino acid sequence of SEQ ID NO:20. In some embodiments, the WT1 consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity over a full length amino acid sequence defined in SEQ ID NO:20. The consensus antigen of WT1 can be the amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the full length amino acid sequence defined in SEQ ID NO:20, as long as residues 312, 317 , 342, and 347 of SEQ ID NO:20 are any residues except cysteine (C) and residues 330, 334, 360, and 364 of SEQ ID NO:20 are any residues except histidine (H). In other embodiments, the WT1 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity over a full length amino acid sequence defined in SEQ ID NO:20, provided that residues 312, 317, 330, 334, 342, 347, 360 and 364 of SEQ ID NO:20 are glycine (G).
The WT1 consensus antigen may be SEQ ID NO:21 of the nucleic acid encoding SEQ ID NO:22. In some embodiments, the WT1 consensus antigen may be the nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity over a full length nucleic acid sequence defined in SEQ ID NO:21 . In some embodiments, the WT1 consensus antigen may be the nucleic acid sequence encoding the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a full length defined amino acid sequence in SEQ ID NO:22.
The WT1 consensus antigen may be the amino acid sequence of SEQ ID NO:22. In some embodiments, the WT1 consensus antigen may be the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity over a full length amino acid sequence defined in SEQ ID NO:22.
[00326] Immunogenic fragments of SEQ ID NO:20 and SEQ ID NO:22 can be provided. Immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, in at least 97%, at least 98%, or at least 99% of SEQ ID NO: 20 and/or SEQ ID NO: 22. In some embodiments, immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the SEQ ID NO:20, as long as residues 312, 317, 342, and 347 of SEQ ID NO:20 are present in the immunogenic fragment, in which case these residues are any residues except cysteine (C), and as long as residues 330 , 334, 360 and 364 of SEQ ID NO:20 are present in the immunogenic fragments, in which case these residues are any residues other than histidine (H). In other embodiments, immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least minus 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:20, provided that residues 312, 317, 330, 334, 342, 347, 360 and 364 of SEQ ID NO:20 are present in the immunogenic fragment, in this case, these residues are glycine (G).
In some embodiments, immunogenic fragments include a leader sequence, for example, an immunoglobulin leader sequence, such as the immunoglobulin E (IgE) leader sequence. In some embodiments, immunogenic fragments are free of a leader sequence.
[00328] Immunogenic protein fragments with amino acid sequences having identity to immunogenic fragments of SED ID NO: 20 and 22 can be provided. Such fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% of the proteins having 95% or greater identity to SEQ ID NO:20 and/or SEQ ID NO:22. Some modalities refer to immunogenic fragments that have 96% or more identity to the immunogenic fragments of the WT1 protein sequences herein. Some modalities refer to immunogenic fragments that have 97% or more identity to the immunogenic fragments of the WT1 protein sequences herein. Some modalities refer to immunogenic fragments that have 98% or more identity to the immunogenic fragments of the WT1 protein sequences herein. Some modalities refer to immunogenic fragments that have 99% or more identity to the immunogenic fragments of the WT1 protein sequences herein. In some embodiments, immunogenic fragments include a leader sequence, for example, an immunoglobulin leader sequence, such as the immunoglobulin leader sequence of IgE. In some embodiments, immunogenic fragments are free of a leader sequence.
[00329] Some embodiments refer to the immunogenic fragments of SEQ ID NO:19 and SEQ ID NO:21. Immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, by at least 97%, at least 98, or at least 99% of SEQ ID NO: 19 and/or SEQ ID NO: 21. In some embodiments, immunogenic fragments include sequences that encode a core sequence, e.g., a sequence immunoglobulin leader such as the IgE main sequence. In some embodiments, immunogenic fragments are free of coding sequences that encode a major sequence.
[00330] Immunogenic nucleic acid fragments with nucleotide sequences having identity to immunogenic fragments of SED ID NO:19 and SEQ ID NO:21 can be provided. Such fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% of the nucleic acids having 95% or greater identity to SEQ ID NO:19 and/or SEQ ID NO:21. Some embodiments refer to immunogenic fragments that have 96% or more identity to the immunogenic fragments of the WT1 nucleic acid sequences herein. Some modalities refer to immunogenic fragments that have 97% or more identity to the immunogenic fragments of the WT1 protein sequences herein. Some embodiments refer to immunogenic fragments that have 98% or more identity to the immunogenic fragments of the WT1 nucleic acid sequences herein. Some embodiments refer to immunogenic fragments that have 99% or more identity to the immunogenic fragments of the WT1 nucleic acid sequences herein. In some embodiments, immunogenic fragments include sequences that encode a core sequence, for example, an immunoglobulin leader sequence, such as the IgE core sequence. In some embodiments, immunogenic fragments are free of coding sequences that encode a major sequence. (17) . gp100
[00331] The vaccine of the present invention may comprise the cancer antigen glycoprotein 100 (gp100; also known as Trp2 and premelanosome protein (PMEL)), a fragment thereof or a variant thereof. gp1000 is encoded by the PMEL gene. It is a 70 kDa type 1 transmembrane glycoprotein of 661 amino acids that plays a central role in melanosome biogenesis as it is involved in the maturation of stage I to II melanosomes. gp100 drives the formation of streaks from within the mutivesicular bodies and is directly involved in the biogenesis of premelanosomes. gp100 is enriched in premelanosomes relative to mature melanosomes, but overexpressed by the proliferation of neonatal melanocytes and during tumor growth. The gp100 protein includes a variety of immunogenic epitopes that are recognized by peripheral blood cytotoxic T lymphocytes of melanoma patients and from tumor-infiltrating lymphocytes.
The gp100 antigen can induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon-gamma (JFN--y) and/or tumor necrosis of alpha factor (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below. (18) . Viral Antigens
[00333] The cancer antigen can be a viral antigen, a fragment thereof or a variant thereof. The viral antigen can be a hepatitis B virus antigen, a hepatitis C virus, or a human papilloma virus (HPV). HPV can be HPV 6, HPV 11, HPV 16 or HPV 18, as discussed below.
The viral antigen may induce antigen-specific T cells and/or high titer antibody responses, thereby inducing or eliciting a targeted or reactive immune response towards the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN--Y) and/or tumor necrosis factor alpha (TNF-α). In other modalities, the induced or provoked immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer that express the antigen, for example, without being limited to factors that negatively regulate the presentation of MHC , factors that positively regulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells , CTLA-4, PD-1, MDSCs, MCP-1 and a checkpoint molecule, which is described in more detail below. (The). Hepatitis B virus antigen
[00335] The viral antigen can be an antigen of the hepatitis B virus (HBV), a fragment thereof or a variant thereof. HBV antigen can be associated with or cause liver cancer. In some embodiments, the HBV antigen can be heterologous nucleic acid molecule(s), such as plasmid(s), that encode one or more of the HBV antigens. HBV antigens can be full-length or immunogenic fragments of full-length proteins.
[00336] The HBV antigen may comprise consensus sequences and/or one or more modifications for enhanced expression. Genetic modifications, including codon optimization, RNA optimization, and the addition of a highly efficient immunoglobulin core sequence to enhance the immunogenicity of the constructs can be included in the modified consensus sequences. The HBV consensus antigen may comprise a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and, in some embodiments, may comprise an HA tag. Immunogens can be designed to elicit stronger and broader cellular immune responses than the corresponding codon optimized immunogens.
[00337] The HBV antigen can be an HBV core protein, an HBV surface protein, an HBV DNA polymerase, an HBV protein encoded by gene X, fragment thereof, variant thereof, or a combination thereof. The HBV antigen can be an HBV genotype A core protein, an HBV genotype B core protein, an HBV genotype C core protein, an HBV genotype D core protein, a core protein of HBV genotype E, an HBV genotype F core protein, an HBV genotype G core protein, an HBV genotype H core protein, an HBV genotype A surface protein, a surface protein of HBV genotype B, an HBV genotype C surface protein, an HBV genotype D surface protein, an HBV genotype E surface protein, an HBV genotype F surface protein, a surface protein of HBV genotype G, an HBV genotype H surface protein, fragment thereof, variant thereof, or a combination thereof. The HBV antigen can be a consensus HBV core protein or a consensus HBV surface protein.
In some embodiments, the HBV antigen may be an HBV genotype A consensus core DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV genotype A core protein or an HBV genotype A consensus core protein sequence.
In other embodiments, the HBV antigen may be an HBV genotype B consensus core DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV genotype B core protein or an HBV genotype B consensus core protein sequence.
[00340] In still other embodiments, the HBV antigen may be an HBV genotype C consensus core DNA sequence construct, an IgE core sequence linked to a consensus sequence for the genotype C core protein HBV or an HBV genotype C consensus core protein sequence.
In some embodiments, the HBV antigen may be an HBV genotype D consensus core DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV genotype D core protein or an HBV genotype D consensus core protein sequence.
In other embodiments, the HBV antigen may be an HBV genotype E consensus core DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV genotype E core protein or an HBV genotype E consensus core protein sequence.
In some embodiments, the HBV antigen may be an HBV F genotype core consensus DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV F genotype core protein or an HBV F genotype consensus core protein sequence.
In other embodiments, the HBV antigen may be an HBV G genotype consensus core DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV G genotype core protein or an HBV genotype G consensus core protein sequence.
In some embodiments, the HBV antigen may be an HBV genotype H consensus core DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV genotype H core protein or an HBV genotype H consensus core protein sequence.
[00346] In still other embodiments, the HBV antigen may be an HBV genotype A consensus surface DNA sequence construct, an IgE core sequence linked to a consensus sequence for the genotype A surface protein of HBV or an HBV genotype A consensus surface protein sequence.
[00347] In some embodiments, further, the HBV antigen may be an HBV genotype B consensus surface DNA sequence construct, an IgE core sequence linked to a consensus sequence for the genotype B surface protein of HBV or an HBV genotype B consensus surface protein sequence.
In other embodiments, the HBV antigen may be an HBV genotype C consensus surface DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV genotype C surface protein or an HBV genotype C consensus surface protein sequence.
[00349] In still other embodiments, the HBV antigen may be an HBV genotype D consensus surface DNA sequence construct, an IgE core sequence linked to a consensus sequence for the genotype D surface protein of HBV or an HBV genotype D consensus surface protein sequence.
[00350] In some embodiments, further, the HBV antigen may be an HBV genotype E consensus surface DNA sequence construct, an IgE core sequence linked to a consensus sequence for the genotype E surface protein HBV or an HBV genotype E consensus surface protein sequence.
In other embodiments, the HBV antigen may be an HBV F genotype consensus surface DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV F genotype surface protein or an HBV F genotype consensus surface protein sequence.
[00352] In still other embodiments, the HBV antigen may be an HBV G genotype consensus surface DNA sequence construct, an IgE core sequence linked to a consensus sequence for the G genotype surface protein of HBV or an HBV genotype G consensus surface protein sequence.
In other embodiments, the HBV antigen may be an HBV genotype H consensus surface DNA sequence construct, an IgE core sequence linked to a consensus sequence for the HBV genotype H surface protein or an HBV genotype H consensus surface protein sequence. (ç). Hepatitis C virus antigen
[00354] The viral antigen can be an antigen of the hepatitis C virus (HCV), a fragment thereof or a variant thereof. The HCV antigen can be associated with or cause liver cancer. In some embodiments, the HCV antigen can be heterologous nucleic acid molecule(s), such as plasmid(s), that encode one or more of the HCV antigens. HCV antigens can be full-length or immunogenic fragments of full-length proteins.
The HCV antigen may comprise consensus sequences and/or one or more modifications for enhanced expression. Genetic modifications, including codon optimization, RNA optimization, and the addition of a highly efficient immunoglobulin core sequence to enhance the immunogenicity of the constructs can be included in the modified consensus sequences. The consensus HCV antigen may comprise a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and, in some embodiments, may comprise an HA tag. Immunogens can be designed to elicit stronger and broader cellular immune responses than the corresponding codon optimized immunogens.
[00356] The HCV antigen may be an HCV nucleocapsid protein (eg core protein), an HCV envelope protein (eg E1 and E2), a non-structural HCV protein (eg NS1 , NS2, NS3, NS4a, NS4b, NS5a and NS5b), a fragment thereof, a variant thereof, or a combination thereof. (ç). Human Papilloma Virus
[00357] The viral antigen can be an HPV antigen, a fragment thereof or a variant thereof. The HPV antigen can be from HPV types 16, 18, 31, 33, 35, 45, 52 and 58, which cause cervical cancer, rectal cancer and/or other types of cancer. The HPV antigen can be HPV types 6 and/or 11, which cause genital warts and are known to cause head and neck cancer. The HPV antigen can be HPV types 16 and/or 18, which cause cervical cancer. The HPV antigen can be from HPV types 6, 11, and/or 16, which cause RRP and anal cancers. The HPV cancer antigen may further be defined by US Patent No. 8,168,769, filed July 30, 2007, US Patent No. 8,389,706 filed January 21, 2010, by US Patent Application No. 13 /271,576, filed October 21, 2011 and by US Patent Application No. 61/777,198, filed March 12, 2013, each of which is incorporated herein by reference in its entirety.
[00358] The HPV antigens can be the E6 or E7 domains of HPV of each HPV type. For example, for HPV type 16 (HPV16), the HPV16 antigen may include the HPV16 E6 antigen, the HPV16 E7 antigen, fragments, variants or combinations thereof. Similarly, the HPV antigen can be HPV 6 E6 and/or E7, HPV 11 E6 and/or E7, HPV 16 E6 and/or E7, HPV 18 E6 and/or E7, HPV 31 E6 and/or E7, HPV 33 E6 and/or E7, HPV 52 E6 and/or E7 or HPV 58 E6 and/or E7, fragments, variants or combinations thereof. (d). herpes virus
[00359] The viral antigen may be a herpes viral antigen. The herpes viral antigen may be an antigen selected from the group consisting of CMV, HSV 1, HSV2, VZV, CeHV1, EBV, roseolovirus, herpes virus associated with Kaposi's sarcoma, or MuHV, and preferably CMV , HSV 1, HSV2, CeHV1 and VZV.
[00360] An HCMV gB consensus protein (SEQ ID NO:26), an HCMV-GM consensus protein (SEQ ID NO:28), an HCMV gN consensus protein (SEQ ID NO:30), a consensus protein HCMV gH (SEQ ID NO:32), an HCMV gL consensus protein (SEQ ID NO:34), an HCMV-gO consensus protein (SEQ ID NO:36), an HCMV-UL128 consensus protein (SEQ ID NO: :38), an HCMV UL130 consensus protein (SEQ ID NO:40), an HCMV-UL-131A consensus protein (SEQ ID NO:42), an HCMV-UL-83 (pp65) consensus protein (SEQ ID NO: 44).
Nucleic acid sequences including sequences encoding SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44. Nucleic acid molecules encoding consensus amino acid sequences were generated. Vaccines can comprise one or more nucleic acid sequences that encode one or more of the consensus versions of the immunogenic proteins selected from this group of sequences generated to optimize stability and expression in humans. Nucleic acid sequence encoding HCMV-gB protein (SEQ ID NO:25), nucleic acid sequence encoding HCMV-gM consensus protein (SEQ ID NO:27), nucleic acid sequence encoding consensus protein HCMV-gN (SEQ ID NO:29), HCMV-gH consensus protein encoding nucleic acid sequence (SEQ ID NO:31), nucleic acid sequence encoding HCMV-gL consensus protein (SEQ ID NO: 33), nucleic acid sequence encoding HCMV-gO consensus protein (SEQ ID NO: 35), nucleic acid sequence encoding HCMV-UL128 consensus protein (SEQ ID NO: 37), nucleic acid sequence encoding encodes the HCMV-UL130 consensus protein (SEQ ID NO: 39), nucleic acid sequence encoding the HCMV-UL-131A consensus protein (SEQ ID NO: 41), nucleic acid sequence encoding the HCMV consensus protein -UL-83 (pp65) (SEQ ID NO: 43). The nucleic acid sequence can additionally have a major IgE attached to the 5' end.
[00362] Due to the evolutionary divergence from clinical isolates and the wide genetic differences between the strains prevalent among circulating humans, consensus amino acid sequences for each of the immunogenic proteins were generated. Consensus amino acid sequences for gB, gM, gH, gL, gE, gI, gK, gC, gD, UL128, UL130, UL-131A and UL-83 (pp65) were based on sequences from human clinical isolates. Due to the great evolutionary divergence of the gN protein, the consensus sequence was generated from only one (gN-4c) of seven serotypes that represents the highest seroprevalence (gN-4). Likewise, in the gO case, consensus amino acid sequences were generated from one (gO-5) of eight serotypes due to that reported specific binding of serotypes to the gN-4c serotype.
[00363] As described above, the herpes viral antigen may be a consensus herpes virus. The consensus herpes viral antigen can be provided with a signal peptide. In some embodiments, the main IgE is linked to the N-terminus. As described herein, when referring to a signal peptide linked to the N-terminus of a consensus sequence, it is specifically intended to include embodiments in which the N-terminal Xaa residue of Consensus sequences is replaced by a signal peptide. That is, as used herein, Xaa should refer to any amino acid or no amino acid. Proteins comprising a consensus sequence set forth herein in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 may comprise the N-terminal Xaa free sequences.
[00364] Amino acid sequences were generated, comprising, in each specific instance, the IgE main sequence at the N-terminus of the herpes virus immunogenic protein consensus sequences. In some embodiments, nucleic acid constructs are provided in which two or more herpes virus antigens are expressed as fusion proteins linked together by proteolytic cleavage sites. A furin proteolytic cleavage site is an example of a proteolytic cleavage site that can bind herpes virus antigens into a fusion protein expressed by a construct. The herpes family cancer viral antigen may further be any antigen disclosed in US Patent Application No. 13/982,457, the contents of which are incorporated by reference in their entirety. 3. Vaccine in combination with immune checkpoint inhibitor
The vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e. an immune checkpoint inhibitor). Immune checkpoint molecules are described below in more detail. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component of the immune system, such as MHC presentation class, T cell presentation and/or differentiation, B cell presentation and/or differentiation, any cytokine, chemokine or signaling and/or differentiation for immune cell proliferation.
Such inhibitor may be a nucleic acid sequence, an amino acid sequence, a small molecule or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. Nucleic acid can also include additional sequences that encode linker or tag sequences that are linked to the immune checkpoint inhibitor by a peptide bond. The small molecule can be one of low molecular weight, for example, less than 800 Daltons, the organic or inorganic compound that can serve as an enzyme substrate, a ligand (or an analogue thereof) linked by a protein or a nucleic acid or a regulator of a biological process. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.
In some embodiments, the immune checkpoint inhibitor can be one or more of: nucleic acid sequences encoding an antibody, a variant thereof, a fragment thereof, or a combination thereof. In other embodiments, the immune checkpoint inhibitor can be an antibody, a variant thereof, a fragment thereof, or a combination thereof. The. Immune Checkpoint Molecule
The immune checkpoint molecule can be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. Nucleic acid can also include additional sequences that encode linker or tag sequences that are linked to the immune checkpoint inhibitor by a peptide bond. The small molecule can be one of low molecular weight, for example, less than 800 Daltons, the organic or inorganic compound that can serve as an enzyme substrate, a ligand (or an analogue thereof) linked by a protein or a nucleic acid or a regulator of a biological process. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. (1) . PD-1 and PD-L1
[00369] The immune checkpoint molecule can be programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), a fragment thereof, a variant thereof, or a combination of the same. PD-1 is a cell surface protein encoded by the PDCD1 gene. PD-1 is a member of the immunoglobulin superfamily and is expressed on T cells and pro-B cells and thus contributes to the fate and/or differentiation of these cells. In particular, PD-1 is a type 1 membrane protein of the CD28/CTLA-4 T cell regulator family and downregulates T cell receptor (TCR) signals, thus downregulating immune responses. PD-1 can down-regulate CD8+ T cell responses, and thus inhibit CD8-mediated cytotoxicity and enhance tumor growth.
[00370] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is up-regulated in macrophages and dendritic cells (DCs) in response to treatments with LPS and GM-CSF and in T cells and B cells from B cell and TCR receptor signaling. PD-L1 is expressed by many tumor cell lines, including myelomas, mast cell tumors and melanomas. B. Anti-immune checkpoint molecule antibody
As described above, the immune checkpoint inhibitor can be an antibody. The antibody may bind or react with an antigen (ie, the immune checkpoint molecule described above). Therefore, the antibody can be considered an anti-immune checkpoint molecule antibody or an immune checkpoint molecule antibody. The antibody may be encoded by a nucleic acid sequence contained in
The antibody may include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region. At least one constant heavy chain region can include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2) and a constant heavy chain region 3 (CH3) and/or a hinge region.
[00373] In some embodiments, the heavy chain polypeptide may include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.
The heavy chain polypeptide may include a tuned complementarity determining region ("CDR"). The CDR fit can contain three hypervariable regions of the VH region. Coming from the N-terminus of the heavy chain polypeptide, these CDRs are denoted "CDR1", "CDR2" and "CDR3", respectively. The heavy chain polypeptide's CDR1, CDR2 and CDR3 may contribute to antigen binding or recognition.
The light chain polypeptide may include a variable light chain region (VL) and/or a constant light chain region (CL). The light chain polypeptide can include a tuned complementarity determining region ("CDR"). The CDR fit can contain three hypervariable regions of the VL region. Coming from the N-terminus of the light chain polypeptide, these CDRs are denoted "CDR1", "CDR2" and "CDR3", respectively. CDR1, CDR2 and CDR3 of the light chain polypeptide can contribute to antigen binding or recognition.
The antibody may comprise a heavy and an adjusted light chain complementarity determining region ("CDR"), respectively, interposed between a heavy chain framework and an adjusted light chain framework ("FR"), which provides support for the CDRs and define the spatial relationship of the CDRs in relation to each other. The fitted CDR can contain three hypervariable regions of a heavy or light chain V region. Coming from the N-terminus of a heavy or light chain, these regions are denoted as "CDR1", "CDR2" and "CDR3", respectively. An antigen binding site, therefore, can include six CDRs, comprising the CDR tuned from each of a heavy and a light chain V region.
The antibody may be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE and IgG. Immunoglobulin can include heavy chain polypeptide and light chain polypeptide. The immunoglobulin heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The immunoglobulin light chain polypeptide can include a VL region and a CL region.
[00378] Additionally, the proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, the two of which (the F(ab) fragments) of each comprise a covalent heterodimer that includes an intact antigen binding site. The pepsin enzyme is able to cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which comprises both antigen binding sites. Therefore, the antibody can be Fab or F(ab')2. Fab can include heavy chain polypeptide and light chain polypeptide. The Fab heavy chain polypeptide can include the VH region and the CH1 region. The Fab light chain can include the VL region and the CL region.
The antibody may be a polyclonal or a monoclonal antibody. The antibody can be a chimeric antibody, a single chain antibody, an affinity maturation antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody may be an antibody from a non-human species that binds to the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from an immunoglobulin molecule. human. (1). PD-1 antibody
The antibody of the anti-immune checkpoint molecule can be an anti-PD-1 antibody (also referred to herein as "PD-1 antibody), a variant thereof, a fragment thereof, or a combination thereof. The antibody PD-1 may be Nivolumab The anti-DP-1 antibody may inhibit PD-1 activity, thereby inducing, eliciting, or enhancing an immune response against a tumor or cancer and slowing tumor growth. (two). PD-L1 antibody
The antibody of the anti-immune checkpoint molecule can be an anti-PD-L1 antibody (also referred to herein as "PD-L1 antibody), a variant thereof, a fragment thereof, or a combination thereof. The antibody anti-DP-L1 can inhibit PD-L1 activity, thereby inducing, eliciting or enhancing an immune response against a tumor or cancer and slowing tumor growth. 4. Vaccine Constructs and Plasmids
The vaccine may include nucleic acid constructs or plasmids encoding the antigens and/or antibodies described above. Nucleic acid constructs or plasmids can include or contain one or more heterologous nucleic acid sequences. Provided herein are generic constructs which can comprise a nucleic acid sequence encoding the antigens described above and/or the antibodies. The genetic construct may be present in the cell as a functional extrachromosomal molecule. The genetic construct can be a linear minichromosome, including centromere, telomeres or plasmids or cosmids. Genetic constructs can include or contain one or more heterologous nucleic acid sequences.
The genetic constructs can be in the form of plasmids that express the antigens described above and/or antibodies in any order.
The genetic construct can also be part of a genome of a recombinant viral vector, including recombinant adenovirus, recombinant adenovirus-associated virus and the recombinant vaccine. The genetic construct can be part of the genetic material of live attenuated microorganisms or recombinant microbial vectors that live in cells.
[00385] Genetic constructs may comprise regulatory elements for gene expression of nucleic acid coding sequences. The regulatory elements can be a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal.
Nucleic acid sequences can form a genetic construct that can be a vector. The vector may be capable of expressing the antigens described above and/or antibodies in a mammalian cell in an amount effective to elicit an immune response in the mammal. The vector can be a recombinant. The vector may comprise a heterologous nucleic acid encoding the antigens and/or antibodies described above. The vector can be a plasmid. The vector may be useful to transfect cells with nucleic acid encoding the above described antigens and/or the antibodies, the transformed host cell being cultured and maintained where expression of the above described antigens and/or antibodies takes place.
[00387] Coding sequences can be optimized for stability and high expression levels. In some cases, codons are selected to reduce the formation of RNA secondary structure such as that formed due to intramolecular binding.
The vector may comprise a heterologous nucleic acid encoding the antigens and/or antibodies described above and may further comprise an initiation codon, which may be upstream of one or more cancer antigen coding sequence(s), which it may be downstream of the coding sequence(s) for the antigens and/or antibodies described above. The start and stop codon can be in-frame with the coding sequence(s) of the antigens and/or antibodies described above. The vector may further comprise a promoter which is operably linked to the antigen and/or antibody coding sequence(s) described above. The promoter operably linked to the coding sequence(s) of the antigens and/or antibodies described above may be a simian virus 40 (SV40) promoter, a murine mammary tumor virus (MMTV) promoter, a virus promoter immunodeficiency virus (HIV) promoter, such as bovine immunodeficiency virus (BIV) long repeat ends (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter ), such as the CMV immediate early promoter, Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine or human metallothionein. The promoter can also be a tissue-specific promoter, such as a natural or synthetic, muscle- or skin-specific promoter. Examples of such promoters are described in US patent application publication No. US20040175727, the contents of which are incorporated herein in its entirety.
[00389] The vector may further comprise a polyadenylation signal, which may be downstream of the sequence(s) encoding the antigens and/or antibodies described above. The polyadenylation signal can be an SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human β-globin polyadenylation signal. The SV40 polyadenylation signal can be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).
[00390] The vector may comprise an enhancer upstream of the antigens and/or antibodies described above. The enhancer may be needed for DNA expression. The enhancer can be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as CMV, HA, RSV or EBV. Polynucleotide function enhancements are described in U.S. Patents No. 5,593,972, No. 5,962,428 and No. WO94/016737, the contents of which are incorporated in their entirety by reference.
[00391] The vector may also include a mammalian origin of replication in order to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The vector may be pVAX1, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may comprise the Epstein-Barr virus origin of replication and the EBNA-1 coding region of the nuclear antigen, which may produce high copy episomal replication no integration. The vector can be pVAX1 or a variant of pVax1, with alterations, such as the plasmid variant described herein. The pVaxl variant plasmid is a 2998 base pair variant of the vector backbone plasmid pVAX1 (Invitrogen, CA Carlsbad). The CMV promoter is located at bases 137-724. The T7 promoter/initiation site is at bases 664-683. Multiple cloning sites are in bases 696-811. Bovine GH polyadenylation signal is in bases 829-1053. The kanamycin resistance gene is at bases 1226-2020. The origin of pUC is in bases 2320-2993.
Based on the pVAX1 sequence provided by Invitrogen, the following mutations were found in the pVAX1 sequence which was used as the backbone of plasmids 1-6 established herein: C>G241^ in the CMV promoter C>T^1942^ main structure, downstream of the bovine growth hormone (bGHpolyA) polyadenylation signal -^2876^ main structure, downstream of the kanamycin gene C>T^3277^ in the origin of replication (Ori) pUC of the high number mutation of copies (see Nucleic Acid Research 1985) G>C^3753^ across the end of Orig pUC upstream of the RNASeH site
Base pairs 2, 3 and 4 are changed from ACT to CTG in the backbone, upstream of the CMV promoter.
[00394] The main structure of the vector can be pAV0242. The vector may be a replication defective adenovirus type 5 (Ad5) vector.
[00395] The vector may also comprise a regulatory sequence, which may be suitable for expression of the gene in a mammalian or human cell to which the vector is administered. The cancer antigen sequence or sequences disclosed herein may comprise a codon, which may allow for more efficient transcription of the coding sequence in the host cell.
[00396] The vector can be pSE420 (Invitrogen, San Diego, California), which can be used for protein production in Escherichia coli (E. coli). The vector can also be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in Saccharomyces cerevisiae yeast strains. The vector may also be the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, California), which can be used for protein production in insect cells. The vector can also be pcDNA I or pcDNA3 (Invitrogen, San Diego, California), which can be used for protein production in mammalian cells, such as Chinese hamster ovary (CHO) cells. The vector can be expression vectors or systems for the production of proteins by routine techniques and available starting materials including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated in its entirety by reference.
In some embodiments, the vector may comprise one or more nucleic acid sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15 and/or 17. 5. Pharmaceutical Compositions of the Vaccine
[00398] The vaccine may be in the form of a pharmaceutical composition. The pharmaceutical composition can comprise the vaccine. Pharmaceutical compositions can comprise about 5 nanograms to about 10 mg of vaccine DNA. In some embodiments, pharmaceutical compositions in accordance with the present invention comprise about 25 nanograms to about 5 mg of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 50 nanograms to about 1 mg of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 0.1 to about 500 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 1 to about 350 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 5 to about 250 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 10 to about 200 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 15 to about 150 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 20 to about 100 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 25 to about 75 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 30 to about 50 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 35 to about 40 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 100 to about 200 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions comprise about 10 micrograms to about 100 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions comprise about 20 micrograms to about 80 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions comprise about 25 micrograms to about 60 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions comprise about 30 nanograms to about 50 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions comprise about 35 nanograms to about 45 micrograms of vaccine DNA. In some preferred embodiments, the pharmaceutical compositions contain about 0.1 to about 500 micrograms of vaccine DNA. In some preferred embodiments, pharmaceutical compositions contain about 1 to about 350 micrograms of vaccine DNA. In some preferred embodiments, pharmaceutical compositions contain about 25 to about 250 micrograms of vaccine DNA. In some embodiments, pharmaceutical compositions contain about 100 to about 200 micrograms of vaccine DNA.
[00399] In some embodiments, pharmaceutical compositions according to the present invention comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95 or 100 nanograms of vaccine DNA. In some embodiments, pharmaceutical compositions can comprise at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 , 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 , 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350 , 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475 , 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700 , 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825 , 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895. 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950 , 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition can comprise at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 , 8, 8.5, 9, 9.5 or 10 mg or more of the vaccine DNA.
[00400] In other embodiments, the pharmaceutical composition may comprise up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms of vaccine DNA. In some embodiments, the pharmaceutical composition can comprise up to and including 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895. 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition can comprise up to and including 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7, 5, 8, 8.5, 9, 9.5 or 10 mg of vaccine DNA.
[00401] The pharmaceutical composition may additionally comprise other agents for formulation purposes, according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, free of pyrogen and free of particulate matter. An isotonic formulation is preferably used. In general, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions, such as phosphate-buffered saline, are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstricting agent is added to the formulation.
[00402] The vaccine may additionally comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, adjuvants, carriers or diluents. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents such as immunostimulating complexes (ISCOMS), incomplete Freund's adjuvant, LPS analogue including monophosphoryl A lipid, muramyl peptides, quinone analogues, vesicles, such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles or other known transfection facilitating agents.
[00403] The transfection facilitating agent is a polyanion, polycation including poly-L-glutamate (LGS) or lipid. The transfection facilitating agent is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg/ml. The transfection facilitating agent may also include surface active agents such as immunostimulating complexes (ISCOMS), incomplete Freund's adjuvant, LPS analogue including monophosphoryl A lipid, muramyl peptides, quinone analogues and vesicles such as squalene and squalene and acid hyaluronic can also be used administered in conjunction with the genetic construct. In some embodiments, DNA vector vaccines can also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see, for example, WO9324640 ), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS) or lipid. The concentration of the transfection agent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ ml, less than 0.100 mg/ml, less than 0.050 mg/ml or less than 0.010 mg/ml.
[00404] The pharmaceutically acceptable excipient can be an adjuvant. Adjuvants can be other genes that are expressed on alternative plasmid or are delivered as proteins in combination with the above plasmid in the vaccine. The adjuvant can be selected from the group consisting of: a-interferon (IFN-a), e-interferon (IFN-β), y- interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM— CSF, epidermal growth factor (EGF), cutaneous T cell attracting chemokine (CTACK), epithelial thymus expressed chemokine (TECK), mucosa-associated epithelial chemokine (ECM), IL-12, IL-15, MHC, CD80 ,CD86 including IL-15 having the signal sequence deleted and optionally including the IgE signal peptide. Adjuvant can be IL—12, IL—15, IL—28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM—CSF, epidermal growth factor (EGF), IL—1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL—18 or a combination thereof. In an exemplary embodiment, the adjuvant is IL-12.
Other genes that may be useful adjuvants include those encoding: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1 , MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL -4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo -1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC 5, TRAIL- R3, TRAIL-R4, RANK, RANK Linker, Ox40, Ox40 Linker, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof. 6. Combination vaccines to treat specific cancers
[00406] The vaccine may be in the form of various combinations of cancer antigens, as described above, to treat specific types of cancer or tumors. Depending on the combination of one or more cancer antigens, several types of cancer or other types of tumor can be targeted with the vaccine. These cancers can include melanoma, blood cancers (eg, leukemia, lymphoma, myeloma), lung carcinomas, esophageal squamous cell carcinomas, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocarcinoma, head and neck , in the brain, anal cancer, small cell lung carcinoma, pancreatic cancer, synovial carcinoma, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papilloatosis, skin cancer, and stomach cancer. Figure 15 provides examples of particular combinations of antigens that can be used to treat specific cancers. The. Melanoma
[00407] The vaccine can combine one or more cancer antigens such as tyrosinase, PRAME or GP100-Trp2 to treat or prevent melanoma (See Figure 15). The vaccine may additionally combine one or more cancer antigen tyrosinase, PRAME or GP100-Trp2 with one or more of the hTERT, NY-ESO-1, MAGE-A1 or WTI cancer antigens to treat or prevent melanoma. Other combinations of cancer antigens can also be applied to treat or prevent melanoma. B. Head and Neck Cancer
[00408] The vaccine may comprise the HPV 16 E6/E7 cancer antigen to treat or prevent head and neck cancer (see Figure 15). The vaccine may also combine the HPV 16 E6/E7 cancer antigen with one or more of the hTERT, NY-ESO-1, MAGE-A1 or WTI cancer antigens to treat or prevent head and neck cancer. Other combinations of cancer antigens can also be applied to treat or prevent head and neck cancer. ç. Recurrent anal/papillary respiratory cancer
[00409] The vaccine can match one or more cancer antigens such as HPV 6, HPV11, HPV16 to treat or prevent recurrent anal or respiratory papillofacial cancer (see Figure 15). The vaccine may further combine one or more HPV 6, HPV11, or HPV16 antigens with one or more hTERT, NY-ESO-1, MAGE-A1 or WTI cancer antigens to treat or prevent recurrent anal or respiratory papillofacial cancer. Other combinations of cancer antigens may also be applied to treat or prevent anal cancer or recurrent respiratory papillomatosis. d. cervical cancer
[00410] The vaccine can combine one or more cancer antigens such as HPV 16 E6/E7 or HPV 18 E6/E7 to treat or prevent cervical cancer (see Figure 15). The vaccine can also combine the HPV 16 E6/E7 cancer antigen with one or more of the hTERT, NY-ESO-1, MAGE-A1 or WTI cancer antigens to treat or prevent cervical cancer. Other combinations of cancer antigens can also be applied to treat or prevent cervical cancer. and. liver cancer
[00411] The vaccine can combine one or more cancer antigens such as HBV core antigen, HBV surface antigen, HCVNS34A, HCVNS5A, HCV NS5B or HCVNS4B to treat or prevent liver cancer (see Figure 15). The vaccine may further combine one or more cancer antigens such as HBV core antigen, HBV surface antigen, HCVNS34A, HCVNS5A, HCV NS5B or HCVNS4B with one or more cancer antigens hTERT, NY-ESO-1, MAGE- A1 or WTI to treat or prevent liver cancer. Other combinations of cancer antigens can also be applied to treat or prevent liver cancer. f. Glioblastoma multiforme
The vaccine may comprise CMV to treat or prevent glioblastoma (see Figure 15). The vaccine can also combine CMV with one or more of the hTERT, NY-ESO-1, MAGE-A1 or WTI cancer antigens to treat or prevent glioblastoma. Other combinations of cancer antigens can also be applied to treat or prevent glioblastoma. g. Prostate
[00413] The vaccine can combine one or more cancer antigens such as PSA, PSMA or STEAP to treat or prevent prostate cancer (see Figure 15). The vaccine may further combine one or more PSA, PSMA or STEAP cancer antigens with one or more hTERT, NY-ESO-1, MAGE-A1 or WTI cancer antigens to treat or prevent prostate cancer. Other combinations of cancer antigens can also be applied to treat or prevent prostate cancer. H. Blood cancer (eg leukemia, lymphoma, myeloma)
[00414] The vaccine can combine one or more cancer antigens such as PRAME, WT-1, hTERT to treat or prevent blood cancers such as leukemia, lymphoma and myeloma (see Figure 51). The vaccine may further combine one or more PRAME, WT-1, or hTERT cancer antigens with one or more NY-ESO-1, or MAGE-A1 cancer antigens to treat or prevent blood cancers such as leukemia, lymphoma, and myeloma. Other combinations of cancer antigens can also be applied to treat or prevent blood cancers such as leukemia, lymphoma and myeloma. 7. Vaccination method
[00415] Provided herein is a method of treating or preventing cancer using pharmaceutical formulations to provide genetic constructs and proteins of one or more of the cancer antigens, as described above, which comprise epitopes make them particularly effective immunogens against which a response immune to one or more cancer antigens can be induced. The method of administering the vaccine, or vaccination, can be provided to induce a therapeutic and/or prophylactic immune response. The vaccination process can generate an immune response in the mammal against one or more of the cancer antigens, as disclosed herein. The vaccine can be administered to an individual to modulate the activity of the mammalian immune system and enhance the immune response. Vaccine administration can be the transfection of the antigen of one or more cancer antigens, as described herein, as a nucleic acid molecule that is expressed in the cell and thus delivered to the cell surface over such a recognized immune system and induces a cellular, humoral, or cellular and humoral response. Vaccine administration can be used to induce or elicit an immune response in mammals against one or more of the cancer antigens as disclosed herein by administering the vaccine to mammals as discussed herein.
[00416] By delivering the vaccine to the mammal, and then the vector into the mammalian cells, the transfected cells will express and secrete one or more of the cancer antigens as disclosed herein. These secreted proteins, or synthetic antigens, will be recognized as foreign by the immune system, which will adjust an immune response that may include: antibodies against one or more cancer antigens and the T cell response, specifically against one or more cancer antigens. In some examples, a mammal vaccinated with the vaccines discussed in this document will have a primed immune system and when challenged with one or more of the cancer antigens, as described here, the primed immune system will allow rapid clearance of the subsequent HBV virus, either through humoral, cellular response, or both. The vaccine can be administered to an individual to modulate the individual's immune system activity, thereby enhancing the immune response.
[00417] Methods of administering the DNA of a vaccine are described in U.S. Patent Nos. 4,945,050 and 5,036,006, both of which are fully incorporated herein by reference.
[00418] The vaccine can be administered to a mammal to elicit an immune response in a mammal. The mammal can be a human, non-human primate, cow, pig, sheep, goat, antelope, bison, water buffalo, bovine, deer, hedgehog, elephant, llama, alpaca, mice, rat, or chicken, and preferably human, cow, pig or chicken.
[00419] The vaccine dose can be between 1 μg to 10 mg of the active component/kg body weight/time and can be between 20 μg and 10 μg of the component/kg body weight/time. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. The number of doses of vaccine for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses. The. Method of Generating a Vaccine Immune Response
The vaccine provided can be used to generate an immune response in a mammal, including therapeutic and prophylactic immune responses. The immune response can generate antibodies and/or killer T cells that are targeted to one or more cancer antigens, as disclosed herein. These antibodies and T cells can be isolated.
Some modalities provide methods for generating immune responses against one or more cancer antigens, as disclosed herein, which comprise administering the vaccine to an individual. Some embodiments provide methods of prophylactically vaccinating against a cancer or tumor that expresses one or more of the cancer antigens, as described above, comprising administering the vaccine. Some embodiments provide methods of therapeutically vaccinating an individual suffering from a cancer or tumor that expresses one or more of the cancer antigens, comprising administering the vaccine. Diagnosis of cancer or tumor expressing one or more of the cancer antigens, as disclosed herein, prior to vaccine administration can be routinely made. B. Cancer treatment method with the vaccine
[00422] The vaccine can be used to generate or elicit an immune response in a mammal that is reactive or targeted to a cancer or tumor (for example, melanoma, head and neck, cervical, liver, prostate, blood cancers , esophageal squamous, gastric) of the mammal or individual with such need. The elicited immune response can prevent cancer or tumor growth.
[00423] The elicited immune response can prevent and/or reduce metastasis of cancer cells or tumors. Consequently, the vaccine can be used in a method that treats and/or prevents cancer or tumors in the mammal or individual who has received the vaccine. Depending on the antigen used in the vaccine, the cancer or tumor-based growth can be any type of cancer, such as, but not limited to, melanoma, blood cancers (eg leukemia, lymphoma, myeloma), lung carcinomas, carcinomas of esophageal squamous cell, bladder cancer, colorectal cancer, esophagus, gastric cancer, hepatocarcinoma, head and neck, brain, anal cancer, non-small cell lung carcinoma, pancreatic cancer, synovial carcinoma, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and stomach cancer.
[00424] In certain embodiments, the vaccine can mediate clearance or prevent the growth of tumor cells by inducing (1) humoral immunity through B cell responses to generate antibodies that block the production of monocyte chemotactic protein-1 (MCP-1). ), thereby retarding myeloid-derived suppressor cells (MDSC) and suppressing tumor growth; (2) increase in cytotoxic T lymphocytes like CD8+ (CTL) to attack and kill tumor cells; (3) increase T-cell helper responses; (4) and enhance inflammatory responses through IFN-Y and TNF-α or, preferably, all of those mentioned above.
[00425] In some embodiments, the immune response may generate a humoral immune response and/or an antigen-specific cytotoxic T lymphocyte (CTL) response that does not cause damage to or inflation of tissues and systems (e.g., neurological or brain system , etc.) in the subject who received the vaccine.
[00426] In some embodiments, the vaccine administered can increase tumor free survival, reduce tumor mass, increase tumor survival, or a combination of these in the individual. The vaccine administered can increase tumor free survival by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% , 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% and 60% in the individual The vaccine administered can reduce the tumor mass by 20%, 21% , 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38 %, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% in the individual after the immunization. The vaccine administered can prevent and block increases in monocyte chemotactic protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells in the individual. In some embodiments, the vaccine administered can prevent and block increases in MCP-1 in the cancerous tissue or tumor in the individual, thereby reducing the vascularization of the cancerous tissue or tumor in the individual.
[00427] The vaccine administered can increase tumor survival by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% , 66%, 67%, 68%, 69% and 70% in the individual. In some embodiments, the vaccine can be administered in the peripheral area (as described in more detail below) to establish an antigen-specific immune response targeting tumor or cancer cells or tissue to clear or eliminate the cancer or tumor expressing one or more cancer antigens without harming or causing disease or death in the individual who received the vaccine.
[00428] The vaccine administered can increase an immune response in an individual by about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times the about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to about 6000 times. In some modalities the vaccine can increase the cellular immune response in the patient about 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times , 3600 fold, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.
[00429] The administered vaccine can increase the levels of interferon gamma (IFN-Y) in the individual by about 50 times to about 6000 times, about 50 times to about 5500 times, about 50 times to about 5000 times , about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times or from about 300 times to 6000 times. In some modalities, the vaccine administered can increase the individual's IFN-y levels by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times , 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.
[00430] The vaccine dose can be between 1 μg to 10 μg of the active component/kg body weight/time and can be between 20 μg and 10 μg of the component/kg body weight/time. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. The number of doses of vaccine for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. (1) Combinational therapies with PD-1 and/or PD-L1 antibodies
The present invention is also directed to a method of increasing an immune response in a mammal using the vaccine as described above. The vaccine as described above may comprise the cancer antigen and a PD1 antibody and/or PDL1 antibody as described above. The combination can be a single formulation or it can be separated and administered in sequence (first cancer antigen and then PDL1 antibody or PD1 antibody or PDL1 antibody first and then cancer antigen). In some modalities, the cancer antigen can be administered to the individual for about 30 seconds, 1 minute, 2 minutes, 3, 4, 5 minutes, 10 minutes, 15 minutes, 20, 25, 30 minutes, 35, 40, 45 minutes , 50, 55, 60 minutes, 0.25 hours, 0.5 hours, 0.75 hours, 1, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours , 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84, 96 hours, 1 day, 2 days, 3 days, 4, 5 days, 6 days, 7 days, 8 days, 9, 10 days, 11, 12 days, 13, 14 days , 15, 16 days, 17, 18 days, 19, 20 days, 21, 22 days, 23, 24 days, 25, 26 days, 27, 28 days, 29 days, 30, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks prior to administering PD-1 antibody or PD-L1 antibody to the subject. In other embodiments, the PD-1 antibody or PD-L1 antibody can be administered to the subject about 30 seconds, 1 minute, 2 minutes, 3, 4, 5 minutes, 10 minutes, 15 minutes, 20, 25, 30 minutes , 35, 40, 45 minutes, 50, 55, 60 minutes, 0.25 hours, 0.5 hours, 0.75 hours, 1, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84, 96 hours, 1 day, 2 days, 3, 4 days, 5 days, 6 days, 7, 8 days, 9, 10 days, 11, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25, 26 days, 27, 28 days, 29 days , 30 days, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks before the cancer antigen is administered to the subject.
The combination of cancer antigen and PD1 antibody or PDL1 antibody induces the immune system more efficiently than a vaccine comprising only the cancer antigen. This more efficient immune response provides increased effectiveness in the treatment and/or prevention of a specific cancer. Depending on the antigen used in the vaccine in combination with the PDL1 antibody or the PD1 antibody, the cancer or tumor-based growth could be any type of cancer, such as, but not limited to, melanoma, blood cancers (eg leukemia , lymphoma, myeloma), lung carcinomas, esophageal squamous cell carcinomas, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocarcinoma, head and neck, brain, anal cancer, non-small cell lung cancer, pancreatic cancer, synovial carcinoma, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer, and stomach cancer.
In some embodiments, the immune response may be increased by about 0.5 times to about 15 times, about 0.5 times to about 10 times, or about 0.5 times to about 8 times. Alternatively, the immune response in the individual receiving the vaccine can be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2, 0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times or at least about 15.0 times.
[00434] In other alternative modalities, still, the immune response in the individual who received the vaccine can be increased by about 50% to about 1500%, about 50% to about 1000% or about 50% to about 800%. In other embodiments, the immune response in the individual receiving the vaccine can be increased by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250 %, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600% , at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1,000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least less about 1,350%, at least about 1450%, or at least about 1500%.
[00435] The vaccine dose can be between 1 μg to 10 mg of the active component/kg body weight/time, and can be between 20 μg to 10 mg of the component/kg body weight/time. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. The number of doses of vaccine for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. (2) Melanoma
[00436] The vaccine can be used to generate or elicit an immune response in a mammal that is reactive or targeted to a melanoma in the mammal or in an individual in need thereof. The elicited immune response can prevent the growth of melanoma. The elicited immune response may reduce melanoma growth. The elicited immune response can prevent and/or reduce metastasis of cancer cells or tumors from a melanoma. Indeed, the vaccine can be used in a method that treats and/or prevents melanoma in the mammal or individual who has received the vaccine.
[00437] In certain embodiments, the vaccine can mediate clearance or prevent the growth of tumor cells by inducing (1) humoral immunity through B cell responses to generate antibodies that block the production of monocyte chemotactic protein-1 (MCP-1). ), thereby retarding myeloid-derived suppressor cells (MDSC) and suppressing tumor growth; (2) increase in cytotoxic T lymphocytes like CD8+ (CTL) to attack and kill tumor cells; (3) increase T-cell helper responses; (4) and enhance inflammatory responses through IFN-Y and TNF-α or, preferably, all of those mentioned above.
[00438] In some embodiments, the vaccine administered can increase melanoma free survival, reduce melanoma mass, increase melanoma survival, or a combination of these in the individual. The vaccine administered can increase melanoma free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% and 45% in the individual The vaccine administered can reduce the melanoma mass by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56% , 57%, 58%, 59% and 60% in the individual after immunization. The vaccine administered can prevent and block increases in monocyte chemotactic protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells in the individual. In some embodiments, the vaccine administered can prevent and block increases in MCP-1 in melanoma tissue in the individual, thereby reducing vascularization of the melanoma tissue in the individual. The vaccine administered can increase melanoma survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43 %, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% and 60% in the individual. 8. Routes of Administration
[00439] The vaccine or pharmaceutical composition can be administered by different routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, through inhalation, through oral, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular administration , intranasal, intrathecal and intraarticular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with standard veterinary practice. The veterinarian can readily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The vaccine can be administered by traditional syringes, needleless injection devices, biolistics ("microprojectile bombardment gene guns"), or other physical methods such as electroporation (EP), hydrodynamic method, or ultrasound.
[00440] The vaccine vector can be administered to the mammal by several known technologies including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome-mediated, nanoparticle-facilitated, recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus and recombinant vaccine. At least one of the vaccine antigens can be administered via DNA injection and in conjunction with in vivo electroporation. The. electroporation
[00441] The vaccine or pharmaceutical composition can be administered by electroporation. Vaccine administration via electroporation can be accomplished using electroporation devices that can be configured to deliver to a desired mammalian tissue a pulse of energy effective to cause reversible pores to become cell membranes, and preferably the pulse power is a constant current similar to a user-predetermined current input. The electroporation apparatus may comprise an electroporation component and an electrode assembly or handling assembly. The electroporation component may include and incorporate one or more of the various elements of electroporation devices, including: controller, current waveform generator, impedance tester, waveform recorder, input element, status reporting element , communication port, memory component, power supply and switch. Electroporation can be performed using an in vivo electroporation device, eg, the CELLECTRA ® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or Elgen electroporator (Inovio Pharmaceuticals, Inc.) to facilitate cell transfection by the plasmid.
[00442] Examples of electroporation devices and electroporation methods that can facilitate the administration of the DNA vaccines of the present invention include those described in US Patent No. 7,245,963 by Draghia-Akli, et al. U.S. Patent Pub. 2005/0052630 submitted by Smith, et al., the contents of which are hereby incorporated by reference in their entirety. Other electroporation devices and electroporation methods that can be used to facilitate the administration of DNA vaccines include those provided in co-owned and co-pending US Patent Application Serial No. 11/874072, filed October 17, 2007, which claims benefit under 35 USC 119(e) for US Provisional Applications Serial No. 60/852,149, filed October 17, 2006 and 60/978,982, filed October 10, 2007, all of which are incorporated by way of of this document in its entirety.
[00443] U.S. Patent No. 7,245,963, by Draghia-Akli, et al., describes modular electrode systems and their use to facilitate the introduction of a biomolecule into cells of a selected tissue in a body or a plant. Modular electrode systems can comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector providing a conductive connection of a programmable constant current pulse controller to the plurality of needle electrodes; and an energy source. An operator can hold the plurality of needle electrodes that are mounted on a support structure and firmly insert them into selected tissue in a body or plant. The biomolecules are then administered via a hypodermic needle into the selected tissue. The programmable constant current pulse controller is activated and constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecule into the cell, between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference in their entirety.
[00444] U.S. Patent Publication US 2005/0052630 filed by Smith, et al. describes an electroporation device that can be used to facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. The electroporation apparatus comprises an electro-kinetic device ("EKD device"), the operation of which is specified by the software or firmware. The EKD device produces a series of programmable constant current pulse patterns between electrodes in an arrangement based on user control and input of pulse parameters and allows for the storage and acquisition of current waveform data. The electroporation apparatus also comprises a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle and a removable guide disk. The entire contents of U.S. Patent Publication 2005/0052630 are fully incorporated herein by reference.
The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S Patent Publication US 2005/0052630 can be adapted for deep penetration not only into tissues such as muscle, but also other tissues or organs. Because of the electrode array configuration, the injection needle (for the deneurological system of the chosen biomolecule) is also fully inserted into the target organ and the injection is administered perpendicular to the target factor, in the area that is previously delineated by electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Pub. No. The electrodes described in US Patent No. 7,245,963 and US Patent Publication 2005/005263 are preferably 20 mm in length and 21 gauge.
[00446] Additionally, contemplated in some embodiments that incorporate electroporation devices and uses thereof, there are electroporation devices that are described in the following patents: US Patent 5,273,525 issued on December 28, 1993, US Patent 6,110,161 issued on 29 August 2000, 6,261,281 issued July 17, 2001 and 6,958,060 issued October 25, 2005 and US Patent 6,939,862 issued September 6, 2005. Additionally, patents covering subject matter provided for in US Patent 6,697 .669, issued on February 24, 2004, which concerns the administration of DNA using any of a variety of devices and US Patent 7,328,064, issued on February 5, 2008, designed for the method of injecting DNA are contemplated. in this document. The above patents are incorporated by reference in their entirety. 9. Vaccine Preparation Method
Methods for preparing DNA plasmids comprising the vaccines discussed herein are described in this document. The DNA plasmids, after the final step of subcloning into the mammalian expression plasmid, can be used to inoculate a cell culture in a large scale fermentation tank, using methods known in the art.
[00448] DNA plasmids for use with the EP devices of the present invention can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using a plasmid manufacturing technique that is described in US Published Application No. ° 20090004716, which was filed May 23, 2007. In some examples, plasmid DNA used in these studies may be formulated in concentrations greater than or equal to 10 mg/mL. Fabrication techniques also include or incorporate various devices and protocols that are commonly known to those of ordinary skill in the art, in addition to those described in US Serial No. 60/939792 including those described in a licensed patent, US Patent No. 7,238,522 , which was issued on July 3, 2007. The above application, US Patent Serial No. 60/939,792 and US Patent No. 7,238,522, respectively, are hereby incorporated in their entirety.
[00449] The present invention has multiple aspects, illustrated by the following non-limiting examples. 10. Examples Example 1 Construction of pTyr
[00450] As shown in Figures 1A and 9, tyrosinase (Tyr) can be found in many different organisms. Therefore, a consensus Tyr was generated through alignment sequences corresponding to Tyr from the organisms shown in FIG. 1A, and by choosing the most common amino acid and/or nucleotide for the consensus Tyr. The corresponding Tyr sequences for each organism were obtained from GenBank (NCBI). As such, the Tyr consensus reflected the conserved elements of Tyr sequences across species.
The nucleic acid sequence encoding the consensus Tyr was adapted to include the IgE core sequence. Specifically, the IgE core sequence was fused in-frame upstream of the Tyr consensus nucleic acid sequence (FIG. 1B). The resulting sequence was then inserted into the pVAX1 expression vector to create a tyrosinase construct or plasmid (pTyr) such that a Kozak sequence precedes the nucleotide sequence encoding the IgE core sequence and the consensus Tyr.
Insertion of the consensus Tyr nucleic acid sequence into pVAX1 was confirmed by restriction enzyme analysis. As shown in FIG. 1C, the consensus Tyr nucleic acid sequence was separated from plasmid pVAX1 on a DNA agarose gel (i.e., lane labeled BamH1/Xho1), thus confirming that vector pVAX1 contained the nucleic acid sequence of Tyr's consensus.
[00453] Consensus Tyr expression was confirmed by transfection of HeLa cells with pTyr. Western blotting with an anti-human Tyr antibody confirmed the expression of the consensus Tyr protein in HeLa cells (FIG. 1D). GPF staining further showed the expression of the consensus Tyr protein in the transfected HeLa cells (FIG. 1E). In the coloring and western blotting experiments, Example 2 Vaccination with pTyr induced a cell-mediated immune response
[00454] The pTyr described above was used to vaccinate mice to assess whether a cellular immune response was induced by pTyr. C57/B6 mice were immunized using the immunization strategy shown in FIG. 2A. Some mice were immunized with pVAX1, while other mice were immunized with pTyr. Mice immunized with pTyr were further divided into the following groups: (1) 5 μg pTyr dosage; (2) 20 µg of pTyr dosage; (3) 30 µg of pTyr dosage; and (4) 60 µg of pTyr dosage.
On day 35 of the immunization strategy, splenocytes were isolated from the C57B/6 mice and evaluated for interferon-Y (IFN-Y) induction by IFN-y ELISpot analysis. As shown in FIG. 2B, dosing 20 μg pTyr induced the highest levels of IFN-Y.
[00456] The cellular immune response to pTyr was further evaluated in immunized Balb/c mice and C57B/6 mice. Mice were immunized with pVAX1 or pTyr. Splenocytes were isolated two weeks after the third immunization and stimulated with the Tyr consensus peptide. After stimulation, the amount of IFN-y secreting splenocytes was calculated as the mean amount of spots in the triplicate stimulant wells. This analysis indicated that C57/B6 mice were suitable for pTyr vaccination (data not shown). Example 3 Vaccination with pTyr increased the cytokines IFN-Y and TNF-α
[00457] The production of cytokines was analyzed in mice immunized with pTyr and pVax1. Mice were immunized using the strategy shown in FIG. 2A. On day 35 of the immunization strategy, cells isolated from the immunized mice were stimulated overnight with Tyr peptides. After stimulation, the analysis of polyfunctional responses was measured by FACS. Specifically, the analysis examined CD8+ and CD4+ T cells. FACS allowed the identification of T cells positive for the cytokines IL-2, TNF-α and IFN-y. Of the CD44 hi cells, a significant percentage of CD8+ T cells produced IFN-y in mice immunized with pTyr compared to mice immunized with pVax1 (FIG. 3). Example 4 Tyr-specific antibodies are produced in response to pTyr vaccination
[00458] The humoral immune response was evaluated in mice vaccinated with pTyr. Specifically, C57BI/6 mice (n=4) were immunized three times at 2-week intervals with pTyr or pVax1. Each immunization consisted of a 20μg/intramuscular injection followed by electroporation with MID-PE. After the third immunization (ie, day 35), serum was collected from the mice and antibody titers were measured by ELISA using IgG-specific HRP-labeled secondary antibodies. Sera were diluted as indicated in FIG. 4A. As shown in FIG. 4A, Tyr-specific antibodies were produced by mice immunized with pTyr. Mice immunized with pTyr are represented by the filled circle in FIG. 4A, while mice immunized with pVax1 are represented by the open triangle in FIG. 4A.
In addition, sera from immunized mice were serially diluted 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640. Each serum dilution was added three times to individual wells (50μg/well) containing Tyr peptides. The peak of increase in Tyr-specific titer, compared to pre-immune serum, was detected for all immunized groups of mice on recheck days 90 and 120. Representative results from three independent experiments at each serial dilution point are shown in FIG. 4B. These data further indicate that immunization with pTyr induced the production of Tyr-specific antibodies in mice immunized with pTyr. Example 5 Mice vaccinated with pTyr increased survival to tumor challenge
pTry was further analyzed to determine if vaccination with pTyr could provide protection from tumors. Specifically, C57BI/6 mice (10 per group) were immunized at 2-week intervals with pTyr or pVax1. Each immunization consisted of a 20μg/intramuscular injection followed by electroporation with MID-PE. One week after the third immunization (ie, day 35), the immunized mice were challenged intradermally with the B16 melanoma until the tumor diameter exceeded 200 mm2.
Subsequently, tumor volume and tumor-free survival were assessed in the immunized groups of mice. As shown in FIG. 5A (a Kaplan-Meier survival curve), mice immunized with pTyr were boosted for tumor-free survival (i.e. 40% at 40 days and after tumor challenge) compared to mice vaccinated with pVax1 ( p = 0.05), who were all dead by day 20 after tumor challenge. Mice immunized with pTyr also had reduced tumor volume (i.e., approximately 50%) compared to mice immunized with pVax1 (FIG. 5B). For FIGS. 5A and 5B, mice immunized with pVax1 are represented by filled squares, whereas mice immunized with pTyr are represented by filled circles. In effect, these data show that vaccination with pTyr provided protection against melanoma, that is, increased tumor-free survival and reduction in tumor volume. Example 6 The MDSC population is reduced in tumors from mice vaccinated with pTyr
MDSC populations were examined in mice immunized with pTyr and in non-immunized mice to examine whether pTyr vaccination altered the levels of MDSCs in the tumors of the respective groups of mice. Specifically, the percentage of Gr-1+ and CD11b cells was examined in immunized mice and in non-immunized mice.
As depicted in Figures 6 and 7, MDSC levels were significantly reduced in tumors from mice immunized with pTyr compared to non-immunized mice (p = 0.0004). The percentage of the MDSC population in non-immunized mice was 40.00 ± 4.826. The percentage of the MDSC population in mice immunized with pTyr was 5.103 ± 0.7718. In effect, these data show that immunization with pTyr reduced MDSC populations within the tumors of mice vaccinated with pTyr. Example 7 MCP-1 levels are reduced by vaccination with pTyr
[00464] MDSCs can secrete MCP-1 cytokines, which promote angiogenesis or vascularization by endothelial cell migration. Given the effect of pTyr vaccination described above pTyr on MDSC levels in tumors, MCP-1 levels were examined after pTyr vaccination.
[00465] As shown in FIG. 8A, MDSCs in B16 melanoma can secrete MCP-1. Therefore, mice immunized with pTyr and mice immunized with pVax1 were challenged with B16 melanoma in order to examine whether pTyr immunization altered MCP-1 levels. Naive mice were included as an additional control. After challenge, MDSCs were isolated directly from tumor tissue, and MCP-1 cytokine levels were analyzed by ELISA. The experiment was performed in triplicate and repeated twice.
[00466] As shown in FIG. 8B, MDSCs in B16 melanoma or tumor tissue significantly secreted MCP-1 (see pVax1 immunized mice). Mice immunized with pTyr, however, do not have a significant increase in MCP-1 levels. Instead, MCP-1 levels in mice immunized with pTyr were about 3-fold lower than in mice immunized with pVax1. Indeed, these data show that pTyr vaccination reduced the level of MCP-1 secretion by MDSCs in the tumors of mice immunized with pTyr. Example 8 pPRAME construction
[00467] A consensus sequence was generated for PRAME and the nucleotide sequence encoding the consensus PRAME antigen was inserted into the Xhol and BamHI restriction enzyme sites of the expression vector or plasmid pVAX (also referred to herein as pVAX1) to render pGX1411 (also referred to herein as pPRAME) (see FIG. 10A).
To confirm that pPRAME resulted in the expression of the consensus PRAME antigen, pVAX and pPRAME were transfected into RD cells and 293T cells. DAPI was used to stain nuclei and the consensus PRIME antigen was also fluorescently stained. This staining, along with a mixture of DAPI and the consensus PRAME antigen staining, are shown in FIG. 10B These stains demonstrated that the consensus PRAME antigen was expressed from pPRAME and no consensus PRAME antigen was detected in the cells transfected with pVAX (ie, negative control).
In addition, western blotting analysis of transfected cell lysates was used to confirm the expression of the consensus PRAME antigen in transfected cells (FIG. 10C). Non-transfected cells and cells transfected with pVAS were used as negative controls (see lanes labeled "control" and "pVAX", respectively, in FIG. 10C). In FIG. 10C, beta-actin detection was used as a loading control. In summary, staining of transfected cells and western blotting of transfected cell lysates demonstrated that the pPRAME vector provided expression of the consensus PRAME antigen in cells. Example 9 Interferon gamma response to pPRAME vaccination
[00470] The pPRAME described above was used to vaccinate mice in order to assess whether the cellular immune response was induced by pPRAME. C57BL/6 mice were separated into groups. A first group was virgin and did not receive pPRAME. The second, third, fourth, fifth and sixth group of mice received 5 μg, 10 μg, 15 μg, 25 μg and 50 μg of pPRAME, respectively.
[00471] After immunization, splenocytes were isolated from C57BL/6 mice and evaluated for interferon gamma (IFN-Y) induction by IFN-Y ELISpot analysis. As shown in Figures 11A and 11B, each dose of pPRAME induced the production or secretion of IFN-Y, differently from naïve negative control mice. In particular, IFN-Y levels were increased by about 3000-fold to about 4500-fold in vaccinated mice compared to non-vaccinated mice. Consequently, these data demonstrated that vaccination with pPRAME, which encodes the consensus PRAME antigen, induced a cellular immune response as evidenced by increased IFN-Y levels compared to non-vaccination. Example 10 Construction of pNY-ESO-1
[00472] A consensus sequence was generated for NY-ESO-1 and the nucleotide sequence encoding the NY-ESO-1 consensus antigen was inserted into the BamHI and Xhol restriction enzyme sites of the expression vector or into plasmid pVAX (also referred to herein as pVAX1) to generate pGX1409 (also referred to herein as pNY-ESO-1) (see FIG. 12A).
To confirm that pNY-ESO-1 resulted in the expression of the consensus NY-ESO-1 antigen, pVAX and pNY-ESO-1 were transfected into the cells. DAPI was used to stain nuclei and the NY-ESO-1 consensus antigen was also fluorescently stained. This staining, along with a mixture of DAPI and the consensus NY-ESO-1 antigen, are shown in FIG. 12B. These stains demonstrated that the NY-ESO-1 consensus antigen was expressed from pNY-ESO-1 and no NY-ESO-1 consensus antigen was detected in the cells transfected with pVAX (ie, negative control).
In addition, western blotting analysis of lysates from 293T and RD transfected cells was used to confirm the expression of the consensus NY-ESO-1 antigen in the transfected cells (FIG. 12C). Non-transfected cells and cells transfected with pVAX were used as negative controls (see lanes labeled "control" and "pVAX", respectively, in FIG. 12C). In FIG. 12C, alpha-actin detection was used as a loading control. In summary, staining of transfected cells and western blotting of transfected cell lysates demonstrated that the pNY-ESO-1 vector provided the expression of the consensus NY-ESO-1 antigen in the cells. Example 11 Interferon gamma response to pNY-ESO-1 vaccination
The pNY-ESO-1 described above was used to vaccinate mice to assess whether a cellular immune response was induced by pNY-ESO-1. C57BL/6 mice were separated into groups. A first group was virgin and did not receive pNY-ESO-1. The second and third group of mice received 25 μg and 50 μg of pNY-ESO-1, respectively.
[00476] After immunization, splenocytes were isolated from C57BL/6 mice and evaluated for interferon gamma (IFN-Y) induction by IFN-Y ELISpot analysis. As shown in FIG. 13, each dose of pPRAME induced IFN-Y production or secretion, differently from naive negative control mice. In particular, IFN-Y levels were increased by about 700-fold to about 1100-fold in vaccinated mice compared to non-vaccinated mice. In effect, these data demonstrated that vaccination with pNY-ESO-1, which encodes the consensus NY-ESO-1 antigen, induced a cellular immune response, as evidenced by increased levels of IFN-Y compared to non-vaccination. Example 12 Interferon gamma response to pNY-ESO-2 vaccination
[00477] A consensus sequence was generated for NY-ESO-2 and the nucleotide sequence encoding the NY-ESO-2 consensus antigen was inserted into the multiple cloning site of the expression vector or into plasmid pVAX (also referred to here as pVAX1) to generate pNY-ESO-2.
[00478] This pNY-ESO-2 was used to vaccinate mice in order to assess whether the cellular immune response was induced by pNY-ESO-2. C57BL/6 mice were separated into groups. A first group was virgin and did not receive pNY-ESO-2. The second and third group of mice received 25 μg and 50 μg of pNY-ESO-2, respectively.
[00479] After immunization, splenocytes were isolated from C57BL/6 mice and evaluated for interferon gamma (IFN-y) induction by IFN-y ELISpot analysis. As shown in FIG. 14, each dose of pNY-ESO-2 induced the production or secretion of IFN-y, differently from naive negative control mice. In particular, IFN-y levels were increased by about 400-fold to about 500-fold in vaccinated mice compared to unvaccinated mice. In effect, these data demonstrated that vaccination with pNY-ESO-2, which encodes the NY-ESO-2 consensus antigen, induced a cellular immune response, as evidenced by increased IFN-y levels compared to non-vaccination.
[00480] It is understood that the detailed description cited above and attached examples are merely illustrative and should not be considered as limitations on the scope of the invention, which is exclusively defined by the appended claims and their equivalents.
[00481] Various changes and modifications in the disclosed modalities will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the invention, may be made without departing from the spirit and scope of the invention.

权利要求:
Claims (26)
[0001]
1. Vaccine, characterized by the fact that it comprises one or more pharmaceutically acceptable excipients and a nucleic acid encoding a consensus growth hormone releasing hormone (GHRH) amino acid sequence (SEQ ID NO:10).
[0002]
2. Vaccine according to claim 1, characterized in that it additionally comprises a nucleic acid encoding one or more antigens selected from the group consisting of: PSA, PSMA, STEAP, PSCA, MAGE A1, gp100, a viral antigen and combinations of the same.
[0003]
3. Vaccine according to claim 2, characterized by the fact that the viral antigen is an antigen of Hepatitis B virus (HBV), Hepatitis C virus (HCV) or Human Papilloma Virus (HPV).
[0004]
4. Vaccine according to claim 3, characterized by the fact that the HBV antigen is an HBV core antigen or an HBV surface antigen or a combination thereof.
[0005]
5. Vaccine according to claim 3, characterized in that the HCV antigen is an HCV NS34A antigen, an HCV NS5A antigen, an HCV NS5B antigen, an HCV NS4B antigen or a combination thereof.
[0006]
6. Vaccine according to claim 3, characterized by the fact that the HPV antigen is an E6 antigen of HPV type 6, an E7 antigen of HPV type 6, an E6 antigen of HPV type 11, an E7 antigen of HPV type 11, an HPV type 16 E6 antigen, an HPV type 16 E7 antigen, an HPV type 18 E6 antigen, an HPV type 18 E7 antigen, or a combination thereof.
[0007]
7. Vaccine according to claim 1, characterized in that it further comprises an immunological checkpoint inhibitor selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody and a combination thereof.
[0008]
8. Vaccine according to claim 1, characterized in that the nucleic acid molecule comprises SEQ ID NO:9.
[0009]
9. Vaccine according to claim 1, characterized in that the nucleic acid is a plasmid.
[0010]
10. Vaccine according to claim 1, characterized in that the nucleic acid is one or more plasmids.
[0011]
11. Vaccine according to claim 1, characterized in that it additionally comprises an adjuvant.
[0012]
12. Vaccine according to claim 11, characterized in that the adjuvant is IL-12, IL-15, IL-28 or RANTES.
[0013]
13. Vaccine according to claim 1, characterized in that it is for use in a method of treating cancer in an individual in need thereof.
[0014]
14. Vaccine according to claim 13, characterized in that the vaccine is for administration by electroporation.
[0015]
15. Vaccine according to claim 13, characterized in that the vaccine is for administration with an immunological checkpoint inhibitor.
[0016]
16. Vaccine according to claim 15, characterized in that the immunological checkpoint inhibitor is selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody and a combination thereof.
[0017]
17. Vaccine according to claim 15, characterized in that the vaccine and immunological checkpoint inhibitor are for administration to the individual in a single formulation.
[0018]
18. Vaccine according to claim 15, characterized in that the vaccine and immunological checkpoint inhibitor are for administration separately to the individual.
[0019]
19. Vaccine according to claim 13, characterized in that the cancer is selected from the group consisting of: melanoma, head and neck cancer, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis ( RRP), anal cancer, a blood cancer and a combination thereof.
[0020]
20. Nucleic acid molecule, characterized in that it comprises a nucleotide sequence encoding a consensus growth hormone releasing hormone (GHRH) amino acid sequence (SEQ ID NO:10).
[0021]
21. Nucleic acid molecule according to claim 20, characterized in that the nucleic acid molecule is a plasmid.
[0022]
22. Nucleic acid molecule according to claim 21, characterized in that the nucleic acid molecule is one or more plasmids.
[0023]
23. Amino acid molecule, characterized in that it comprises a consensus amino acid sequence as defined in SEQ ID NO:10.
[0024]
24. Nucleic acid molecule according to claim 20, characterized in that the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 9.
[0025]
25. Nucleic acid molecule according to any one of claims 20 or 24, characterized in that the nucleic acid is a plasmid.
[0026]
26. Vaccine characterized in that it comprises a nucleic acid molecule as defined in any one of claims 20 or 24 and 25.

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JP2021100944A|2021-07-08|

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WO2014144885A3|2014-12-04|

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MX2015011486A|2016-02-03|

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HK1216991A1|2016-12-16|

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AU2017213514A1|2017-08-31|

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WO2014144885A2|2014-09-18|

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JP6769865B2|2020-10-14|

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EP2968607B1|2019-07-24|

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JP2019112418A|2019-07-11|

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MX2019013756A|2020-01-15|

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AU2017213514B2|2019-08-15|

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CA2898126A1|2014-09-18|

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BR112015022367A2|2017-10-10|

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EA201992251A1|2020-05-08|

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AU2019261784A1|2019-11-28|

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AU2014228405A1|2015-08-13|

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-10-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-11-10| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-03-30| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-22| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/03/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361799952P| true| 2013-03-15|2013-03-15|

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US61/799,952|2013-03-15|

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PCT/US2014/029479|WO2014144885A2|2013-03-15|2014-03-14|Cancer vaccines and methods of treatment using the same|

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