Patent Application
20230063041
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 7, 2021, is named 25133-8-104384-101_SL.txt and is 51,554 bytes in size.
The present invention relates to high-titer hybrid virus vectors which produce virus-like vesicle (VLVs) and compositions and methods thereof for targeting a malignancy or infectious disease. VLVs are a capsid-free, self-replicating artificial virus platform carrying positive-strand capped and polyadenylated RNA encoding an in vitro evolved Semliki Forest virus (SFV) RNA-dependent RNA replicase and the vesicular stomatitis virus (VSV) glycoprotein. The VLVs of the present invention and pharmaceutical compositions thereof encode for polynucleotide and/or polypeptide therapeutic agents useful in methods for the treatment, prophylaxis, and prevention of malignancies and infectious diseases.
Oncolytic viruses may target and specifically replicate in cancer cells resulting in the lysis and/or death of the cancer cells. These viruses may express transgenes that can direct the virus to cancer cells, increase their potential to activate T cell infiltration or activity, or imbue multiple other activities that enhance oncolysis.
Many forms of oncolytic viruses have been composed and clinically tested. However, only a single oncolytic virus has been approved for medical use: talimogene laherparepvec (T-VEC), a genetically modified herpesvirus that expresses a transgene, granulocyte-macrophage colony stimulating factor (GM-CSF) for use in malignant melanoma. Unfortunately, T-VEC has had minimal commercial success due to its limited activity. The lack of success of T-VEC and many other types of oncolytic viruses is likely due to multiple factors, including:
The viruses that have been used for oncolytic activity include paroviruses, adenoviruses, herpesvirus (as above with T-VEC), poxviruses, picornovirus, alphaviruses, retroviruses, paramyxoviruses, rhabdoviruses, and reoviruses. However, each of these types of viruses have one or more of the deficiencies listed above that have tempered their success.
Virus-like vesicles (VLVs) are a capsid-free, self-replicating artificial virus platform carrying positive-strand capped and polyadenylated RNA encoding an in vitro evolved Semliki Forest virus (SFV) RNA-dependent RNA replicase and the vesicular stomatitis virus (VSV) glycoprotein. VLVs are able to overcome many of the deficiencies harbored by natural virus vectors. As an artificial virus that lacks an encoded capsid protein, VLVs are non-pathogenic. The lack of a capsid protein also avoids any limit to the size of RNA packaged into the VLVs, therefore, allowing a very large capacity for packaging RNA and expression of transgene antigens or proteins. Being derived from animal viruses that are not to be endemic in human populations, pre-existing neutralizing immunity is likely rare or absent. VLVs can be grown to high titers enabling large scale manufacturing. These features of VLVs are ideal for an oncolytic virus.
U.S. Pat. No. 9,987,353 by Robek et al., issued Jun. 5, 2018 and incorporated by reference herein in its entirety, relates to compositions and methods for therapeutic immunization for treatment of chronic hepatitis B using a VLV composition produced from a high-titer hybrid-hepatitis B virus vector.
U.S. Pat. No. 10,435,712 by Rose et al., issued Oct. 8, 2019 and incorporated by reference herein in its entirety, relates to compositions and methods for immunization using high-titer hybrid-virus vectors. The high-titer hybrid virus vectors producing evolved VLV compositions therein comprise alphavirus nonstructural proteins with multiple mutations.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease comprising the following operably linked sequence elements:
a) a first DNA sequence comprising a DNA promoter sequence,
b) a second DNA sequence encoding alphavirus non-structural protein polynucleotide sequences,
c) a third DNA sequence encoding an alphavirus subgenomic RNA promoter,
d) a fourth DNA sequence comprising at least one sequence domain each independently selected from the group consisting of a sequence domain encoding a cytokine agonist polypeptide a sequence domain encoding a cytokine antagonist polypeptide a sequence domain encoding a short hairpin RNA (shRNA), and combinations thereof,
e) a fifth DNA sequence encoding a vesiculovirus glycoprotein.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the DNA promoter sequence of the first DNA sequence comprises a constitutive promoter for RNA-dependent RNA polymerases.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the DNA promoter sequence comprises a constitutive promoter for bacteriophage RNA polymerases.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the promoter sequence is a cytomegalovirus (CMV) promoter sequence.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the alphavirus non-structural protein polynucleotide sequences of the second DNA sequence are Semliki Forest virus (SFV) non-structural protein polynucleotide sequences.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the SFV non-structural protein polypeptide sequences encoded by the second DNA sequence comprise one or more amino acid mutations.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the SFV non-structural protein polypeptide sequences comprise a sequence having at least about 70% sequence identity to SEQ ID NO. 13.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the SFV non-structural protein polypeptide sequences comprise SEQ ID NO. 13.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the alphavirus non-structural protein polynucleotide sequences of the second DNA sequence comprise alphavirus RNA-dependent polymerase.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the alphavirus RNA-dependent polymerase is a Semliki Forest virus (SFV) RNA-dependent polymerase.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the alphavirus subgenomic RNA promoter of the third DNA sequence is a Semliki Forest virus (SFV) subgenomic RNA promoter.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the fourth DNA sequence comprises at least one sequence domain encoding a cytokine agonist or antagonist polypeptide each independently selected from the group consisting of IL-2, IL-7, IL-15, IL-18, IL-19, IL-35, IL-21, GM-CSF, IL-17, Flt3L, and combinations thereof.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the fourth DNA sequence comprises at least one sequence domain encoding an shRNA polynucleotide checkpoint inhibitor each independently selected from the group consisting of PD-L2, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, CD90, BTLA, CD160, PD-1, and combinations thereof.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the fourth DNA sequence comprises three sequence domains.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the fourth DNA sequence comprises:
a) a sequence domain encoding IL-12,
b) a sequence domain encoding IL-17RA-DN; and
c) a sequence domain encoding PD-L1-shRNA.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the fourth DNA sequence comprises:
a) a sequence domain having at least 70% sequence identity with SEQ ID NO. 6,
b) a sequence domain having at least 70% sequence identity with SEQ ID NO. 8; and
c) a sequence domain having at least 70% sequence identity with SEQ ID NO. 10.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein each sequence domain of the fourth DNA sequence is preceded by a sequence independently selected from the group consisting of a subgenomic promoter, a sequence encoding a 2A peptide sequence, or combinations thereof.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the high-titer hybrid virus vector is trivalent; and wherein the fourth DNA sequence consists of three sequence domains.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the fourth DNA sequence consists of:
a) a subgenomic promoter sequence and a sequence domain encoding IL-12,
b) a subgenomic promoter sequence and a sequence domain encoding IL-17RA-DN; and
c) a subgenomic promoter sequence and a sequence domain encoding PD-L1-shRNA.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the fourth DNA sequence consists of:
a) a subgenomic promoter sequence and a sequence domain having at least 70% sequence identity with SEQ ID NO. 6,
b) a subgenomic promoter sequence and a sequence domain having at least 70% sequence identity with SEQ ID NO. 8; and
c) a subgenomic promoter sequence and a sequence domain having at least 70% sequence identity with SEQ ID NO. 10.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the fourth DNA sequence further comprises a sequence domain encoding a polynucleotide or polypeptide inhibitor of an immunosuppressive regulator selected from the group consisting of TDO, IDO1, IDO2, and combinations thereof.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein each of the sequence domains of the fourth DNA sequence are expressed under control of single or multiple subgenomic promoters resulting in single or multiple subgenomic RNA.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein each of the sequence domains of the fourth DNA sequence are co-translated from the shared subgenomic RNA that includes ribosome skipping 2A peptides.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease further comprising a sixth DNA sequence comprising an expression cassette expressing single or multiple specific antigens of the malignancy or infectious disease; and
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the expression cassette expresses specific antigens of a tumor or infectious agent to induce humoral and/or cellular immune responses to the antigens.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein the vesiculovirus glycoprotein of the fifth DNA sequence comprises envelope glycoprotein of vesicular stomatitis Indiana virus, vesicular stomatitis New Jersey virus, Chandipura virus or structurally related vesiculoviruses.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein titers of at least 5×107 plaque forming units (pfu) per mL of virus like vesicles (VLVs) are obtained.
In some embodiments, the invention relates to a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease, wherein titers of at least 1×108 plaque forming units (pfu) per mL of virus like vesicles (VLVs) are obtained.
In some embodiments, the invention relates to a high-titer hybrid virus vector for generating oncolytic virus-like vesicles (VLVs) comprising the following operably linked sequence elements:
a) a first DNA sequence comprising a DNA promoter sequence,
b) a second DNA sequence encoding alphavirus non-structural protein polynucleotide sequences,
c) a third DNA sequence encoding an alphavirus subgenomic RNA promoter,
d) a fourth DNA sequence comprising an expression cassette expressing single or multiple specific antigens of the malignancy or infectious disease; and
e) a fifth DNA sequence encoding a vesiculovirus glycoprotein.
In some embodiments, the invention relates to virus-like vesicles (VLVs) containing replicon RNA generated by a high-titer hybrid virus vector for generating oncolytic virus-like vesicles (VLVs).
In some embodiments, the invention relates to virus-like vesicles (VLVs) containing replicon RNA generated by a high-titer hybrid virus vector for generating oncolytic virus-like vesicles (VLVs), wherein the replicon RNA is positive-strand capped and polyadenylated.
In some embodiments, the invention relates to a composition comprising virus-like vesicles (VLVs) produced by a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease comprising the following operably linked sequence elements:
a) a first DNA sequence comprising a DNA promoter sequence,
b) a second DNA sequence encoding alphavirus non-structural protein polynucleotide sequences,
c) a third DNA sequence encoding an alphavirus subgenomic RNA promoter,
d) a fourth DNA sequence comprising at least one sequence domain each independently selected from the group consisting of a sequence domain encoding a cytokine agonist polypeptide a sequence domain encoding a cytokine antagonist polypeptide a sequence domain encoding a short hairpin RNA (shRNA), and combinations thereof,
e) a fifth DNA sequence encoding a vesiculovirus glycoprotein.
In some embodiments of the invention, the VLVs are self-replicating.
In some embodiments of the invention, the VLVs are oncolytic.
In some embodiments of the invention, the VLVs are capsid-free.
In some embodiments, the invention relates to a method of producing virus-like vesicles (VLVs) for treatment, prophylaxis, or prevention of malignancy or infectious disease comprising the steps of:
a) generating a high-titer virus vector comprising at least one sequence domain each independently selected from the group consisting of a sequence domain encoding a cytokine agonist polypeptide, a sequence domain encoding a cytokine antagonist polypeptide, a sequence domain encoding a short hairpin RNA (shRNA), and combinations thereof,
b) transfecting BHK-21 cells with the high-titer virus vector of step (a),
c) incubating the transfected BHK-21 cells of step (b) in a buffer solution for a suitable time and at a suitable temperature to propagate VLVs; and
d) isolating the VLVs from the BHK-21 cells and buffer solution by a technique selected from the group consisting of ultrafiltration, centrifugation, tangential flow filtration, affinity purification, ion exchange chromatography, and combinations thereof; wherein the isolating of step (d) yields VLVs of a high titer.
In some embodiments, the invention relates to a method of treating and preventing malignancy in a subject, the method comprising administering a therapeutically effective amount of a composition comprising virus-like vesicles (VLVs) produced by a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease to a mammalian or human subject in need thereof.
In some embodiments, the invention relates to a method of treating a subject having an infectious disease, the method comprising administering a therapeutically effective amount of a composition comprising virus-like vesicles (VLVs) produced by a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease to a mammalian or human subject in need thereof.
In some embodiments, the invention relates to a method of immunizing a subject against an infectious disease, the method comprising administering a therapeutically effective amount of a composition comprising virus-like vesicles (VLVs) produced by a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease to a mammalian or human subject in need thereof.
In some embodiments, the invention relates to a method of downregulating genes associated with malignancy, the method comprising administering a therapeutically effective amount of a composition comprising virus-like vesicles (VLVs) produced by a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease to a mammalian or human subject in need thereof.
In some embodiments, the invention relates to a method of downregulating genes associated with malignancy, the method comprising administering a therapeutically effective amount of a composition comprising virus-like vesicles (VLVs) produced by a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease to a mammalian or human subject in need thereof, wherein the gene is selected from the group consisting of PD-L1, CXCL1, CXCL2, and combinations thereof.
In some embodiments, the invention is related to methods wherein the mammalian subject in need thereof is a human or animal.
In some embodiments, the invention relates to the use of a composition in the manufacture of a medicament for the treatment, prophylaxis, or prevention of a malignancy or infectious disease in a mammalian or human subject in need thereof, said composition being a composition comprising virus-like vesicles (VLVs) produced by a high-titer hybrid virus vector for treatment, prophylaxis, or prevention of malignancy or infectious disease comprising the following operably linked sequence elements:
a) a first DNA sequence comprising a DNA promoter sequence,
b) a second DNA sequence encoding alphavirus non-structural protein polynucleotide sequences,
c) a third DNA sequence encoding an alphavirus subgenomic RNA promoter,
d) a fourth DNA sequence comprising at least one sequence domain each independently selected from the group consisting of a sequence domain encoding a cytokine agonist polypeptide a sequence domain encoding a cytokine antagonist polypeptide a sequence domain encoding a short hairpin RNA (shRNA), and combinations thereof.
The present invention relates to the unexpected discovery that virus-like vesicles (VLVs) can be armed with one or multiple sequences encoding cytokine agonists, cytokine antagonists, short hairpin RNAs, and other polynucleotides or polypeptides, each of which being useful for treatment, prophylaxis, and prevention of cancer, malignancy or infection disease. The VLVs of the present invention are produced from high-titer hybrid virus vectors encoding the same. Virus-like vesicles (VLVs) are a capsid-free, self-replicating artificial virus platform carrying positive-strand capped and polyadenylated RNA encoding an alphavirus non-structural protein and a vesiculovirus glycoprotein.
In some aspects, the VLVs of the present invention useful for treatment, prophylaxis, and prevention of cancer, malignancy are further complemented by the unexpected discovery that the VLVs are oncolytic. Tumor cells, cancer cells, malignant cells, or cells with characteristics of malignancy allow VLV replication as a lytic process due to unchecked VLV replication, while VLV replication does not proceed unchecked in normal cells. In some examples of normal cells, VLV replication is restricted by the recognition of dsRNA by pattern recognition receptors and induction of type I interferons, which blocks VLV replication in the normal cells. If type I interferon pathways are impaired, such as in cancer or tumor cells, VLV can proceed unchecked. Thereby, they allow VLV replication proceed as a lytic process: once VLV replication is started it causes host cell protein synthesis shut-off and causes apoptosis. Even if apoptosis pathways are deficient in tumor cells, cells with high burden of VLV are lysed.
Therefore, the VLVs of the present invention can have at least two beneficial effects on tumor cells, cancer cells, and malignancies by their production of one or multiple cytokine agonists, cytokine antagonists, short hairpin RNAs, and other polynucleotides or polypeptides useful for the treatment, prophylaxis, and/or prevention of a malignancy, and by further having oncolytic activity during their self-replication. Said cytokine agonists, cytokine antagonists, short hairpin RNAs, and other polynucleotides or polypeptides useful for the treatment, prophylaxis, and/or prevention of a malignancy or infection disease which are produced in a host cell as replication or translation products the RNA of the VLV may be referred to as “therapeutic agents”.
In one embodiment of this invention, the VLVs are competent for oncolysis of several types of cancer cells in vitro (
In another embodiment of this invention, VLVs can be armed with IL-12, a cytokine that has anticancer activities. When armed with IL-12, VLV can infect MC38 mouse colon cancer cell line and baby hamster kidney (BHK21) cells, and express secreted IL-12 protein in vitro (
In yet another embodiment, VLVs can be armed with a dominant-negative (DN) mutant of IL-17A-receptor (IL-17RA) (
In another embodiment, VLV can be armed with shRNAs against PD-L1, the ligand for Programmed-Death-1 receptor, an immune check point protein that attenuates the activity of CD8+ T cells when ligand and receptor expressed on separate cells interact. When PD-L1 expressing BHK21 cells are infected with VLVs encoding PD-L1-shRNA, the expression of PD-L1 is suppressed by the shRNA in a specific manner (no suppression by a scrambled version (SCR) of the shRNA) (
In another embodiment, trivalent VLVs can be made that express IL-12, IL-17RA-DN, and PD-L1-shRNA (CARG2020) (
In a further embodiment, VLVs are shown to be oncolytic (
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In addition to the materials and methods described herein, any methods and materials similar or equivalent to those described herein, or any methods and materials suitable to carry out the scope of the invention, may be used in the practice or testing of the present invention In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
As used herein, the articles “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein, the terms “control,” or “reference” are used interchangeably, and refer to a value that is used as a standard of comparison.
“Vaccination” refers to the process of inoculating a subject with an antigen to elicit an immune response in the subject, that helps to prevent or treat the disease or disorder the antigen is connected with. The term “immunization” is used interchangeably herein with vaccination.
The term “immunogenicity” as used herein, refers to the innate ability of an antigen or organism to elicit an immune response in an animal when the antigen or organism is administered to the animal. Thus, “enhancing the immunogenicity” refers to increasing the ability of an antigen or organism to elicit an immune response in an animal when the antigen or organism is administered to an animal. The increased ability of an antigen or organism to elicit an immune response can be measured by, among other things, a greater number of antibodies that bind to an antigen or organism, a greater diversity of antibodies to an antigen or organism, a greater number of T-cells specific for an antigen or organism, a greater cytotoxic or helper T-cell response to an antigen or organism, a greater expression of cytokines in response to an antigen, and the like.
The term “activation”, as used herein, refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change. Within the context of T cells, such activation refers to the state of a T cell that has been sufficiently stimulated to induce cellular proliferation. Activation of a T cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physico-chemical process.
The term “activated T cell” means a T cell that is currently undergoing cell division, cytokine production, performance of regulatory or cytolytic effector functions, and/or has recently undergone the process of “activation.”
“Humoral immunity” or “humoral immune response” both refer to B-cell mediated immunity and are mediated by highly specific antibodies, produced and secreted by B-lymphocytes (B-cells).
“Prevention” refers to the use of a pharmaceutical compositions for the vaccination against a disorder.
“Adjuvant” refers to a substance that is capable of potentiating the immunogenicity of an antigen. Adjuvants can be one substance or a mixture of substances and function by acting directly on the immune system or by providing a slow release of an antigen. Examples of adjuvants are aluminium salts, polyanions, bacterial glycopeptides and slow release agents as Freund's incomplete.
“Delivery vehicle” refers to a composition that helps to target the antigen to specific cells and to facilitate the effective recognition of an antigen by the immune system. The best-known delivery vehicles are liposomes, virosomes, microparticles including microspheres and nanospheres, polymers, bacterial ghosts, bacterial polysaccharides, attenuated bacterias, virus like particles, attenuated viruses and ISCOMS.
“Incorporated into” or “encapsulated in” refers to an antigenic peptide that is within a delivery vehicle, such as microparticles, bacterial ghosts, attenuated bacteria, virus like particles, attenuated viruses, ISCOMs, liposomes and preferably virosomes.
As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise a protein or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
A “fusion protein” as used herein refers to a protein wherein the protein comprises two or more proteins linked together by peptide bonds or other chemical bonds.
The proteins can be linked together directly by a peptide or other chemical bond, or with one or more amino acids between the two or more proteins, referred to herein as a “spacer”.
In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
The term “RNA” as used herein is defined as ribonucleic acid. The term “dsRNA” is used to define double-stranded RNA.
“Transform”, “transforming”, and “transformation” is used herein to refer to a process of introducing an isolated nucleic acid into the interior of an organism.
The term “treatment” as used within the context of the present invention is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for the disease or disorder. As used herein, the term “treatment” and associated terms such as “treat” and “treating” means the reduction of the progression, severity and/or duration of a disease condition or at least one symptom thereof. The term ‘treatment’ therefore refers to any regimen that can benefit a subject. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviative or prophylactic effects. References herein to “therapeutic” and “prophylactic” treatments are to be considered in their broadest context. The term “therapeutic” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylactic” does not necessarily mean that the subject will not eventually contract a disease condition. Thus, for example, the term treatment includes the administration of an agent prior to or following the onset of a disease or disorder thereby preventing or removing all signs of the disease or disorder. As another example, administration of the agent after clinical manifestation of the disease to combat the symptoms of the disease comprises “treatment” of the disease.
The term “biological” or “biological sample” refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. Frequently the sample will be a “clinical sample” which is a sample derived from a patient. Such samples include, but are not limited to, bone marrow, cardiac tissue, sputum, blood, lymphatic fluid, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
The term “equivalent,” when used in reference to nucleotide sequences, is understood to refer to nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions- or deletions, such as allelic variants; and will, therefore, include sequences that differ from the nucleotide sequence of the nucleic acids described herein due to the degeneracy of the genetic code.
“Hybridization” refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing. Two single-stranded nucleic acids “hybridize” when they form a double-stranded duplex. The region of double-strandedness can include the full-length of one or both of the single-stranded nucleic acids, or all of one single stranded nucleic acid and a subsequence of the other single stranded nucleic acid, or the region of double-strandedness can include a subsequence of each nucleic acid. Hybridization also includes the formation of duplexes which contain certain mismatches, provided that the two strands are still forming a double stranded helix. “Stringent hybridization conditions” refers to hybridization conditions resulting in essentially specific hybridization. The term “specific hybridization” of a probe to a target site of a template nucleic acid refers to hybridization of the probe predominantly to the target, such that the hybridization signal can be clearly interpreted. As further described herein, such conditions resulting in specific hybridization vary depending on the length of the region of homology, the GC content of the region, the melting temperature “Tm” of the hybrid. Hybridization conditions will thus vary in the salt content, acidity, and temperature of the hybridization solution and the washes.
The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. An “isolated cell” or “isolated population of cells” is a cell or population of cells that is not present in its natural environment.
As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. ESTs, chromosomes, cDNAs, mRNAs, and rRNAs are representative examples of molecules that may be referred to as nucleic acids.
The term “variant,” when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to that of a gene or the coding sequence thereof. This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants. The polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants may encompass “single nucleotide polymorphisms” (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
The term “silent mutation” as used herein refers to a change in the sequence or identity of nucleotide bases which constitute DNA, without a subsequent change in the amino acid sequence of the function of the overall protein.
The term “ameliorating” or “treating” means that the clinical signs and/or the symptoms associated with a disease are lessened as a result of the actions performed. The signs or symptoms to be monitored will be well known to the skilled clinician.
As used herein, by “combination therapy” is meant that a first agent is administered in conjunction with another agent. “In combination with” or “In conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in combination with” refers to administration of one treatment modality before, during, or after delivery of the other treatment modality to the individual. Such combinations are considered to be part of a single treatment regimen or regime.
“Titers” are numerical measures of the concentration of a virus or viral vector compared to a reference sample, where the concentration is determined either by the activity of the virus, or by measuring the number of viruses in a unit volume of buffer. The titer of viral stocks are determined, e.g., by measuring the infectivity of a solution or solutions (typically serial dilutions) of the viruses, e.g., on HeLa cells using the soft agar method (see, Graham & Van Der eb (1973) Virology 52:456-467) or by monitoring resistance conferred to cells, e.g., G418 resistance encoded by the virus or vector, or by quantitating the viruses by UV spectrophotometry (see, Chardonnet & Dales (1970) Virology 40:462-477). In some embodiments, the titer is provided in units of plaque forming units (pfu) per mL (pfu/mL).
The term “high-titer” as used herein describes a titer of at least about 1×107 pfu/mL in some embodiments, or at least about 5×107 pfu/mL in some embodiments, or at least about 1×108 pfu/mL in some embodiments.
As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with other chemical components, such as carriers, stabilizers, diluents, adjuvants, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
The language “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
The term “antibody” or “Ab” as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies useful in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab and F(ab).sub.2, as well as single chain antibodies (scFv) and humanized antibodies (Harlow et al., 1998, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). An antibody may be derived from natural sources or from recombinant sources. Antibodies are typically tetramers of immunoglobulin molecules.
The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
“Heterologous antigens” used herein to refer to an antigen that is not endogenous to the organism comprising or expressing an antigen. As an example, a virus vaccine vector comprising or expressing a viral or tumor antigen comprises a heterologous antigen. The term “Heterologous protein” as used herein refers to a protein that elicits a beneficial immune response in a subject, irrespective of its source.
As defined herein, an “Alphavirus” is a member of the Group IV Togaviridae family of viruses. Alphaviruses include, but are not limited to Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong'nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Sagiama virus, Salmon pancreas disease virus, Semliki Forest virus, Sindbis virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus and Whataroa virus.
As defined herein, an “alphavirus non-structural protein” can be any of nsp1, nsp2, nsp3 and nsp4, as well as combinations and variants thereof.
As defined herein, an “alphavirus structural protein” can be selected from the group consisting of an alphavirus capsid protein and at least one spike protein.
The term “specifically binds”, “selectively binds” or “binding specificity” refers to the ability of the humanized antibodies or binding compounds of the invention to bind to a target epitope present on VSV with a greater affinity than that which results when bound to a non-target epitope. In certain embodiments, specific binding refers to binding to a target with an affinity that is at least 10, 50, 100, 250, 500, or 1000 times greater than the affinity for a non-target epitope.
As used herein, the term “effective amount” or “therapeutically effective amount” means the amount of the virus like particle generated from vector of the invention which is required to prevent the particular disease condition, or which reduces the severity of and/or ameliorates the disease condition or at least one symptom thereof or condition associated therewith.
The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In the present disclosure, the term “vector” includes an autonomously replicating virus.
The terms “2A” or “2A peptide” is a self-processing viral peptide. The 2A peptide can separate different protein coding sequences in a single ORF transcription unit (Ryan et al., 1991, J Gen Virol 72:2727-2732). Initially, the 2A peptide cleavage was thought to be mediated by an autoproteolytic event and 2A peptides were called “self-cleaving peptides.” Ultimately, a ribosomal-skip mechanism was proposed, and 2A and 2A-like sequences are now referred to as CHYSELs (cis-acting hydrolase elements) rather than self-cleaving peptides (Donnelly et al., 2001, J Gen Virol 82:1013-1025) Linking proteins with 2A or 2A-like peptide sequences results in cellular expression of multiple, discrete proteins (in essentially equimolar quantities) derived from a single ORF (de Felipe et al., 2006, Trends Biotechnol 24:68-75).
As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. There are numerous expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art that may be used in the compositions of the invention. “Operably linked” should be construed to include RNA expression and control sequences in addition to DNA expression and control sequences. “Operably linked” should further not be construed to define any specific ordering of sequence elements that are defined to be operably linked in order to form a recited construct, such as a vector, and it can be appreciated that operably linked sequence elements can be assembled in any order such that the construct comprising operably linked sequences is useful, even if crudely, for its intended purpose.
“Oncolytic” as used herein refers to the property of a virus or VLV to infect and kill cancer cells via an apoptotic mechanism. Likewise, “oncolytic effect” refers to the property of a virus or VLV to induce oncolysis
A “subject” or “patient”, as used herein, may be a human of non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In some embodiments, the subject is human.
“Vesiculovirus glycoprotein” as used herein is any suitable vesiculovirus glycoprotein that promotes the formation of a virus-like vesicle when incorporated into an SFV replicon encoding SFV non-structural proteins. In some embodiments, the vesiculovirus glycoprotein is a vesicular stomatitis virus (SFV) glycoprotein.
“Malignancy” as used herein refers to the state or presence of cancer or tumors and is intended to refer to any cancerous state. The term “malignancy” is also intended to describe cells that have a propensity to become cancerous or may develop a propensity to become cancerous, or cells that have a characteristic of malignancy. The term “malignancy” is used interchangeably herein with “cancer” and “tumor”.
“Therapeutic agent” as used herein refers to any component of or encoded by the RNA of the VLV, such as cytokine agonists, cytokine antagonists, short hairpin RNAs, and other polynucleotides or polypeptides useful for the treatment, prophylaxis, and/or prevention of a malignancy or infectious disease which is produced in a host cell as replication or translation products the RNA of the VLV. In some embodiments, the VLV may comprise or encode more than one or multiple therapeutic agents. In some embodiments where an oncolytic effect alone is desired, a VLV may not encode any therapeutic agents. The high-titer virus vector used to produce the VLV will comprise DNA sequences encoding the therapeutic agents and may comprise subgenomic promoters (SGPs), 2A sequences, or other sequences associated with the DNA sequences encoding the therapeutic agents.
“Armed” as used herein refers to the state of a VLV or high-titer virus vector comprising or encoding one or more therapeutic agents. For example, a “VLV armed with IL-12” would comprise sequence elements encoding IL-12. It can also be appreciated that a given VLV has a corresponding high-titer hybrid virus vector that encodes it, and it is therefore appropriate to refer to a “high-titer virus vector armed with IL-12” or a “vector armed with IL-12” to the same end.
“Percent Identity” is a term used to describe sequence relationships between two or more polynucleotides of polypeptides. In some cases, a sequence of given sequence (e.g. see Sequence Listing) is termed the “reference sequence” to which the contemplated variant sequence is compared. In general, the comparison is made along the length of the reference sequence after a optimal alignment of the reference sequence and the contemplated variant. Computer-implemented algorithms such as those utilized by the BLAST family of programs may be used to produce an optimal alignment for comparison purposes. Any of the sequences disclosed in the present invention can have about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity or homology to the reference sequence.
The high-titer hybrid virus vectors of the present invention comprise five operably linked sequence elements: a first DNA sequence comprising a DNA promoter sequence; a second DNA sequence encoding alphavirus non-structural protein polynucleotide sequences; a third DNA sequence encoding an alphavirus subgenomic RNA promoter; a fourth DNA sequence comprising at least one sequence domain each independently selected from the group consisting of a sequence domain encoding a cytokine agonist polypeptide a sequence domain encoding a cytokine antagonist polypeptide a sequence domain encoding a short hairpin RNA (shRNA), and combinations thereof; and a fifth DNA sequence encoding a vesiculovirus glycoprotein. When the vector is propagated in cell culture, high titers of virus like vesicles (VLVs) are obtained, for example, titers of at least 1×107 plaque forming units (pfu) per ml are obtained.
Methods of making the high-titer hybrid-virus vector of the invention are described in detail in the Experimental Examples Section herein.
In one aspect the alphavirus is Semliki Forest virus (SFV). In another aspect the subgenomic RNA promoter is an Semliki Forest virus (SFV) promoter. In another aspect, the alphavirus non-structural protein comprises an RNA-dependent RNA replicase. In another aspect, the Semliki Forest virus (SFV) non-structural protein comprises a SFV RNA-dependent RNA replicase.
In one aspect, the VSV encoding the VSV G protein can be from any VSV serotype known in the art. Non-limiting examples of VSV serotypes include the Indiana (IND-VSV) serotype and New Jersey (NJ-VSV) serotype. In an embodiment, the NJ-VSV serotype glycoprotein is defined by a DNA sequence comprising SEQ ID NO. 11 and a polypeptide sequence comprising SEQ ID NO. 12. In another embodiment, the NJ-VSV serotype glycoprotein is defined by a DNA sequence or polypeptide sequence comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity or homology to SEQ ID NOs. 11 or 12.
In one aspect, although the cytomegalovirus immediate early promoter is exemplified herein, the invention should not be construed to be limited to this promoter sequence. Promoter sequences that are useful in the invention include any promoter that induces high levels of gene expression. Such promoters may include, but are not limited to those disclosed elsewhere herein.
In a further aspect of the composition of the invention comprises DNA encoding a heterologous protein inserted between the subgenomic alphavirus promoter and DNA encoding the VSV G protein wherein the DNA encoding the heterologous protein is operably linked to DNA encoding a T2A peptide from Thosea asigna virus (Szymczak et al., 2004. Nature Biotechnology 22:589-594) which is in turn operably linked to DNA encoding the VSV G protein. In this way, expression of the heterogeneous protein in the resulting VLVs of the invention is effectively tied to expression of the VSV G protein, the latter being essential for replication of the vector. Thus, expression of the heterologous protein is stabilized and the continued presence of the gene expressing this protein in the hybrid vector is assured.
In some embodiments, the 2A peptide is selected from the group consisting of equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A), Thosea asigna virus (T2A) and any 2A peptide or fragment thereof known in the art. In further embodiments, the 2A peptide is a T2A peptide or any T2A fragment thereof known in the art.
In some embodiments, the heterologous gene can be under the control of an RNA virus promoter sequence that may not necessarily be an alphavirus subgenomic promoter sequence. Such modifications and variations of the RNA promoter sequence driving expression of the heterologous promoter sequences will become apparent to those skilled in the art as they practice the invention. The vector may also include conventional control elements which are operably linked to the heterologous gene in a manner which permits its transcription, translation and/or expression in a cell infected with the hybrid-virus vector produced by the invention.
The high-titer hybrid virus vectors comprise, among other elements, a DNA sequence comprising at least one sequence domain each independently selected from the group consisting of a sequence domain encoding a cytokine agonist polypeptide a sequence domain encoding a cytokine antagonist polypeptide a sequence domain encoding a short hairpin RNA (shRNA), and combinations thereof. The VLV produced therefrom comprises RNA containing, in the case of an shRNA, or encoding, in the case of a polypeptide, the elements that have a therapeutic effect when delivered to or are produced within a target cell. Such elements are termed “therapeutic agents” and are useful in the treatment, prophylaxis, and prevention of malignancy or infectious disease. In some embodiments, the therapeutic agent is a heterologous protein.
An important aspect of the present invention is that a given high-titer hybrid virus vector may encode several different therapeutic agents. In some aspects, multiple polypeptide therapeutic agents can be encoded by a given high-titer hybrid virus vector. In some aspects, multiple polynucleotide or shRNA therapeutic agents can be encoded by a given high-titer hybrid virus vector. In further aspects, a given high-titer hybrid virus vector can encode one or more polypeptide therapeutic agents and one or more polynucleotide or shRNA therapeutic agents.
A VLV or composition comprising VLVs produced from the high-titer hybrid virus vector comprises dsRNA that includes certain therapeutic agents, such as shRNAs, or encodes polypeptide therapeutic agents. In some embodiments, the VLV or composition comprising VLVs includes or encodes a single therapeutic agents. In some embodiments, the VLV or composition comprising VLVs includes or encodes multiple therapeutic agents. In some embodiments, the VLV or composition comprising VLVs includes or encodes from 1-10 therapeutic agents, or from 1-7 therapeutic agents, or from 1-5 therapeutic agents, or from 1-3 therapeutic agents. In some embodiments, the VLV or composition comprising VLVs is trivalent, meaning that three therapeutic agents are included or encoded.
It can be understood that any therapeutic agent useful for the treatment, prophylaxis, and prevention of a malignancy and/or infectious disease could be incorporated into the high-titer hybrid virus vectors and VLVs of the present invention. In some embodiments, the therapeutic agents are associated with a malignancy or cancer. In some embodiments, the therapeutic agents are cytokine agonist or antagonist polypeptides. In some embodiments, the therapeutic agents are shRNA polynucleotide checkpoint inhibitors.
The encoded cytokine agonist or antagonist polypeptide therapeutic agents may be any such polypeptide that is useful for, or plays a role in, the treatment, prophylaxis, and/or prevention of a malignancy or cancer. In some embodiments, the encoded cytokine agonist or antagonist polypeptides can be any of IL-2, IL-7, IL-15, IL-18, IL-19, IL-35, IL-21, GM-CSF, IL-17, Flt3L, or combinations thereof.
In some embodiments, the shRNA polynucleotide checkpoint inhibitors can be a polynucleotide selected from any of PD-L2, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, CD90, BTLA, CD160, PD-1, or combinations thereof. In further embodiments, the shRNA polynucleotide checkpoint inhibitors can be any such polynucleotide that is useful for, or plays a role in, the treatment, prophylaxis, and/or prevention of a malignancy or cancer.
Table 1 provides descriptions for exemplary sequences of the present invention. In some aspects, a therapeutic agent polynucleotide or polypeptide sequence comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity or homology to any of the reference sequence described herein (see, e.g., Sequence Listing). Typically, a contemplated variant will maintain at least a portion of the biological activity of the reference sequence.
In some embodiments, the high-titer hybrid virus vector is trivalent and encodes IL-12, IL-17RA-DN, and PD-L1 shRNA. In another embodiment, the high-titer hybrid virus vector comprises a sequence domain having at least about 70% sequence identity with SEQ ID NO. 6, a sequence domain having at least about 70% sequence identity with SEQ ID NO. 8, and a sequence domain having at least about 70% sequence identity with SEQ ID NO 10.
The high-titer hybrid virus vectors of the present invention encode, among the other elements, alphavirus non-structural proteins. The alphavirus nonstructural proteins of the present invention can be any such proteins that are suitable for carrying out the scope and methods of the invention. In some embodiments, the alphavirus nonstructural proteins comprise an RNA-dependent RNA replicase. In other embodiments, the alphavirus nonstructural proteins are Semliki Forest Virus (SFV) non-structural proteins. In some embodiments, the SFV non-structural proteins comprise a SFV RNA-dependent RNA replicase.
In some embodiments of carrying out the invention, the alphavirus nonstructural proteins comprise mutations that enhance the titer of VLVs produced in the methods of the invention. See U.S. Pat. No. 10,435,712 by Rose et al., incorporated by reference herein in its entirety, which relates to compositions and methods for immunization using high-titer hybrid-virus vectors. The high-titer hybrid virus vectors producing evolved VLV compositions therein comprise SFV alphavirus nonstructural proteins with at least two mutations that improve the titer of VLVs produced.
In some embodiments of carrying out the invention, the alphavirus nonstructural proteins of the present invention are SFV alphavirus nonstructural proteins comprising at least one mutation as described in Table 2. The mutations listed in Table 2 are shown bolded and underlined in SEQ ID NO. 13 in Table 1. In Table 2, the amino acid mutations shown are relative to the wild type sequence. That is, when each of the mutations in Table 2 are present, the SFV nonstructural protein sequence is defined as SEQ ID NO. 13. SEQ ID NO. 13 comprises four SFV nonstructural protein segments, i.e. nsP1 (amino acids 1-537), nsP2 (amino acids 538-1136), nsP3 (amino acids 1137-1818), and nsP4 (amino acids 1819-2432).
The high-titer hybrid virus vectors of the present invention encoding for mutated alphavirus nonstructural proteins can be prepared with any suitable polynucleotide sequences for producing the desired amino acid sequences. It can be appreciated that the nucleotide sequences utilized to generate the polypeptide sequences of the present invention can potentially or optionally include mutations, including silent mutations, or alternate codons that function to produce the associated amino acid sequence. It would be readily apparent to a skilled practitioner whether or not a given polynucleotide sequence would produce a given polypeptide sequence.
In some embodiments, the high-titer hybrid virus vectors of the present invention comprise a DNA sequence encoding a SFV nonstructural protein with at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% similarity to SEQ ID NO: 13. In further embodiments, the high-titer hybrid virus vectors of the present invention comprise a DNA sequence encoding a SFV nonstructural protein comprising SEQ ID NO: 13.
The vectors of the invention are useful in a variety of applications for generating VLVs useful for the treatment, prophylaxis, and/or prevention of a malignancy or infection disease. The vectors are further useful for immunizing a subject against a malignancy or disease, and/or treating, preventing or diminishing risk of malignancy or infectious disease in a subject.
The invention includes a method of immunizing a subject against an heterologous protein associated with an infectious disease. The method comprises administering to the subject a composition comprising virus like vesicles (VLVs) produced by the high-titer hybrid virus vector, wherein the high-titer hybrid virus vector comprises DNA encoding a heterogeneous gene. Expression of the heterogeneous gene induces an immune response to the heterologous protein associated with an infectious disease encoded thereby in the subject. The invention further includes a method of treating a subject in need thereof where the method comprises administering to the subject a composition comprising virus like vesicles (VLVs) produced by the high-titer hybrid virus vector of the invention, wherein the high-titer hybrid virus vector comprises DNA encoding a heterogeneous gene and wherein expression of the heterogeneous gene provides benefit to the subject. In one aspect, the invention includes a method of generating a memory T cell immune response to a heterologous protein in a subject. In another aspect, generating an adaptive B cell immune response to a heterologous protein in a subject.
Also included in the invention is a method of diminishing the risk that a subject will develop an infectious disease. The method comprises administering to the subject a composition comprising the virus like vesicles (VLVs) produced by the high-titer hybrid virus vector, wherein the high-titer hybrid virus vector comprises DNA encoding a heterogeneous gene. Expression of the heterogeneous gene induces an immune response to the heterologous protein associated with the infectious disease encoded thereby in the subject, thereby diminishing the risk that the subject will develop an infectious disease associated with the heterologous protein.
The invention also includes a method of selecting high-titer RNA virus like vesicles (VLVs). This selection is achieved by passaging in a cell culture a hybrid-virus vector comprising an RNA sequence encoding a viral structural protein, wherein the viral structural protein does not comprise an alphavirus structural protein and by screening the generated VLVs for high-titer VLVs using extensive passaging of VLVs in cell culture, direct mutagenesis of the VLVs or any other selection method known in the art. In one aspect the hybrid-virus vector RNA sequence encodes an alphavirus non-structural protein. In another aspect the non-structural protein is a Semliki Forest virus (SFV) non-structural protein.
The VLVs of the invention may be formulated as a pharmaceutical composition. Such a pharmaceutical composition may be in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
In one embodiment, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1×105 and 1×109 PFU.
In one embodiment, the pharmaceutical compositions useful for practicing the method of the invention may comprise an adjuvant. Suitable adjuvants contemplated by this invention include but are not limited to Freund's complete adjuvant, Freund's incomplete adjuvant, Quil A, Detox, ISCOMs or squalene.
Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it is understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment.
The regimen of administration may affect what constitutes an effective amount. For example, the VLVs produced by the high-titer hybrid virus vector of the invention or compositions thereof may be administered to the subject in a single dose, in several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions of the present invention to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat the disease in the subject. An effective amount of the composition necessary to achieve the intended result will vary and will depend on factors such as the disease to be treated or prevented, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. In particular embodiments, it is especially advantageous to formulate the composition in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the composition and the heterologous protein to be expressed, and the particular therapeutic effect to be achieved.
One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Routes of administration of any of the compositions\of the invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
In some embodiments a kit is provided for treating, preventing, or ameliorating an a given disease, disorder or condition, or a symptom thereof, as described herein wherein the kit comprises: a) a compound or compositions as described herein; and optionally b) an additional agent or therapy as described herein. The kit can further include instructions or a label for using the kit to treat, prevent, or ameliorate the disease, disorder or condition. In yet other embodiments, the invention extends to kits assays for a given disease, disorder or condition, or a symptom thereof, as described herein. Such kits may, for example, contain the reagents from PCR or other nucleic acid hybridization technology (microarrays) or reagents for immunologically based detection techniques (e.g., ELISpot, ELISA).
The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods.
The VLV double promoter (dp) vector was linearized with Ascl/SbfI restriction digestion to insert VSV New Jersey G fragment (Carolina Chiale et. al vaccines 2020, 8, 279). A DNA fragment encoding envelope glycoprotein from New Jersey vesiculovirus was gene-synthesized from Synbio Technologies and infused into Ascl and SbfI cloning sites downstream of the second sub-genomic promoter. The resulted VLV dp VSV New Jersey G vector digested with BamHI/PacI and used to clone IL12 and CARG2020 inserts downstream of the first sub-genomic promoter.
To clone single chain IL-12 into dp-IL12 with VSV New Jersey G construct, mouse IL12 p40 and IL12p35 fragments were synthesized as string fragments from Thermo Fisher Scientific. The p40 and p35 fragments were PCR amplified using primers pair modified with elastin linker sequence extensions ((gttcctggagtaggg/gtcccaggtgtgggc (VPGVGVPGVG)) and flanking BamHI and PacI cloning sites. All PCR fragments were amplified with Q5® high-fidelity DNA polymerase, purified from the gel, and infused into VLV dp VSVG New Jersey vector BamHI/PacI cloning sites downstream to the first sub-genomic promoter.
To combine IL17RA ECD HA tag and three shRNAs fragment, the extracellular domain (ECD) part of 17A receptor domain (N-terminal 972 bp fragment) was PCR amplified with modified reverse primer contain HA-tag sequence (YPYDVPDYA). The mouse three shRNAs sequences targeting PDL1 gene at 486,373 and 9 positions were synthesized from Synbio Technologies. To infuse IL17RA ECD HA tag fragment with shRNAs fragment, both fragments were purified from the gel and infused into VLV dp VSVG New Jersey vector BamHI/PacI cloning sites downstream to the first sub-genomic promoter.
To clone final Triple sub-genomic promoter (Tp) CARG-2020 VSV New Jersey G construct, a full-length IL12 fragment, and an IL17 RA ECD HA tag/shRNAs fragment with sub-genomic promoter sequence (as generated above) were amplified using Q5® high-fidelity DNA polymerase. All PCR fragments were purified from the gel and infused into VLV dp VSVG New Jersey vector BamHI/PacI cloning sites downstream to the first sub-genomic promoter. This cloning step creates an extra sub-genomic promoter between IL12 and IL17RA ECD.
All primers to amplify DNA fragments were designed according to NEBuilder instruction and infused according to NEB HiFi DNA Assembly Kit Protocol (E5520S). The recombinant clones screened for positive inserts by DNA restriction digestion. The final positive clones were further confirmed by DNA sequencing (GENEWIZ, NJ).
A high-titer hybrid virus vector (Dp-G(NJ)-IL12) was constructed with several sequence elements in order to produce VLVs competent for the production of IL-12 polypeptide (SEQ ID NO. 2), i.e. a DNA promoter sequence, a DNA sequence encoding Semliki Forest Virus non-structural protein (SFV nsp1-4), a DNA sequence encoding vesicular stomatitis virus glycoprotein (VSV-G; SEQ ID NO: 11), a DNA sequence encoding IL-12 (SEQ ID NO: 1) as shown in
BHK-21 cells were transfected with the Dp-G(NJ)-IL12 high-titer hybrid virus vector using standard protocols. The transfected cells were incubated in buffer for 48 hours for VLV production to proceed. Non-transfected cells were incubated as a control. After the incubation period, the supernatants were tested for the presence of IL-12 in an anti-IL-12 Western blot, and IL-12 was observed in the supernatant of the transfected cells but not in the control.
The quantity of IL-12 secreted after 48 hours of incubation (See Example 1) was determined using a UV-Visible spectroscopic method. A standard curve was prepared using various dilutions of IL-12p70 in the concentration range from 0 to 500 pg/m L. The calibration data curve is shown below in Table 2.
A linear best fit obtained the equation shown in Table 3. The supernatant optical density (OD) was measured at a 4,000-fold dilution to obtain an OD fraction 450/750 nm of 0.3945 corresponding to 0.745 μg of IL-12 per mL of supernatant. This value corresponds to 2 μg of IL-12 per million transfected cells produced.
MC38 cells were transplanted subcutaneously into a syngeneic mouse strain to allow tumors to grow under the skin of mice. VLVs producing IL-12 (See example 1; VLV-IL-12) were produced and prepared as a composition in PBS buffer for injection. A PBS buffer not containing the VLVs was used as the control. The experimental design and timeline are shown in
A high-titer hybrid virus vector (Dp-G(NJ)-IL17RA) was constructed with several sequence elements in order to produce VLVs competent for the production of dominant negative IL-17RA-DN extracellular domain (ECD) polypeptide (SEQ ID NO. 4), i.e. a DNA promoter sequence, a DNA sequence encoding Semliki Forest Virus non-structural protein (SFV nsp1-4), a DNA sequence encoding vesicular stomatitis virus glycoprotein (VSV-G; SEQ ID NO: 11), a DNA sequence encoding IL-17RA-DN (SEQ ID NO. 3) as shown in
BHK-21 cells were transfected with the Dp-G(NJ)-IL17RA high-titer hybrid virus vector using standard protocols. The transfected cells were incubated in buffer for 48 hours for VLV production to proceed. Non-transfected cells were incubated as a control. After the incubation period, the supernatants were tested for the presence of IL-17RA-DN in a Western blot, and IL-17RA-DN was observed in the supernatant of the transfected cells but not in the control. Additionally, Western blots of BHK-21 cell lysates indicated the presence of IL-17RA-DN in cells transfected with the vector.
Four high-titer hybrid virus vectors were constructed, each with several sequence elements in order to produce VLVs competent for the production of the encoded shRNA sequences, i.e. a DNA promoter sequence, a DNA sequence encoding Semliki Forest Virus non-structural protein (SFV nsp1-4), a DNA sequence encoding vesicular stomatitis virus glycoprotein (VSV-G; SEQ ID NO. 11), and a DNA sequence encoding an shRNA sequence or sequences as shown in
VLVs of the four different vectors were produced in BHK-21 cells as described in Examples 1 and 4. PD-L1 expressing BHK-21 cells were then treated with the produced VLVs. While the VLVs expressing the scrambled (Scr) shRNA did not show suppression of PD-L1 suppression while each of the VLVs expressing the shRNA 486 sequence, the shRNA 373 sequence, or the shRNA 486/373 showed suppression of PD-L1. These results indicate that VLVs can be engineered to express one or more shRNAs effective against gene targets associated with cancer and malignancy.
A high-titer hybrid virus vector (Tp-IL12-1L17RA/shRNA-G(NJ); CARG2020) was constructed with several sequence elements in order to produce VLVs competent for the production of all three of IL-12 polypeptide (SEQ ID NO. 2), dominant negative IL-17RA-DN extracellular domain (ECD) polypeptide (SEQ ID NO. 4), and PD-L1 checkpoint inhibitor shRNA 486/373/9, i.e. a DNA promoter sequence, a DNA sequence encoding Semliki Forest Virus non-structural protein (SFV nsp1-4), a DNA sequence encoding vesicular stomatitis virus glycoprotein (VSV-G; SEQ ID NO. 11), a DNA sequence encoding IL-12 (SEQ ID NO. 1), a DNA sequence encoding IL-17RA-DN (SEQ ID NO. 3), and a DNA sequence encoding shRNA 486/373/9 (SEQ ID NO. 5), as shown in
VLVs of the trivalent CARG2020 vectors were produced in BHK-21 cells as described in Examples 1 and 4. The polypeptide products were detected using Western blots, indicating that the trivalent VLVs are able to produce IL-12, IL-17RA-DN, and VSV glycoprotein (VSVG). In vivo PD-L1 downregulation in PD-L1 expressing BHK-21 cells indicated that CARG2020 VLVs (containing shRNA 486/373/9) downregulated PD-L1 more efficiently than the control VLV IL-12 lacking shRNA.
The experimental design and results are shown in
To test the effect of CARG-2020 and VLV-IL-12 in tumor immunity, we treated MC38 tumors with 2 doses via intratumoral injection (
Normal murine fibroblasts NIH 3T3 were labeled with CFSE dye (green) whereas tumorigenic fibroblasts of line L929 that causes fibrosarcoma were labeled with eFluor670 (red). The cells were co-cultured and either mock treated (control) or treated with VLV at multiplicity of infection (MOI) of 10. Fluorescence microscopy was used to view the cells after 72 hours of incubation had elapsed. The microscopy images indicate that most of the L929 are undergoing apoptosis caused by the replication of the VLV, while normal NIH 3T3 were not lysed.
Oncolytic identification was performed by flow cytometry analysis. The kit for live/dead cell staining (eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermofisher Scientific, 65-0865-14) was used following manufacture user manual. Briefly, cells were harvested by trypsinization and washed two times with cold PBS, centrifuged at 1500 rpm for 5 min at 4° C., triplicity resuspended the cells using 96 well plate with 100 ul of cells for each well (5×104 cells/well), spin down and wash 2 times with cold 1×PBS. Add 100 ul of the live/dead stain (diluted 1:1000 in PBS) to each well. Incubate in dark at 4° C. for 15 minutes; wash 2 times with 200 ul FACS buffer. Add 100 ul fixative solution and incubate in the dark for 45 min at 4° C., Wash cells in permeabilization buffer and stain first with the primary antibodies for 1 hr. After 2-3 washes, stain in secondary Ab for 30 min. Wash 2 times with FACS buffer and resuspend cells in 200 ul FACS buffer, Transfer to cluster tubes for Flow cytometry (LSR II Flow Cytometer at Cell Sorter Core Facility (The Health Center at UCNN) analysis.
The results shown in
The results in
VLVs are observed to be oncolytic toward malignancies and cancer cells but not toward normal cells. The oncolytic pathway was further investigated using Western blot (
The results indicate that infection of the HCT116 cancer cell line with 5 MOI of VLVs results in induced the activation of caspase 8 and caspase 9, expression of tumor necrosis factor (TNF), and cleavage of poly(ADP) ribose polymerase (PARP indicating that oncolysis of cancer cells occurs through apoptotic mechanisms (
The entire disclosure of each of the patent documents, including certificates of correction, patent application documents, scientific articles, governmental reports, websites, and other references referred to herein is incorporated by reference herein in its entirety for all purposes. In case of a conflict in terminology, the present specification controls.
The invention can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are to be considered in all respects illustrative rather than limiting on the invention described herein. In the various embodiments of the compositions and methods of the present invention, where the term comprises is used with respect to the compositions or recited steps of the methods, it is also contemplated that the compositions and methods consist essentially of, or consist of, the recited compositions or steps or components. Furthermore, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
In the specification, the singular forms also include the plural forms, unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification will control.
Furthermore, it should be recognized that in certain instances a composition can be described as being composed of the components prior to mixing, or prior to a further processing step such as drying, binder removal, heating, sintering, etc. It is recognized that certain components can further react or be transformed into new materials.
All percentages and ratios used herein are on a volume (volume/volume) or weight (weight/weight) basis as shown, or otherwise indicated.
This application is a national phase entry under 35 U.S.C. § 371 of International Application PCT/US2021/012834, which claims priority under 35 U.S.C § 119(e) to U.S. Provisional Application No. 62/959,435, filed Jan. 10, 2020, all of which are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/012834 | 1/8/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62959435 | Jan 2020 | US |
