Viruses for inducing antigen presentation

Abstract

The present disclosure relates to a genetically modified virus comprising at least one nucleic acid molecule encoding a cytokine that recruits and / or activates T cells and a class II major histocompatibility complex transactivator (CIITA), as well as pharmaceutical compositions comprising said genetically modified virus and methods of treating cancer with said genetically modified virus.

Description

VIRUSES FOR INDUCING ANTIGEN PRESENTATION

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 570,340 filed March 27, 2024, the contents of which are incorporated herein by reference.

[0003] FIELD

[0004] The present invention relates to genetically modified viruses for inducing antigen presentation in infected cells.

[0005] BACKGROUND

[0006] Genetically modified viruses have been produced for use in treating cancer. For example, oncolytic viruses are viruses that preferentially infect and / or kill cancer cells. Various approaches have been undertaken to develop oncolytic viruses for the effective targeting of cancer cells in the treatment of cancer.

[0007] Although oncolytic viruses have been developed, there has been varying success in improving the host immune response against virus -infected tumors. There thus remains a need for improving anti-tumor responses in the context of viral anti-cancer strategies.

[0008] SUMMARY

[0009] In an embodiment, there is provided a genetically modified virus, comprising at least one nucleic acid encoding (i) a cytokine that recruits and / or activates T cells; and (ii) a class II major histocompatibility complex trans activator (CIITA).

[0010] In an embodiment, there is provided a pharmaceutical composition comprising the genetically modified virus as described herein and a pharmaceutically acceptable carrier.

[0011] In an embodiment, there is provided a method of treating cancer in a subject, comprising administering the genetically modified virus as described herein or the pharmaceutical composition as described herein to the subject.

[0012] In an embodiment, there is provided a use of the genetically modified virus as described herein or the pharmaceutical composition as described herein for treating cancer in a subject.

[0013] In an embodiment, there is provided a use of the genetically modified virus as described herein in the preparation of a medicament for treating cancer in a subject.

[0014] In an embodiment, there is provided a genetically modified virus as described herein or a pharmaceutical composition as described herein for use in treating cancer in a subject.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Embodiments will be described, by way of example only, with reference to the accompanying figures wherein:

[0017] Figure 1: CIITA expression in U2OS: non-infected (lane 1), and infected with CIITA-encoding (lane 3) and control (lane 4) viruses.

[0018] Figure 2: Human IL-12 (hIL-12) expression from control and CIITA- and hIL-12- encoding (P2700) viruses.

[0019] Figure 3: Enhanced tumor antigen recognition in infected human sarcoma cell line U2OS and T-cell activation leading to increased IFN-gamma release after co-culture with T- cells.

[0020] Figure 4: U2OS and T-cell co-culture, with control or and P2700 virus pretreatment.

[0021] DETAILED DESCRIPTION

[0022] The present invention relates to genetically modified virus that is capable of inducing antigen presentation in cells that are infected by the genetically modified virus. The genetically modified virus encodes class II major histocompatibility complex transactivator (CIITA) and inflammatory cytokines, which can induce antigen presentation by infected cells and activate T cells. Infection by the genetically modified virus of a cancer cell and subsequent expression of transgenes enhances antigen presentation by the cancer cell and stimulates T-cell activation, leading to cancer cell killing and resulting in anti-cancer immunity. In some embodiments, the genetically modified virus may encode other transgenes such as antigens and / or checkpoint inhibitors to direct the body’s cellular machinery to recognize tumor antigens encoded in the virus and produce cellular and humoral immunity against cancer cells.

[0023] Genetic Modification

[0024] As used herein, the term “genetically modified” or “genetically engineered” refers to altering genetic material using molecular biology techniques known in the art such as, but not limited to, molecular cloning, recombinant DNA methods, and gene editing by knowntechniques such as with the use of restriction endonucleases, Gibson assembly, zinc finger nucleases, TALENs, and Crispr-Cas systems such as Crispr-Cas9. Genetic modification includes addition, deletion, substitution, modification, and / or mutation of genetic material. Numerous techniques for the genetic modification of virus are known and routinely used in the art (Mackett et al., J. Virol. (1984), 49(3):857-864; Falkner, F. and Moss, B., J. Virol. (1990), 64(6):3108- 3111; Domi, A., and Moss, B., PNAS (2002), 99(19): 12415-12420; Yuan et al., J. Virol. (2015), 89(9):5176-5179). As used herein, the term “modified” may refer to a nucleic acid molecule or a polypeptide that contains one or more changes in the nucleotide or amino acid sequence compared to the un-modified wild-type version of said nucleic acid molecule or polypeptide.

[0025] As used herein, the term “wild-type” carries the ordinary meaning in the art of an organism, nucleic acid molecule, or polypeptide that can be found naturally occurring in the absence of a modification as described herein. A naturally occurring, wild-type nucleic acid molecule or polypeptide can be modified to differ in sequence, structure, and / or biological properties as compared to the un-modified wild-type version of the nucleic acid molecule or polypeptide.

[0026] As used herein, the term “nucleic acid molecule” may refer to a large sequence of nucleotides encoding multiple cassettes, operons, genes, and / or reading frames (e.g. a chromosomes, a plasmid, an artificial chromosome, a viral genome) but may also refer to a smaller sequence of nucleotides encoding one or a small number (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of cassettes, operons, genes and / or reading frames (e.g. a transgene or a cassette) wherein the smaller sequence of nucleic acids may be isolated or may be comprised in a larger sequence of nucleotides (e.g. . a chromosomes, a plasmid, an artificial chromosome, a viral genome). Further, as used herein, the term "nucleic acid molecule" is intended to include unmodified DNA or RNA or modified DNA or RNA. The nucleic acid molecules of the disclosure may contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms.

[0027] As used herein, the term “exogenous” refers to an element that has been introduced into a nucleic acid molecule, such as a viral genome. An exogenous nucleic acid molecule is introduced into a genome by a method of genetic modification. An exogenous nucleic acid molecule may code for the expression of an RNA and / or a protein. An exogenous nucleic acid molecule may have been derived from the same species (homologous) or from adifferent species (heterologous). An exogenous nucleic acid molecule may comprise a homologous sequence that is altered such that it is introduced into a genome in a form that is not naturally found in the genome. For example, an exogenous nucleic acid molecule that is homologous may contain mutations or be integrated into a different region of the genome, relative to the endogenous version of the nucleic acid molecule.

[0028] The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences.

[0029] Nucleic acid sequences as described herein can be provided in nucleic acid molecules in different arrangements or combinations. As used herein with reference to nucleic acid molecules that encode a gene or polypeptide, the term “at least one nucleic acid molecule encoding” means that all encoded sequences are collectively comprised in one or more nucleic acid molecules. In some embodiments, all encoded sequences are comprised in separate nucleic acid molecules, all encoded sequences are comprised in the same nucleic acid molecule, or combinations where some encoded sequences are comprised in separate nucleic acid molecules and some encoded sequences are comprised together in the same nucleic acid molecule. For example, in the genetically modified virus of the present invention, the nucleic acid sequences encoding the cytokine and the CIITA may be provided in the same nucleic acid molecule or in separate nucleic acid molecules. Where more than one sequence that encodes a cytokine and / or CIITA is provided in the same nucleic acid molecule, the sequences can be provided in separate expression cassettes, or together in the same expression cassette. Where two or more sequences are in the same expression cassette, they may form a polycistronic mRNA for subsequent translation, wherein each of the sequences has its own ribosomal binding site for translation. Where two or more sequences are in the same expression cassette, they can be provided in the same open reading frame so as to produce a fusion protein. Two or more sequences that encode a fusion protein can be separated by linker sequences that encode restriction nuclease recognitionsites or self-cleaving peptide linkers. Nucleic acid molecules encoding genes or polypeptides can include, without limitation, plasmids, artificial chromosomes, episomes, viral genomes, transgenes, and expression cassettes inserted into known vectors or viral genomes.

[0030] The term “operably linked”, as used herein, refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner. For example, a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the transcriptional regulatory sequence or promoter facilitates aspects of the transcription of the coding sequence. The skilled person can readily recognize aspects of the transcription process, which include, but are not limited to, initiation, elongation, attenuation and termination. In general, an operably linked transcriptional regulatory sequence j oined in cis with the coding sequence, but it is not necessarily directly adjacent to it.

[0031] The word “expression” as used herein refers to the translation of a polypeptide (for example a cytokine of CIITA) encoded by a nucleic acid (such as an exogenous nucleic acid molecule) and / or transcription of an RNA transcript (for example, mRNA or an interfering RNA, such as microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA) or Dicer-substrate small-interfering RNA (DsiRNA)) encoded by a nucleic acid (such as an exogenous nucleic acid molecule). As used herein, “increased expression” refers to an increase in the amount of translated polypeptide and / or RNA transcript encoded by a nucleic acid (such as an exogenous nucleic acid). The increased expression may be relative to a wild-type virus or a cell infected by a wild-type virus.

[0032] As used herein, the term “exogenous” refers to an element that has been introduced into a nucleic acid molecule, such as a genome or chromosome. An exogenous nucleic acid molecule is introduced into a genome by a method of genetic modification. An exogenous nucleic acid molecule may code for the expression of an RNA and / or a protein. An exogenous nucleic acid molecule may have been derived from the same species (homologous) or from a different species (heterologous). An exogenous nucleic acid molecule may comprise a homologous sequence that is altered such that it is introduced into a genome in a form that is not naturally found in the genome. For example, an exogenous nucleic acid molecule that is homologous may contain mutations or be integrated into a different region of the genome, relative to the endogenous version of the nucleic acid molecule.

[0033] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences.To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions multiplied by 100%). The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin, S. and Altschul, S., PNAS (1990), 87(6):2264-2268, modified in Karlin, S. and Altschul, S., PNAS (1993), 90(12):5873-5877. Such an algorithm is incorporated into the BLAST programs. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a given nucleic acid molecule. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a given polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. (1997), 25(17):3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers, E. and Miller, W., Bioinformatics (1988), 4(1): 11-17. Such an algorithm is incorporated in the ALIGN program which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0034] Viruses

[0035] In some embodiments, the genetically modified virus is a genetically modified poxvirus. Poxviruses are viruses of the family Poxviridae, comprising a family of double-stranded DNA viruses. Poxviridae comprises the genera Avipoxvirus, Capripoxvirus, Centapoxvirus, Cervidpoxvirus, Crocodylipoxvirus , Leporipoxvirus , Macropopoxvirus, Molluscipoxvirus, Mustelpoxvirus, Orthopoxvirus, Oryzopoxvirus, Parapoxvirus, Pteropopoxvirus, Salmonpoxvirus, Sciuripoxvirus, Suipoxvirus, Vespertilionpoxvirus, Yatapoxvirus, Alphaentomopoxvirus , Betaentomopoxvirus , Deltaentomopoxvirus, Diachasmimorpha entomopoxvirus, and Gammaentomopoxvirus . Four genera of poxviruses are known to infect humans: Molluscipoxvirus, Orthopoxvirus, Parapoxvirus , and Yatapoxvirus . The Molluscipoxvirus comprises the molluscum contagiosum virus (MCV). The Orthopoxvirus comprises the variola virus, vaccinia virus, cowpox virus, buffalopox virus, horsepox virus, rabbitpox virus, taterapox virus, camelpox virus, ectromelia virus, alaskapox virus, skunkpox virus, racconpox virus, and monkeypox virus. The Parapoxvirus comprises the orf virus, pseudocowpox virus, and bovine popular stomatitis virus. The Yatapoxvirus comprises the tanapox virus, and yaba monkey tumor virus.

[0036] In some embodiments, the genetically modified virus is a vaccinia virus. Vaccinia virus is a member of the orthopoxvirus of the Poxviridae family. Vaccinia is a double-stranded DNA virus with a genome of about 190 kb and encoding for approximately 250 genes, including C12L which encodes a interleukin- 18 binding protein; C2L and F3L which each encode a kel ch- like protein; NIL which encodes a BCL-2 inhibitor; N2L which encodes a TLR signaling inhibitor; MIL, B18R, and B20R which each encode an Ankyrin repeat protein; M2L, K1L and K7R which each encode aNF-KB inhibitor; K2L which encodes a serine protease inhibitor; K3L which encodes a PKR inhibitor; K4L which encodes a phospholipase-D; K5L and K6L which each encode a monoglyceride lipase; F1L which encodes a caspase-9 inhibitor; F2L which encodes a dUTPase; B16R which encodes an IL- 1 -beta-inhibitor; B19R which encodes a interferon-alpha / beta-receptor-like secreted glycoprotein; A56R which encodes a glycoprotein; J2R which encodes the viral thymidine kinase (TK); A28L, H2R, A21L, L5R, G3L, O3L, A16L, G9R, J5L, L1R, and F9L which encode proteins of the entry / fusion complex; and A26L, A27L, D8L, H3L which encode attachment proteins. Vaccinia virus replicates only in the cytoplasm of the host cell, and the large genome codes for various enzymes and proteins needed for viral DNA replication (Greseth, M. and Traktman, P., Annu. Rev. Virol. (2022), 9:239-259).

[0037] Many strains of vaccinia virus are known in the art and may be used in the present invention. In some embodiments, the genetically modified vaccinia virus of the present invention is a genetically modified LISTER strain, modified vaccinia Anakara (MV A) strain, Copenhagen strain, Wyeth strain, Western Reserve strain, or Tian Tan strain of vaccinia virus. In someembodiments, the genetically modified vaccinia virus of the present invention is a genetically modified MVA strain of vaccinia virus.

[0038] Cytokine

[0039] The genetically modified virus of the present invention comprises at least one nucleic acid molecule that encodes a cytokine that recruits and / or activates T cells. By “activates T cells” it is meant that the cytokine activates T cell mediated immune responses, as opposed to the activity of regulatory cytokines that inhibit T cell mediated immune responses. Functions of cytokines that recruit and / or activate T cells include recruiting, activating, proliferating, and / or promoting viability of T cells. In some embodiments, the cytokine comprises IL-12, IL-18, IL- 15, IL-7, and / or IL-2.

[0040] In some embodiments, the cytokine is IL-12. Active IL-12 comprises a heterodimer of an IL-12a (p35) subunit and an IL-12b (p40) subunit. In some embodiments, the IL-12 comprises the IL-12a amino acid sequence of SEQ ID NO: 1 (UniProtKB / Swiss-Prot: P29459.2); and the IL-12b amino acid sequence of SEQ ID NO: 2 (UniProtKB / Swiss-Prot: P29460.1).

[0041] CIITA

[0042] The genetically modified virus of the present invention comprises at least one nucleic acid molecule that encodes a class II major histocompatibility complex transactivator (CIITA, also known as C2TA). CIITA promotes the expression of MHC class II molecules, which present antigen to T cells. T cells recognizing antigen in the context of MHC class II presentation are activated in an antigen-specific manner to effect T cell mediated immune responses. Induction of MHC Class II expression on infected cancer cells allows them to present antigen to stimulate local anti-tumor T cell mediated immune responses. In some embodiments, the infected cancer cells will present endogenous antigen derived from polypeptides produced by the cancer cell, such as a tumor antigen. In some embodiments, the genetically engineered virus of the present invention may further comprise a nucleic acid sequence that encodes an antigen, such as for example a tumor antigen, and in such embodiments the encoded antigen can be presented by the infected cancer cell to activate T cells.

[0043] In some embodiments, the CIITA comprises the amino acid sequence of SEQ ID NO: 3 (UniProtKB / Swiss-Prot: P33076.3); or of SEQ ID NO: 4 (NCBI Reference Sequence: NP_000237.2).

[0044] Checkpoint Inhibitors and Co-Stimulatory Molecules

[0045] In some embodiments, the genetically modified virus of the present invention further encodes a checkpoint inhibitor and / or a co-stimulatory molecule.

[0046] As used herein, a “checkpoint inhibitor” refers to a molecule that totally or partially reduces, inhibits, interferes with or modulates one or more checkpoint proteins. Checkpoint proteins regulate cell activation or function. Numerous checkpoint proteins are known, such as for example CTLA-4 and its ligands CD80 and CD86; PD-1 and its ligands PD- L1 and PD-L2; CD27; CD28; CD40; CD122; CD137; CD137 / 4-1BB; ICOS; IL-10; 0X40 TGF-beta; TOR receptor; glucocorticoid-induced TNFR-related protein GITR; killer-cell immunoglobulin-like receptor (KIR); lymphocyte activation gene-3 (LAG3); V-domain Ig suppressor of T cell activation (VISTA) T-cell immunoglobulin domain and mucin domain 3 (TIM-3); and indoleamine 2,3-dioxygenase (IDO), as well as their ligands and / or receptors.

[0047] In some embodiments, the checkpoint inhibitor is an antibody or an antigen binding fragment thereof that is an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1, CD279), CTLA-4 (CD154), PD-L2 (B7-DC, CD273), LAG3 (CD223), TIM3 (HAVCR2, CD366), 41BB (CD137), 2B4, A2aR, B7H1, B7H3, B7H4, B- and T-lymphocyte attenuator (BTLA), CD2, CD27, CD28, CD30, CD33, CD40, CD70, CD80, CD86, CD160, CD226, CD276, DR3, GAL9, GITR, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), Killer inhibitory receptor (KIR), LAG-3, LAIR1, LIGHT, MARCO (macrophage receptor with collageneous structure), phosphatidylserine (PS), OX-40, Siglec-5, Siglec-7, Siglec-9, Siglec-11, SLAM, TIGIT, TIM3, TNF-a, VISTA, VTCN1, or any combination thereof.

[0048] In some embodiments, the checkpoint inhibitor is an inhibitor of PD-L1 or PD-1. In an embodiment, the inhibitor of PD-L1 or PD-1 may be an anti-PD-1 or anti-PD-Ll antibody or an antigen binding fragment thereof, such as but not limited to those disclosed in WO 2015 / 103602. In some embodiments, the anti-PD-1 antibody or anti-PD-Ll antibody is selected from nivolumab, pembrolizumab, pidilizumab, atezolizumab, or durvalumab.

[0049] In some embodiments, the immune response checkpoint inhibitor is an inhibitor of CTLA-4. In an embodiment, the inhibitor of CTLA-4 may be an anti-CTLA-4 antibody or an antigen binding fragment thereof, such as but not limited to ipilimumab and tremelimumab.

[0050] An “antibody” refers to a polypeptide or protein that consists of or comprises antibody domains, which are understood as constant and / or variable domains of the heavy and / or light chains of immunoglobulins, with or without a linker sequence. The term “antibody” refers to an intact antibody. In an embodiment, an “antibody” may comprise a full-lengthimmunoglobulin molecule, including e.g. polyclonal, monoclonal, chimeric, humanized and / or human versions having full length heavy and / or light chains. The term “antibody” encompasses any and all isotypes and subclasses, including without limitation the major classes of IgA, IgD, IgE, IgG and IgM, and the subclasses IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The term “chimeric antibody” as used herein refers to a recombinant protein that contains the variable domains (including the complementarity determining regions (CDRs)) of an antibody derived from one species, such for example a rodent, while the constant domains of the antibody are derived from a different species, such as a human. A “humanized antibody” as used herein refers to a recombinant protein in which the CDRs from an antibody from one species (e.g. a rodent) are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains, including human framework region (FR) sequences. The constant domains of the humanized antibody are likewise derived from a human antibody. As used herein, the term “antigen binding fragment” or “functional fragment”, used interchangeably, with respect to an antibody, refers to an antigen binding portion of an antibody. In this context, by “functional” it is meant that the fragment maintains its ability to bind to the target antigen. The binding affinity of the antigen binding fragment may be equivalent to, or greater than, that of parent antibody, or may be less than the parent antibody, but nevertheless the antigen binding fragment maintains a specificity and / or selectivity for the target antigen. Antigen binding fragments of antibodies include, without limitation, a portion of an antibody such as a F(ab')2, a F(ab)2, a Fab', a Fab, a Fab2, a Fab?. a single domain antibody. Regardless of structure, an antigen binding fragment of an antibody binds with the same antigen that is recognized by the full antibody. The term “antigen binding fragment” or “functional fragment”, used interchangeably, in relation to antibodies, also includes isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker (“scFv proteins”). As used herein, the term “antigen binding fragment” or "functional fragment" does not include fragments such as Fc fragments that do not contain antigen binding site.

[0051] As used herein, a “co-stimulatory molecule” refers to a molecule that provides a secondary signal (z.e. a signal secondary to the activation signal from an antigen) to an immune cell to promote immune cell activation. Numerous co-stimulatory molecules are known, such as for example CD28, CD80 (B7.1). ICOS, CD226, 41-BB, 0X40, CD27, CD40, and BAFF, or a ligand or receptor of any thereof. In some embodiments, the co-stimulatory molecule is a co-stimulatory receptor, a co-stimulatory ligand, or an agonist antibody or fragment thereof that binds to a co-stimulatory receptor.

[0052] Pharmaceutical Compositions

[0053] In some embodiments, the genetically modified virus of the present invention is comprised in a pharmaceutical composition.

[0054] Pharmaceutical compositions comprising the genetically modified virus of the present invention can be prepared using methods known in the art using physiologically acceptable carriers, excipients, and / or or stabilizers (Remington The Science and Practice of Pharmacy 23rd Edition, Adejare, A. Ed. (2020) and in a desired form such as a solution, an emulsion, an aerosol, a capsule, a tablet, or a lyophilized formulation.

[0055] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical substances is well known in the art. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions. The phrase “pharmaceutically acceptable” refers to carriers and compositions that do not result in unnecessary allergy or toxicity when administered to a subject.

[0056] As used herein, an “excipient” is a molecule or substance that is included in a composition to improve the non-therapeutic properties of a composition such as its stability, pH, tonicity, shelf-life, color, melting point, viscosity, or other non-therapeutic properties. Many pharmaceutical excipients are known in the art and may be used in compositions of the present invention (Pramanick et al., Pharma Times (2013), 45(3):65-77). In some embodiments, the excipient comprises a bulking agent, a cytoprotectant, a buffering agent, a tonicity agent, a preservative, a surfactant, an antioxidant, or a polymer.

[0057] For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. Sterile aqueous solutions that can be employed will be known to those of skill in the art.

[0058] Treatment

[0059] The genetically modified virus of the present invention may be used in the treatment of cancer in a subject.

[0060] “Treating” or “treatment of’ as used herein, refers to an approach for obtaining beneficial or desired results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilisation of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset, and amelioration or palliation of the disease state. “Treating” can also mean prolonging survival of a patient beyond that expected in the absence of treatment and can also mean inhibiting the progression of disease temporarily or preventing the occurrence of disease. “Treating” may also refer to a reduction in the size of a tumor mass, reduction in tumor aggressiveness, etc.

[0061] As used herein, a "therapeutically effective amount" means an amount of the pharmaceutical composition or genetically modified virus effective to provide a therapeutic benefit to a subject. In some embodiments, a therapeutically effective amount of the composition is an amount capable of inducing a clinical response in a subject in the treatment of a particular disease or disorder such as cancer. Determination of a therapeutically effective amount of the composition is well within the capability of those skilled in the art. The therapeutically effective amount may vary according to a variety of factors such as the subject’s condition, weight, sex and age.

[0062] As used herein, the terms “cancer”, “cancer cells”, “tumor” and “tumor cells” may be used interchangeably to refer to cells or masses of cells that exhibit abnormal growth, characterized by a significant loss of control of cell proliferation or cells that have been immortalized. The term “cancer” or “tumor” includes metastatic as well as non-metastatic cancer or tumors. As used herein, a “solid tumor” refers to a tumor that does not contain cysts of liquid areas. A cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.

[0063] As used herein, the term “oncolytic,” refers to the capacity of virus, such as a genetically modified virus, to target cancer cells, inhibit cancer cell growth, destroy cancer cells and / or aid in the destruction of cancer cells by the immune system. As used herein "oncolytic” activity refers to inhibition or suppression of tumor and / or malignant and / or cancerous cell growth; regression of tumor and / or malignant and / or cancerous cell growth; cell death of tumor and / or malignant and / or cancerous cells or prevention of the occurrence of additional tumor and / or malignant and / or cancerous cells. As used herein, "inhibiting or suppressing tumorgrowth" refers to reducing the rate of growth of a tumor, halting tumor growth completely, causing a regression in the size of an existing tumor, eradicating an existing tumor and / or preventing the occurrence of additional tumors.

[0064] The genetically modified virus of the present invention may be used for treating cancer in a subject and / or in the preparation of a medicament for treating cancer in a subject. In some embodiments, the cancer comprises one or more solid tumors. In some embodiments, the cancer is selected from ovarian cancer, renal cancer, lung cancer, pancreatic cancer, skin cancer, stomach cancer, liver cancer, hepatic cell cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, uterine cancer, breast cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, testicular cancer, head and neck cancer, brain cancer, thymic cancer, sarcoma, melanoma, and bone cancer.

[0065] In some embodiments, the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer,pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom macroglobulinemia.

[0066] The genetically modified virus of the present invention may be delivered directly or in pharmaceutical compositions containing carriers, excipients, and / or stabilizers, as is known in the art. The genetically modified virus or pharmaceutical composition of the present invention may be administered to a subject by conventional techniques, such as intravenously, intramuscularly, intraperitoneally, intra-cerebrally, subcutaneously, intra-articularly, intrasynovially, intrathecally, trans dermally, intranasally, inhalation, or intra-tumorally. In some embodiments, the genetically modified virus or pharmaceutical composition of the present invention is administered adjacent to a tumor or directly into a tumor, optionally by injection.

[0067] Intratumoral injection, or injection directly into the tumor vasculature may be provided for discrete, solid, accessible tumors. Local, regional or systemic administration also may be used to expose tumor cells to the genetically modified virus. The genetically modified virus may be administered in multiple injections to the tumor or adjacent to the tumor.Continuous administration also may be applied where appropriate, for example, by implanting a catheter into a tumor or into tumor vasculature. Generally, the dose of the genetically modified virus or pharmaceutical composition via continuous perfusion may be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.

[0068] Particular embodiments of the disclosure include, without limitation, the following:1. A genetically modified virus, comprising at least one nucleic acid molecule encoding: (i) a cytokine that recruits and / or activates T cells; and (ii) a class II major histocompatibility complex transactivator (CIITA).2. The genetically modified virus of embodiment 1, wherein the cytokine is IL-12, IL-18, IL-15, IL-7, and / or IL-2.3. The genetically modified virus of embodiment 2, wherein the IL-12 comprises: (i) the amino acid sequence of SEQ ID NO: 1; and (ii) the amino acid sequence of SEQ ID NO: 2.4. The genetically modified virus of any one of embodiments 1-3, wherein the CIITA comprises the amino acid sequence of SEQ ID NO: 3 or 4.5. The genetically modified virus of any one of embodiments 1-4, wherein the virus is a poxvirus.6. The genetically modified virus of embodiment 5, wherein the poxvirus is a vaccinia virus.7. The genetically modified virus of embodiment 6, wherein the vaccinia virus is a LISTER strain, modified vaccinia Anakara (MV A) strain, Copenhagen strain, Wyeth strain, Western Reserve strain, or Tian Tan strain of vaccinia virus.8. The genetically modified virus of embodiment 6, wherein the vaccinia virus is a modified vaccinia Ankara (MV A) strain of vaccinia virus.9. The genetically modified virus of any one of embodiments 1-8, wherein the virus induces antigen presentation on MHC Class II molecules by a cell infected by the virus.10. The genetically modified virus of embodiment 9, wherein the virus induces presentation of an endogenous antigen on MHC Class II molecules by a cell infected by the virus.11. The genetically modified virus of any one of embodiments 1-10, wherein the at least one nucleic acid molecule further encodes an antigen.12. The genetically modified virus of embodiment 11, wherein the antigen is a tumor antigen.13. The genetically modified virus of any one of embodiments 1-12, wherein the at least one nucleic acid molecule further encodes a checkpoint inhibitor and / or a co-stimulatory molecule.14. The genetically modified virus of embodiment 13, wherein the checkpoint inhibitor is an antibody or an antigen binding fragment thereof.15. The genetically modified virus of embodiment 14, wherein the antibody or antigen binding fragment thereof binds to PD-1, PD-L1, or CTLA-4.16. The genetically modified virus of embodiment 15, wherein the checkpoint inhibitor is atezolizumab and / or nivolumab.17. A pharmaceutical composition comprising the genetically modified virus of any one of embodiments 1-16 and a pharmaceutically acceptable carrier.18. A method of treating cancer in a subject, comprising administering the genetically modified virus of any one of embodiments 1-16 or the pharmaceutical composition of embodiment 17 to the subject.19. Use of the genetically modified virus of any one of embodiments 1-16 or the pharmaceutical composition of embodiment 17 for treating cancer in a subject.20. Use of the genetically modified virus of any one of embodiments 1-16 in the preparation of a medicament for treating cancer in a subject.21. A genetically modified virus of any one of embodiments 1-16 or a pharmaceutical composition of embodiment 17 for use in treating cancer in a subject.22. The method, use, or genetically modified virus for use of any one of embodiments 18-21, wherein the cancer comprises a solid tumor.23. The method, use, or genetically modified virus for use of any one of embodiments 18-21, wherein the cancer is selected from ovarian cancer, lung cancer, pancreatic cancer, skin cancer, stomach cancer, liver cancer, hepatic cell cancer, colorectal cancer, esophageal cancer, uterine cancer, breast cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, testicular cancer, head and neck cancer, brain cancer, thymic cancer, lymphoma, leukemia, and bone cancer / osteo sarcoma.

[0069] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps

[0070] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the othercomponents or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0071] All publications and patents cited herein are incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the case of any conflict between a definition of a term in the present disclosure and a definition in a cited publication or patent, the definition provided in the present disclosure is to be used in describing the present invention.

[0072] The present invention will now be described by way of non-limiting examples having regard to the appended drawings.

[0073] EXAMPLE 1

[0074] Genetically modified recombinant vaccinia virus (MVA strain) P2700 encodes CIITA and IL-12 genes. Nucleic acid sequences encoding IL-12a and IL-12b were linked by a sequence encoding a P2A linker. Control virus is a recombinant vaccinia encoding a green fluorescent protein.

[0075] As shown in Figure 1: CIITA expression in U2OS cells: non-infected (lane 1), and infected with CIITA-encoding (lane 3) and control (lane 4) viruses. Bio-Rad precision plus dual color protein standard was used (Lane 2). Predicted band size: 130 kDa. CIITA Ab@ 1:500 (Abeam #ab70060) in 5% Milk in IX TBST (0.1%) - Incubated at 4°C overnight; 2° Ab: Goat anti-rabbit HRP @ 1:2000 (Invitrogen # 65-6120) in 5% Milk in IX TBST (0.1%) - Incubated at RT for 1 h.

[0076] As shown in Figure 2, human IL-12 expression from control and P2700 viruses: IL-12 expression in the supernatants (ELISA for hIL-12). Supernatants from non-infected, control virus infected and two clones of P2700 infected U2OS cells were collected and hIL-12 measured by ELISA. hIL12 was quantified by ELISA using the R & D systems’ Human IL-12 p70 DuoSet ELISA kit (Cat# DY1270).

[0077] As shown in Figure 3: Enhanced tumor antigen recognition in human sarcoma cell line U2OS and T-cell activation leading to increased IFN-gamma release after co-culture with T-cells. U2OS cells infected with P2700 virus or control virus were incubated with T cells to measure T cell activation as indicated by IFN-gamma secretion by activated T cells. As shown in Figure 4: U2OS and T-cell co-culture. Enhanced cancer cell killing of human sarcoma cell line U2OS was observed when infected with P2700 and co-cultured with T-cells. Minimum cell killing was exhibited with no viral infection or with infection with control virus.

Claims

CLAIMS:

1. A genetically modified virus, comprising at least one nucleic acid molecule encoding:(i) a cytokine that recruits and / or activates T cells; and(ii) a class II major histocompatibility complex transactivator (CIITA).

2. The genetically modified virus of claim 1, wherein the cytokine is IL-12, IL-18, IL-15, IL-7, and / or IL-2.

3. The genetically modified virus of claim 2, wherein the IL-12 comprises:(i) the amino acid sequence of SEQ ID NO: 1; and(ii) the amino acid sequence of SEQ ID NO: 2.

4. The genetically modified virus of any one of claims 1-3, wherein the CIITA comprises the amino acid sequence of SEQ ID NO: 3 or 4.

5. The genetically modified virus of any one of claims 1-4, wherein the virus is a poxvirus.

6. The genetically modified virus of claim 5, wherein the poxvirus is a vaccinia virus.

7. The genetically modified virus of claim 6, wherein the vaccinia virus is a LISTER strain, modified vaccinia Anakara (MV A) strain, Copenhagen strain, Wyeth strain, Western Reserve strain, or Tian Tan strain of vaccinia virus.

8. The genetically modified virus of claim 6, wherein the vaccinia virus is a modified vaccinia Ankara (MV A) strain of vaccinia virus.

9. The genetically modified virus of any one of claims 1-8, wherein the virus induces antigen presentation on MHC Class II molecules by a cell infected by the virus.

10. The genetically modified virus of claim 9, wherein the virus induces presentation of an endogenous antigen on MHC Class II molecules by a cell infected by the virus.

11. The genetically modified virus of any one of claims 1-10, wherein the at least one nucleic acid molecule further encodes an antigen.

12. The genetically modified virus of claim 11, wherein the antigen is a tumor antigen.

13. The genetically modified virus of any one of claims 1-12, wherein the at least one nucleic acid molecule further encodes a checkpoint inhibitor and / or a co-stimulatory molecule.

14. The genetically modified virus of claim 13, wherein the checkpoint inhibitor is an antibody or an antigen binding fragment thereof.

15. The genetically modified virus of claim 14, wherein the antibody or antigen binding fragment thereof binds to PD-1, PD-L1, or CTLA-4.

16. The genetically modified virus of claim 15, wherein the checkpoint inhibitor is atezolizumab and / or nivolumab.

17. A pharmaceutical composition comprising the genetically modified virus of any one of claims 1-16 and a pharmaceutically acceptable carrier.

18. Use of the genetically modified virus of any one of claims 1-16 or the pharmaceutical composition of claim 17 for treating cancer in a subject.

19. The use of claim 18, wherein the cancer comprises a solid tumor.

20. The use of claim 18, wherein the cancer is selected from ovarian cancer, lung cancer, pancreatic cancer, skin cancer, stomach cancer, liver cancer, hepatic cell cancer, colorectal cancer, esophageal cancer, uterine cancer, breast cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, testicular cancer, head and neck cancer, brain cancer, thymic cancer, lymphoma, leukemia, and bone cancer / osteo sarcoma.

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