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Definitions

• the present disclosure relates generally to the fields of oncology, virology and immunotherapy. It concerns poxviruses, specifically the highly attenuated modified vaccinia virus Ankara (MVA), and a recombinant modified vaccinia Ankara virus with deletion of vaccinia virulence factor E3 (MVAAE3L), each further modified to express human Fms-like tyrosine kinase 3 ligand (Flt3L) or GM-CSF.
• the disclosure relates to use of the foregoing recombinant viruses as cancer immunotherapeutic agents.
• the foregoing recombinant poxviruses can also be used in combination with immune checkpoint blockade therapy. Background
• Immunotherapy has become an evolving area of research and an additional option for the treatment of certain types of cancers.
• the immunotherapy approach rests on the rationale that the immune system may be stimulated to identify tumor cells, and target them for destruction.
• tumors develop a number of immunomodulatory mechanisms to evade antitumor immune responses.
• immune inhibitory cytokines such as TGF- ⁇
• immune cells such as CD4 + T regulatory cells and macrophages
• Tumors have also the ability to bias CD4 + T cells to express the regulatory phenotype.
• the overall result is impaired T-cell responses and impaired induction of apoptosis or reduced anti-tumor immune capacity of CD8 + cytotoxic T cells. Additionally, tumor-associated altered expression of MHC class I on the surface of tumor cells makes them 'invisible' to the immune response (4)(Garrido et al. Cancer Immunol. Immunother. 59(10), 1601-1606 (2010)). Inhibition of antigen-presenting functions and dendritic cell (DC) additionally contributes to the evasion of anti-tumor immunity (5)(Gerlini et al. Am. J. Pathol. 165(6), 1853-1863 (2004)).
• Immune checkpoints have been implicated in the tumor-mediated downregulation of anti- tumor immunity and used as therapeutic targets. It has been demonstrated that T cell dysfunction occurs concurrently with an induced expression of the inhibitory receptors, CTLA-4 and programmed death 1 polypeptide (PD-1), members of the CD28 family of receptors.
• PD-1 is an inhibitory member of the CD28 family of receptors that in addition to PD-1 includes CD28, CTLA-4, ICOS and BTLA.
• Poxvirus-based oncolytic therapy has the advantage of killing cancer cells through a combination of cell lysis, apoptosis, and necrosis. It also triggers innate immune sensing pathway that facilitates the recruitment of immune cells to the tumors and the development of anti-tumor adaptive immune responses.
• the current oncolytic vaccinia strains in clinical trials are replicative strains. They use wild-type vaccinia with deletion of thymidine kinase to enhance tumor selectivity, and with expression of transgenes such as granulocyte macrophage colony stimulating factor (GM-CSF) to stimulate immune responses (/0)(Breitbach et al., Curr Pharm Biotechnol 13, 1768-1772 (2012)).
• GM-CSF granulocyte macrophage colony stimulating factor
• the composition further comprises a second amount of a replication competent recombinant attenuated vaccinia virus with deletion of thymidine kinase encoding and expressing human Flt3L, wherein the second amount contributes to augmenting the induced or enhanced or promoted immune response.
• the composition further comprises a third amount of inactivated MVA wherein the third amount contributes to augmenting the induced or enhanced or promoted immune response.
• the subject is a human.
• the present disclosure relates to a method for treating a malignant tumor in a subject, the method comprising delivering to tumor cells of the subject a virus selected from the group consisting of MVA-hFlt3L , MVAAE3L-hFlt3L and a combination thereof in an amount effective to induce the immune system of the subject to mount an immune response against the tumor or to enhance or promote an ongoing immune response of said subject against the tumor and conjointly administering to the subject a second amount of an immune checkpoint blocking agent or an immune checkpoint agonist effective to block immune suppressive mechanisms within the tumor.
• a virus selected from the group consisting of MVA-hFlt3L , MVAAE3L-hFlt3L and a combination thereof in an amount effective to induce the immune system of the subject to mount an immune response against the tumor or to enhance or promote an ongoing immune response of said subject against the tumor and conjointly administering to the subject a second amount of an immune checkpoint blocking agent or an immune checkpoint agonist effective to block immune suppressive mechanisms within the tumor
• the conjoint administration enhances effector T-cell responses; in some embodiments, the conjoint administration enhances memory T cell responses. In some embodiments, the conjoint administration significantly increases survival, achieves at least, inhibition of growth of the tumor including metastatic tumor compared to either monotherapy,
• the immune checkpoint blocking agent is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA4 inhibitors, inhibitory antibodies against LAG- 3 (lymphocyte activation gene 3), TIM3 (T cell Immunoglobulin and Mucin-3), B7-H3, and TIGIT (T-cell immunoreceptor with Ig and ITIM domains); and the immune checkpoint agonist is selected from the group consisting of anti-ICOS antibody anti-OX40 antibody agonist antibody against 4- IBB (CD 137) and against GITR.
• the tumor is primary or metastatic melanoma or primary or metastatic colon carcinoma.
• the virus is delivered and the immune checkpoint blocking agent is administered each according to its own administration schedule of spaced apart intervals.
• a first dose of the virus is delivered first and after a lapse of time a first dose of the immune checkpoint blocking agent is administered.
• the present disclosure relates to a kit comprising: 1) A first component comprising a composition according the present disclosure;
• MVA-hFlt3L or MVAAE3L-hFlt3L strain can be used as cancer immunotherapeutic agent.
• intratumoral delivery of MVAAE3L-hFlt3L is more efficacious in eradiating tumors and generating antitumoral immunity than MVAAE3L.
• intratumoral delivery of MVA- hFlt3L is more efficacious in eradiating tumors and generating antitumoral immunity than MVA.
• patients can be treated with MVA-hFlt3L or MVAAE3L- hFlt3L or both in order to achieve improved treatment results.
• Viral genomic DNAs were analyzed by PCR to verify the insertions of transgenes and deletion of TK, and to make sure there were no contaminating patent viruses, MVA or MVAAE3L.
• the PCR products including the inserted transgenes were sequenced to make sure the inserted genes have the correct sequences.
• adenocarcinoma cells (1 x 10 5 ).
• Figure 4 is a series of graphical representations of data collected after intratumoral injection of MVAAE3L-hFlt3L demonstrating that it is effective in inducing proliferation and activation of CD8 + T cells in both injected and non-injected tumors in a bilateral melanoma model.
• (B) is a series of representative flow cytometry plots of CD8 + cells expressing Granzyme B in injected and non-injected tumors.
• (D) is a series of representative flow cytometry plots of CD8 + cells expressing Ki-67 in injected and non-injected tumors.
• Figure 5 is a series of graphical representations of data collected after intratumoral injection of MVAAE3L-hFlt3L demonstrating that it is effective in inducing proliferation and activation of CD4 + T cells in both injected and non-injected tumors.
• (A) consists of two plots of the percentage of CD4 + Granzyme B + cells in injected and non- injected tumors of mice treated with PBS, MVAAE3L, MVAAE3L-mGM-CSF, MVAAE3L- hFlt3L, or Heat-MVA (*, p ⁇ 0.05, **, p ⁇ 0.01, ****, p ⁇ 0.0001).
• Figure 6 is a series of graphical representations of data collected after intratumoral injection of MVAAE3L-hFlt3L showing that it is effective in inducing reduction of CD4 + FoxP3 + regulatory T cells in both injected and non-injected tumors.
• B is a series of representative flow cytometry plots of CD4 + cells expressing FoxP3 in injected and non-injected tumors.
• Figure 7 is a series of bar graphs showing that intratumoral injection of MVAAE3L-hFlt3L is effective in the recruitment of CD45 + cells and CD8 + cells, and increases the ratio of CD8 + /Treg as well as of conventional CD4 + /Treg in both injected and non-injected tumors.
• A, B two plots of absolute numbers of tumor-infiltrating CD45 + and CD8 + cells respectively per gram of injected and non-injected tumors of mice treated with PBS, MVAAE3L, MVAAE3L-mGM-CSF, MVAAE3L-hFlt3L, or Heat-MVA (*, p ⁇ 0.05, **, p ⁇ 0.01, ns: non-significant).
• FIG 8 is a series of graphic representations of flow cytometry plots of F4/80 + TAMs and CD24 + DCs in injected and non-injected tumors.
• TAMs and DCs are CD45 + MHC-II hi Ly6C 10 . They were further separated by CD24 and F4/80 expression patterns.
• TAMs are F4/80hiCD24 l0 , where CD24 + DCs express high levels of CD24.
• Figure 9 is a series of graphic representations showing that intratumoral injection of MVAAE3L-hFlt3L reduces tumor-associated macrophages (TAM) in both injected and non-injected tumors.
• TAM tumor-associated macrophages
• CD103* and CDl lb + DCs in injected and non-injected tumors after virus treatment can be further separated by their expression of CDl lb and CD103.
• CDl lb + DCs express a high level of CDl lb
• CD103 + DCs express a high level of CD103.
• Figure 11 is a series of graphical representations showing that intratumoral injection of M V ⁇ 3 L-hFlt3 L leads to the reduction of CD24 + , CD103 + and CDl lb + dendritic cells in the injected tumors and CD24 + , CD103 + dendritic cells in the non-injected tumors.
• A, B Percentages of CD24 + DCs out of CD45 + cells in both injected (B) and non- injected (A) tumors of WT mice treated with PBS, MVAAE3L, MVAAE3L-mGM-CSF, MVAAE3L-hFlt3L, or Heat-MVA as well as ⁇ 3 ⁇ 3 _ " mice treated with PBS or MVAAE3L- Flt3L (**, p ⁇ 0.01, ***, p ⁇ 0.001, ****, p ⁇ 0.0001).
• C, D Percentages of CD103 + DCs out of CD45 + cells in both injected (D) and non-injected (C) tumors (*, p ⁇ 0.05, **, p ⁇ 0.01, ***, p ⁇ 0.001, ****, p ⁇ 0.0001).
• Figure 12 is a series of graphic representations of flow cytometry plots of myeloid cell populations in injected and non-injected tumors after virus treatment.
• CD45 + tumor infiltrating myeloid cells were further separated by their expression of CDl lb and Ly6C.
• Ly6C + cells can be further divided into several populations, including Ly6C hl CDl lb + inflammatory monocytes, Ly6C hl CDl lb " cells, as well as Ly6C inl CDl lb + cells (likely neutrophils).
• Figure 13 is a series of graphic representations of data showing that intratumoral injection of MVAAE3L-hFlt3L leads to the influx of Ly6C + CDl lb " and Ly6C + CDl lb + cells in both injected and non-injected tumors.
• A, B Percentages of Ly6C hl CDl lb + cells out of CD45 + cells in both injected (B) and non-injected (A) tumors of WT mice treated with PBS, MVAAE3L, MVAAE3L-mGM-CSF, MVAAE3L-hFlt3L, or Heat-MVA as well as Batfi " ' mice treated with PBS or MVAAE3L-Flt3L (*, p ⁇ 0.05, **, p ⁇ 0.01, ****, p ⁇ 0.0001).
• C, D Percentages Ly6C hi CDllb- cells of out of CD45 + cells in both injected (B) and non-injected (A) tumors (*, p ⁇ 0.05, **, p ⁇ 0.01, ***, p ⁇ 0.001).
• Figure 14 is a series of graphical representations of data showing that intratumoral injection of MVAAE3L-hFlt3L is more effective than MVAAE3L in delaying the growth of both virus-injected and non-injected (contralateral) tumors in a bilateral MC38 tumor implantation model.
• (A) consists of 12 plots of volume of injected and non-injected tumor over days after injection with PBS, MVAAE3L, or MVAAE3L-hFlt3L viruses in a bilateral MC38 tumor implantation model. As described before, the viruses were only injected into the larger tumors on the right flank.
• Figure 15 shows a series of graphical representations of intratumoral injection of MVAAE3L or MVAAE3L-hFlt3L in a 4T1 murine triple negative breast carcinoma (TNBC) bilateral implantation model.
• 4T1 cells 2.5 x 10 5
• 5 x 10 4 cells were implanted to the left flank of BALB/c mice.
• the right side tumors about 3 mm in diameter
• PBS PBS
• MVAAE3L (2 x 10 7 pfu
• MVAAE3L-hFlt3L 2 x 10 7 pfu
• (A) are a series of graphs of respective tumor volumes (injected and non- injected) over days after injection with PBS, MVAAE3L, or MVAAE3L-hFlt3L viruses in a bilateral 4T1 tumor implantation model.
• Figure 17 is a series of graphical representations of data showing the combination of intratumoral injection of MVA-hFlt3L is effective for delaying the growth or eradicating tumors in a bilateral B16-F10 murine melanoma implantation model.
• A Volumes of injected and contralateral non-injected tumors over days after injections with PBS, or MVA-hFlt3L, or MVAAE3L-hFlt3L in a bilateral B16-F10 murine melanoma implantation model.
• Figure 17 shows that the combination of intratumoral injection of MVAAE3L-hFlt3L with systemic delivery of anti-CTLA-4, anti-PD- 1, or anti-PD-Ll antibodies increases the overall response and cure rates in a B16-F10 bilateral implantation model.
• B Volumes of injected and contralateral non-injected tumors over days after intratumoral injections with MVAAE3L-hFlt3L in the presence of intraperitoneal delivery of anti-CTLA-4, anti-PD-1, or anti-PD-Ll antibodies in a bilateral B16-F10 murine melanoma implantation model.
• Figure 18 is a series of graphical representations of data showing that Intratumoral injection with MVAAE3L-hFlt3L leads to the generation of antitumor CD8 + T cell immunity against autologous and heterologous tumors, which is enhanced in the presence of anti-CTLA-4 antibody.
• A is a scanned image of ELISPOT in triplicates. Pooled CD8 + T cells isolated from 3 spleens of naive mice were used as a negative control (Naive). Pooled CD8 + T cells isolated from 3 spleens of MVAAE3L-hFlt3L-treated mice without anti-CTLA- 4 (-Ab) showed reactivity against both irradiated B 16-F10 and MC38.
• Figure 19 is a series of graphical representations of data showing that intratumoral injection with Heat-inactivated MVA induces higher levels of type I IFN and inflammatory cytokines and chemokines gene expression in both injected and non-injected tumors than MVA in a bilateral B16-F10 tumor implantation model. Shown here are graphs of quantitative real-time PCR analyses of Ifnb, 116, Ccl4, Ccl5, Cxcl9, and CxcllO gene expression in both injected and non-injected B16-F10 tumors treated with either PBS, MVA or Heat-MVA.
• FIG 20 is a series of graphic representations showing that intratumoral injection with Heat- inactivated MVA induces anti-melanoma CD8 + T cell responses in the tumor draining lymph nodes (TDLNs).
• TDLNs tumor draining lymph nodes
• A are a series of representative flow cytometry plots of TRP-2 tetramer positive CD8 + T cells in tumor draining lymph nodes in a B 16-F10 melanoma model treated with either PBS or Heat-inactivated MVA.
• (B) is a graph of the percentages of TRP-2 tetramer positive CD8 + T cells in WT and Batf3 _ " mice with B16-F10 melanomas treated with either PBS or Heat-MVA. Each sample were from lymph nodes pooled from 2-3 mice treated with the same condition. (*, p ⁇ 0.05).
• FIG. 21 is a series of graphic representations showing that intratumoral injection with MVAAE3L-hFlt3L is effective in the treatment of large established B16-OVA melanoma in a unilateral tumor implantation model.
• A is a schematic diagram of a unilateral tumor implantation model with large established B 16-OVA model.
• B16-OVA melanoma cells (5 x 10 s cells) were implanted intradermally to the right flanks of C57B/6 mice. 8-9 days after tumor implantation, mice were intratumorally injected with 2 x 10 7 pfu of MVAAE3L-hFlt3L, Heat-MVA, poly (I:C), or PBS mock control twice weekly. Tumor sizes were measured and the survival of mice was monitored.
• Figure 22 is a series of graphic representations showing that the combination of intratumoral injection with Heat-inactivated MVA and systemic delivery of immune checkpoint antibodies improves antitumor efficacy in a large established melanoma unilateral implantation model.
• A is a schematic diagram of a unilateral tumor implantation model with large established B16-F10 model.
• B16-F10 melanoma cells (5 x 10 5 cells) were implanted intradermally to the right flanks of C57B/6 mice. 8-9 days after tumor implantation, mice were intratumorally injected with 2 x 10 7 pfu of Heat-MVA twice weekly in the presence or absence of intraperitoneal delivery of anti-CTLA-4, anti-PD-1, or anti-PD-Ll antibodies.
• (B) Volumes of injected tumors over days after injections with PBS, or with Heat-MVA in the presence of isotype control, or anti-CTLA-4, or anti-PD-1, or Anti-PD-Ll.
• cancer refers to a class of diseases of humans and animals characterized by uncontrolled cellular growth. Unless otherwise explicitly indicated, the term “cancer” may be used herein interchangeably with the terms “tumor,” “malignancy,” “hyperproliferation” and “neoplasm(s);” the term “cancer cell(s)” is interchangeable with the terms “tumor cell(s),” “malignant cell(s),” “hyperproliferative cell(s),” and “neoplastic cell(s)”.
• solid tumors include, but are not limited to: soft tissue sarcoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor and other bone tumors (e.g., osteosarcoma , malignant fibrous histiocytoma), leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
• Some of the most common solid tumors for which the compositions and methods of the present disclosure would be useful include: head-and-neck cancer, rectal adenocarcinoma, glioma, medulloblastoma, urothelial carcinoma, pancreatic adenocarcinoma, uterine (e.g., endometrial cancer, fallopian tube cancer,) ovarian cancer, cervical cancer prostate adenocarcinoma, non-small cell lung cancer (squamous and adenocarcinoma), small cell lung cancer, melanoma, breast carcinoma, ductal carcinoma in situ, renal cell carcinoma, and hepatocellular carcinoma, adrenal tumors (e.g., adrenocortical carcinoma), esophageal, eye (e.g., melanoma, retinoblastoma), gallbladder, gastrointestinal, Wilms' tumor, heart, head and neck, laryngeal and hypopharyngeal, oral (e.
• Metalastasis refers to the spread of cancer from its primary site to neighboring tissues or distal locations in the body. Cancer cells (including cancer stem cells) can break away from a primary tumor, penetrate lymphatic and blood vessels, circulate through the bloodstream, and grow in in normal tissues elsewhere in the body. Metastasis is a sequential process, contingent on tumor cells (or cancer stem cells) breaking off from the primary tumor, traveling through the bloodstream or lymphatics, and stopping at a distant site. Once at another site, cancer cells re-penetrate through the blood vessels or lymphatic walls, continue to multiply, and eventually form a new tumor (metastatic tumor). In some embodiments, this new tumor is referred to as a metastatic (or secondary) tumor.
• Immuno response refers to the action of one or more of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, etc.
• An immune response may include a cellular response, such as a T cell response that is an alteration (modulation, e.g., significant enhancement, stimulation, activation, impairment, or inhibition) of cellular, i.e., T cell function.
• a T cell response may include generation, proliferation or expansion, or stimulation of a particular type of T cell, or subset of T cells, for example, effector CD4 + , CD4 + helper, effector CD8 + , CD8 + cytotoxic, or natural killer (NK) cells.
• T cell subsets may be identified by detecting one or more cell receptors or cell surface molecules (e.g., CD or cluster of differentiation molecules).
• a T cell response may also include altered expression (statistically significant increase or decrease) of a cellular factor, such as a soluble mediator (e.g., a cytokine, lymphokine, cytokine binding protein, or interleukin) that influences the differentiation or proliferation of other cells.
• a soluble mediator e.g., a cytokine, lymphokine, cytokine binding protein, or interleukin
• Type I interferon is a critical regulator of the innate immunity (52)(Huber et al. Immunology 132(4):466-474 (2011)). Animal and human studies have shown a role for IFN- ⁇ ⁇ in directly influencing the fate of both CD4 + and CD8 + T cells during the initial phases of antigen recognition and anti- tumor immune response.
• IFN Type I is induced in response to activation of dendritic cells, in turn a sentinel of the innate immune system.
• An immune response may also include humoral (antibody) response.
• Helper T cell refers to a CD4 + T cell; helper T cells recognize antigen bound to MHC Class ⁇ molecules. There are at least two types of helper T cells, Thl and Th2, which produce different cytokines.
• Cytotoxic T cell refers to a T cell that usually bears CD8 molecular markers on its surface (CD8 + ) and that functions in cell-mediated immunity by destroying a target cell having a specific antigenic molecule on its surface. Cytotoxic T cells also release Granzyme, a serine protease that can enter target cells via the perforin-formed pore and induce apoptosis (cell death). Granzyme serves as a marker of cytotoxic phenotype. Other names for cytotoxic T cell include CTL, cytolytic T cell, cytolytic T lymphocyte, killer T cell, or killer T lymphocyte.
• cytotoxic T cells may include virus-infected cells, cells infected with bacterial or protozoal parasites, or cancer cells. Most cytotoxic T cells have the protein CD8 present on their cell surfaces. CD8 is attracted to portions of the Class I MHC molecule. Typically, a cytotoxic T cell is a CD8 + cell.
• 'Tumor-infiltrating leukocytes refers to white blood cells of a subject afflicted with a cancer (such as melanoma), that are resident in or otherwise have left the circulation (blood or lymphatic fluid) and have migrated into a tumor.
• a cancer such as melanoma
• Immuno checkpoint inhibitor or “immune checkpoint blocking agent” or “immune checkpoint blockade inhibitor” refers to molecules that completely or partially reduce, inhibit, interfere with or modulate the activity of one or more checkpoint proteins.
• Checkpoint proteins regulate T-cell activation or function.
• Checkpoint proteins include, but are not limited to, CD28 receptor family members, CTLA-4 and its ligands CD80 and CD86; PD- 1 and its ligands PDL1 and PDL2; LAG3, B7-H3, B7-H4, TIM3, ICOS, II DLBCL, BTLA or any combination of two or more of the foregoing (53).
• Nonlimiting examples contemplated for use herein include ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A, BMS-936559, MEDI4736, MSB 00107180, or any combination thereof.
• Parenter when used in the context of administration of a therapeutic substance or composition includes any route of administration other than administration through the alimentary tract. Particularly relevant for the methods disclosed herein are intravenous (including for example through the hepatic portal vein for hepatic delivery), intratumoral or intrathecal administration.
• Antibody refers to an immunoglobulin molecule which specifically binds to an antigen or to an antigen-binding fragment of such a molecule.
• antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive (antigen-binding) fragments or portions of intact immunoglobulins.
• the antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies (scFv) humanized antibodies, chimeric antibodies, human recombinant antibodies and bi- and tri- specific antibodies.
• Oncolytic virus refers to a virus that preferentially infects cancer cells, replicates in such cells, and induces lysis of the cancer cells through its replication process.
• Nonlimiting examples of naturally occurring oncolytic viruses include vesicular stomatitis virus, reovirus, as well as viruses engineered to be oncoselective such as adenovirus, Newcastle disease virus and herpes simplex virus (See, e.g., Nemunaitis, J. Invest New Drugs. 17(4):375-86 (1999); Kirn, DH et al. Nat Rev Cancer. 9(l):64-71(2009); Kirn et al. Nat. Med. 7:781 (2001 ); Coffey et al.
• Vaccinia virus infects many types of cells but replicates preferentially in tumor cells due to the fact that tumor cells have a metabolism that favors replication, exhibit activation of certain pathways that also favor replication and create an environment that evades the innate immune system, which also favors viral replication.
• MVA and MVAAE3L do not fit the definition of oncolytic viruses as they do not produce an antitumor effect primarily by replicating inside tumor cells and causing apoptosis. (Nor do they fit the classic definition of vaccines as these viruses do not express tumor antigens.
• MVA means "modified vaccinia Ankara” and refers to a highly attenuated strain of vaccinia derived from the Ankara strain and developed for use as a vaccine and vaccine adjuvant.
• the original MVA was isolated from the wild-type Ankara strain by successive passage through chicken embryonic cells, Treated thus, it lost about 15% of the genome of wild-type vaccinia including its ability to replicate efficiently in primate (including human) cells. (57) (Mayr et al., Monbl Bakteriol B 167, 375-390 (1978)).
• the smallpox vaccination strain MVA marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defense mechanism. MVA is considered an appropriate candidate for development as a recombinant vector for gene or vaccination delivery against infectious diseases or tumors.
• 5S (Verheust et al., Vaccine 30(16), 2623-2632 (2012)). MVA has a genome of 178 kb in length and a sequence first disclosed in (59)(Antoine et al., Virol. 244(2): 365-396 (1998)). Sequences are also disclosed in Genbank U94848.1. Clinical grade MVA is commercially and publicly available from Bavarian Nordic A S Kvistgaard, Denmark. Additionally, MVA is available from ATCC, Rockville, MD and from CMCN (Institut Pasteur Collection Nationale des Microorganismes) Paris, France.
• MVAE3L means a deletion mutant of MVA which lacks a functional E3L gene and is infective but non replicative and it is further impaired in its ability to evade the host's immune system. It has been used as a vaccine vector (by others) to transfer tumor or viral antigens. This mutant MVA E3L knockout and its preparation have been described for example in U.S. Patent 7,049,145.
• Subject means any animal (mammalian, human or other) patient that can be afflicted with cancer and when thus afflicted is in need of treatment.
• “Pharmaceutically acceptable excipient” refers to substances and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human.
• the term includes all inert, non-toxic, liquid or solid fillers or diluents, as long as they do not react with the therapeutic substance of the invention in an inappropriate negative manner, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, preservatives and the like, for example liquid pharmaceutical carriers e.g., sterile water, saline, sugar solutions, Tris buffer, ethanol and/or certain oils.
• an effective amount respectively of the MVA or MVAAE3L is an amount that (administered for a suitable period of time and at a suitable frequency) reduces the number of cancer cells; or reduces the tumor size or eradicates the tumor; or inhibits (i.e., slows down or stops) cancer cell infiltration into peripheral organs; inhibits (i.e., slows down or stops) metastatic growth; inhibits (stabilizes or arrests) tumor growth; allows for treatment of the tumor, and/or induces and promotes an immune response against the tumor.
• an appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation in light of the present disclosure. Such determination will begin with amounts found effective in vitro and amounts found effective in animals. The therapeutically effective amount will be initially determined based on the concentration or concentrations found to confer a benefit to cells in culture. Effective amounts can be extrapolated from data within the cell culture and can be adjusted up or down based on factors such as detailed herein. Effective amounts of the viral constructs are generally within the range of about 10 5 to about 10 10 plaque forming units (pfu), although a lower or higher dose may be administered. In a preferred embodiment, the dosage is about 10 6 -10 9 pfu. Typically, a unit dosage is administered in a volume within the range from 1 to 10 ml.
• the equivalence of pfu to virus particles can differ according to the specific pfu titration method used. Generally, pfu is equal to about 5 to 100 virus particles.
• a therapeutically effective amount the hFlt3L transgene bearing viruses can be administered in one or more divided doses for a prescribed period of time and at a prescribed frequency of administration.
• therapeutically effective amount of hFU3L bearing viruses in accordance with the present disclosure may vary according to factors such as the disease state, age, sex, weight, and general condition of the subject, and the potency of the viral constructs to elicit a desired immunological response in the particular subject for the particular cancer.
• therapeutically effective amount refers to an amount of a composition comprising MVA-hFlt3L or MVAAE3L-hFlt3L sufficient to reduce, inhibit, or abrogate tumor cell growth, thereby reducing or eradicating the tumor, or sufficient to inhibit, reduce or abrogate metastatic spread either in vitro, ex vivo or in a subject or to elicit and promote an immune response against the tumor that will eventually result in one or more of metastatic spread reduction, inhibition and/or abrogation as the case may be.
• the reduction, inhibition, or eradication of tumor cell growth may be the result of necrosis, apoptosis, or an immune response or a combination of two or more of the foregoing (however, the precipitation of apoptosis for example may not be due to the same factors as observed with oncolytic viruses).
• the amount that is therapeutically effective may vary depending on such factors as the particular virus used in the composition, the age and condition of the subject being treated, the extent of tumor formation, the presence or absence of other therapeutic modalities, and the like.
• the dosage of the composition to be administered and the frequency of its administration will depend on a variety of factors, such as the potency of the active ingredient, the duration of its activity once administered, the route of administration, the size, age, sex and physical condition of the subject, the risk of adverse reactions and the judgment of the medical practitioner.
• the compositions are administered in a variety of dosage forms, such as injectable solutions.
• terapéuticaally effective amount for an immune checkpoint blocking agent shall mean an amount of an immune checkpoint blocking agent sufficient to reverse or reduce immune suppression in the tumor microenvironment and to activate or enhance host immunity in the subject being treated.
• immune checkpoint blocking agents approved, in clinical trials or still otherwise under development including inhibitory antibodies against CD28 inhibitor such as CTLA-4 (cytotoxic T lymphocyte antigen 4) (e.g., ipilimumab), anti- PD-1 (programmed Death 1) inhibitory antibodies (e.g., nivolumab, pembrolizumab, pidilizumab, lambrolizumab), and anti-PD-Ll (Programmed death ligand 1) inhibitory antibodies (MPDL3280A, BMS-936559, MEDI4736, MSB 00107180), as well as inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3 (T cell Immunoglobulin and Mucin-3), B7-H3,
• the tumor expresses the particular checkpoint but in the context of the present invention this is not strictly necessary as immune checkpoint blocking agents block more generally immune suppressive mechanisms within the tumors, elicited by tumor cells, stromal cells, and tumor-infiltrating immune cells.
• the CTLA4 inhibitor ipilimumab when administered as adjuvant therapy after surgery in melanoma is administered at 1-2 mg mL over 90 minutes for a total infusion amount of 3 mg/kg every three weeks for a total of 4 doses.
• This therapy is often accompanied by severe even life-threatening immune-mediated adverse reactions, which limits the tolerated dose as well as the cumulative amount that can be administered. It is anticipated that it will be possible to reduce the dose and/or cumulative amount of ipilimumab when it is administered conjointly with MVA-hFlt3L or MVAAE3L-hFlt3L.
• CTLA4 inhibitor's dose if it is administered directly to the tumor conjointly with one or both the foregoing MVA viruses. Accordingly, the amounts provided above for ipilimumab will be a starting point for determining the particular dosage and cumulative amount to be given to a patient in conjoint administration but dosing studies will be required to determine optimum amounts.
• Pembrolizumab is prescribed for administration as adjuvant therapy in melanoma diluted to 25 mg/mL. It is administered at a dosage of 2 mg/kg over 30 minutes every three weeks. Again, this would be a starting point for determining dosage and administration in the conjoint administration with MVA-hFlt3L or MVAAE3L-hFlt3L.
• Nivolumab is prescribed for administration at 3 mg kg as an intravenous infusion over 60 minutes every two weeks, providing a similar starting point in determining dosage and administration regimen of this and other checkpoint inhibitors conjointly with MVA-hFlt3L or MVAAE3L-hFlt3L described herein or conjointly with Heat-MVA (inactivated MVA which inactivation can be heat-induced or UV radiation-induced) in amounts generally within the same range as the viruses and vital constructs of MVA and MVA AE3L.
• Immune stimulating agents such as agonist antibodies have also been explored as immunotherapy for cancers.
• anti-ICOS antibody binds to the extracellular domain of ICOS leading to the activation of ICOS signaling and T cell activation.
• the immune stimulating agonist antibodies can be used systemically in combination with intratumoral injection of MVA-hFlt3L or MVAAE3L-hFlt3L (or inactivated MVA). Alternatively, the immune stimulating agonist antibodies can be used conjointly with MVA- hFlt3L or MVAAE3L-hFlt3L via intratumoral delivery either simultaneously or sequentially.
• “Pharmaceutically acceptable carrier and/or diluent” or “pharmaceutically acceptable excipient” includes without limitation any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for biologically active substances is well known in the art. Further details of excipients are provided below. Supplementary active ingredients, such as antimicrobials, for example antifungal agents, can also be incorporated into the compositions.
• “Delivering” used in connection with depositing the MVA-hFlt3L or MVAAE3L-hFlt3L (or Heat-MVA in conjoint administration with immune checkpoint blockade inhibitors, especially for large established tumors) of the present disclosure in the tumor microenvironment whether this is done by local administration to the tumor (intratumoral) or by for example intravenous route.
• the term focuses on MVA-hFlt3L or MVAAE3L-hFlt3L that reaches the tumor itself.
• Conspan administration herein refers to administration of a second therapeutic modality in combination with MVA-hFlt3L or MVAAE3L-hFlt3L for example an immune checkpoint blocking agent administered in close temporal proximity with MVA- hFlt3L or MVAAE3L-hFlt3L.
• a PD-l/PDL-1 inhibitor and/or a CTLA4 inhibitor in more specific embodiments, an antibody
• a PD-l/PDL-1 inhibitor and/or a CTLA4 inhibitor in more specific embodiments, an antibody
• useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter.
• a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
• the phrases “operatively positioned,” “operatively linked,” “under control,” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
• expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
• expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA) from a transcribed gene.
• a biologically-active polypeptide product or inhibitory RNA e
• the inventors generated recombinant MVA and MVAAE3L viruses expressing human Flt3L, with the goal of delivering Flt3L to the tumor microenvironment to facilitate recruitment, differentiation and function of immune cells, including CD103 + /CD8oc dendritic cells (DCs).
• DCs CD103 + /CD8oc dendritic cells
• kits comprising one or more compositions comprising one or more of the recombinant MVAs described herein.
• the kit can comprise one or multiple containers or vials of the recombinant MVA, together with instructions for the administration of the recombinant MVA to a subject to be treated.
• the instructions may indicate a dosage regimen for administering the composition or compositions as provided below.
• mice with bilateral tumors were treated with intratumoral injection of MVAAE3L-hFlt3L to the larger tumors on the right flank and intraperitoneal delivery of immune checkpoint blockade antibodies twice weekly, including anti-CTLA-4 (100 ⁇ per mouse), anti-PD-1 (250 ⁇ g per mouse), anti-PD-Ll (250 ⁇ g per mouse), or isotype control (100 ⁇ g per mouse).
• the tumor sizes were measured and the tumors were injected twice a week. The survival of mice was monitored.
• STING Gt/Gt , BatO " ' ' mice and WT age-matched controls were used for bilateral B16-F10 melanoma implantation, and treated with PBS or Heat-MVA to the larger tumors on the right flank of the mice.
• the commercial sources for reagents were as follows: Therapeutic anti-CTLA4 (clone 9H10 and 9D9), anti-PDl (clone RMP1-14), anti-PD-Ll (clone 10F.9G2) were purchased from BioXcell; Antibodies used for flow cytometry were purchased from eBioscience (CD45.2 Alexa Fluor 700, CD3 PE-Cy7, CD4 APC-efluor780, CD8 PerCP-efluor710), Invitrogen (CD4 QDot 605, Granzyme B PE-Texas Red, Granzyme B APC).
• the inventors generated recombinant MVA or MVAAE3L viruses comprising a TK-deletion with and without expressing human Flt3L or murine GM-CSF under the vaccinia synthetic early/late promoter (Pse/I) using standard recombinant virus technology.
• the inventors constructed a plasmid containing specific gene of interest (SG) under the control of the vaccinia Pse/1 as well as the E. coli xanthine-guanine phosphoribosyl transferase gene (gpt) under the control of vaccinia P7.5 promoter flanked by the thymidine kinase (TK) gene on either side ( Figure 1).
• BHK21 cells were infected with MVA or MVAAE3L at a MOI of 0.05 for 1 h, and then were transfected with the plasmid DNAs described above. The infected cells were collected at 48 h. Recombinant viruses were selected through further culturing in gpt selection medium including MPA, xanthine and hypoxanthine, and plaque purified (Lorenzo et al., 2004). PCR analysis was performed to identify recombinant viruses with loss of part of the TK gene and with and without murine GM-CSF, or human Flt3L, ( Figure 2).
• PCR was used to verify the correct insertions in the recombinant viruses MVA-mGM-CSF, MVAAE3L-mGM-CSF, MVA-hFlt3L, and MVAAE3L-hFlt3L.
• Primer pair mGM-CSF- Fl/Rl was used to amplify a 310 bp DNA fragment from mGM-CSF gene inserted in recombinant viruses MVA-mGM-CSF or MVAAE3L-mGM-CSF.
• TK-F4/R4 can amplify a 304 bp DNA fragment from MVA or MVAAE3L, but not in MVA-mGM-CSF, MVAAE3L-mGM-CSF, MVA-hFlt3L, or MVAAE3L-hFlt3L viruses due to the deletion of TK-R4 primer locus.
• TK-R4 (SEQ ID NO:3): TCCTTCGTTTGCCATACGCT;
• TK-F5 (SEQ ID NO:4): GAACGGGACTATGGACGCAT;
• TK-R5 (SEQ ID NO:5): TCGGTTTCCTC ACCC A ATCG ; pCB-R3 (SEQ ID NO:6): ACCTGATGGATAAAAAGGCG; mGMCSF-Fl (SEQ ID NO:7): GGCATTGTGGTCTACAGCCT; mGMCSF-Rl (SEQ ID NO:8): GTGTTTC AC AGTCCGTTTCCG ; hFlt3L-Fl (SEQ ID NO:9): A ACGACCTATCTCCTCCTGC ; hFlt3L-Rl (SEQ ED NO:10): GGGCTGAAAGGCACATTTGG.
• MVAAE3L-hFlt3L is more effective than MVAAE3L-mGM-CSF or MVAAE3L in eradicating or delaying the growth of both injected and non-injected tumors in a bilateral B10-F10 melanoma model
• the inventors investigated the effects of intratumoral injection of MVAAE3L-hFlt3L, MVAAE3L-mGM-CSF, and MVAAE3L on metastatic growth using a murine B16-F10 melanoma bilateral implantation model.
• B 16-F10 melanoma cells were implanted intradermally to the left and right flanks of C57B/6 mice (5 x 10 5 to the right flank and 1 x 10 5 to the left flank). 7-8 days after tumor implantation, the inventors intratumorally injected MVAAE3L-hFlt3L, MVAAE3L-mGM-CSF, and MVAAE3L (2 x 10 7 pfu) or PBS to the ' larger tumors on the right flank twice weekly. The tumor sizes were measured and the survival of mice was monitored (Figure 3A).
• mice treated with MVAAE3L-mGM-CSF also had a median survival of 25 days and 1/9 mice were cured of melanoma.
• Intratumoral injection of MVA ⁇ E3L ⁇ hFh3L is effective in the proliferation and activation of CD8 + and CD4 + T cells and reduction of regulatory T cells in both injected and non-injected tumors.
• MVAAE3L, MVAAE3L-mGM-CSF, MVAAE3L-hFlt3L, or Heat-MVA in B16-F10 melanomas leads to activation and proliferation of CD8 + and CD4 + T cells
• 2.5 x 10 5 B 16-F10 melanoma cells were intradermally implanted to the left flank and 5 x 10 5 B 16-F10 melanoma cells to the right flank of 6-8 weeks old C57B/6 mice.
• MVAAE3L, MVAAE3L- mGM-CSF, MVAAE3L-hFlt3L, or Heat-MVA or PBS was injected into the larger tumors on the right flank.
• Ki-67 + CD8 + T cells increased from 58.3% in PBS-treated tumors to 83.2% in MVAAE3L-hFlt3L-treated tumors (p ⁇ 0.01; Fig. 4C, 4D).
• MVAAE3L- hFlt3L p ⁇ 0.01; PBS vs. MVAAE3L, ⁇ 0.05; MVAAE3L-hFlt3L vs. MVAAE3L Fig. 5A, 5B).
• Ki-67 + CD4 + T cells from 40% in PBS-treated tumors to 59.6% in MVAAE3L-treated tumors and 66.6% in MVAAE3L-hFH3L-treated tumors (p ⁇ 0.001; PBS vs. MVAAE3L-hFlt3L, p ⁇ 0.01; PBS vs. MVAAE3L, /? ⁇ 0.05; MVAAE3L- hFlt3L vs. MVAAE3L, Fig. 5 C, D).
• CD4 + Foxp3 + T cells decreased from 45.1% in PBS-treated tumors to 26.6% in MVAAE3L-hFlt3L-treated tumors (p ⁇ 0.001; Fig. 6A, 6B).
• CD4 + Foxp3 + T cells decreased from 51.6% in PBS-treated mice to 37.1% in MVAAE3L-hFlt3L-treated tumors (p ⁇ 0.01 ; Fig. 6A, 6B).
• the inventors determined the absolute numbers of CD45 + cells, CD8 + cells in both injected and non-injected tumors after virus treatment. It was found that in the injected tumors, intratumoral injection of MVAAE3L-hFlt3L or MVAAE3L increased the CD45 + cells from 3.8 x 10 6 /g to 1.6 x 10 7 /g or 1.3 x 10 7 /g, respectively (P ⁇ 0.01, MVAAE3L-hFlt3L vs. PBS; P ⁇ 0.01, MVAAE3L vs. PBS; Fig. 7A).
• intratumoral injection of MVAAE3L-hFlt3L or MVAAE3L at the contralateral tumors increased the CD45 + cells from 3.3 x 10 6 /g to 9.5 x 10 7 /g or 5.4 x 10 7 /g, respectively (P ⁇ 0.05, MVAAE3L-hFlt3L vs. MVAAE3L; P ⁇ 0.05, MVAAE3L-hFlt3L vs. PBS; Fig. 7A).
• the inventors also found that in the injected tumors, intratumoral injection of MVAAE3L- hFU3L or MVAAE3L increased the CD8 + cells from 2.9 x 10 5 /g to 2.9 x 10 6 /g or 2.0 x 10 6 /g, respectively (P ⁇ 0.01, MVAAE3L vs. PBS; P ⁇ 0.001, MVAAE3L-hFlt3L vs. PBS; Fig. 7B).
• intratumoral injection of MVAAE3L-hFlt3L or MVAAE3L at the contralateral tumors increased the CD8 + cells from 2.8 x 10 5 /g to 1.6 x 10 6 /g or 1.1 x 10 6 /g, respectively (P ⁇ 0.001, MVAAE3L-hFlt3L vs. PBS; Fig. 7B).
• the ratios of CD8 + T cells over regulatory T cells (Tregs, defined as CD4 + FoxP3 + cells) and Tconv (CD4 + Foxp3 " cells) over Tregs was also assessed. It was observed that in the injected tumors, intratumoral injection of MVAAE3L-hFlt3L or MVAAE3L increased the ratios of CD8 + Treg from 2.8 to 18.6 or 12.5 (P ⁇ 0.01, MVAAE3L vs. PBS; P ⁇ 0.001, MVAAE3L- hFlt3L vs. PBS, P ⁇ 0.05, MVAAE3L vs. MVAAE3L-hFlt3L; Fig. 7C).
• the inventors also found that in the non-injected tumors, intratumoral injection of MVAAE3L-hFlt3L or MVAAE3L at the contralateral tumors increased the ratios of CD8 + /Treg from 3.4 to 11 or 7.8 (P ⁇ 0.01, MVAAE3L vs. PBS; P ⁇ 0.01, MVAAE3L-hFlt3L vs. PBS; Fig. 7C).
• Tumor-associated macrophages are tumor infiltrating myeloid cells that express the following surface markers CD45 + MHC-H + F4/80 hi CD24 l0 (Broz, et al. Cancer Cell, 26(5):638-52, 2014).
• the inventors analyzed the percentage of TAMs among CD45+ cells in both injected and non-injected tumors. They observed that intratumoral injection of
• MVAAE3L-hFlt3L reduced the percentages of TAMs out of CD45 + cells from 21.4% to 0.7% in injected tumors (P ⁇ 0.0001, MVAAE3L-hFlt3L vs. PBS; Fig. 8, 9B) and from 22% to 3.2% in non-injected tumors (P ⁇ 0.0001 , MVAAE3L-hFlt3L vs. PBS; Fig. 8, 9A).
• MVAAE3L- hFlt3L is more effective than MVAAE3L in reducing TAMs in non-injected tumors (P ⁇ 0.05, MVAAE3L-hFlt3L vs. MVAAE3L; Fig. 8, 9A).
• mice In the Batf3 " ' " mice, the percentages of TAMs out of CD45 + cells in PBS-treated mice were 5.4% in injected tumors and 7.2% in the non-injected tumors (P ⁇ 0.01, injected tumors in WT PBS vs. Batf3 _ " PBS; Fig. 8, 9B; P ⁇ 0.05, non-injected tumors in WT PBS vs. Batf3 7" PBS; Fig. 8, 9B).
• Intratumoral injection of MVAAE3L-hFlt3L further reduced the percentages of TAMs out of CD45 + cells to 0.5% in the injected tumors and 1.5% in the non-injected tumors (P ⁇ 0.0001, injected tumors in BatfT' " MVAAE3L-hFlt3L vs. Batf3 ⁇ ' " PBS; Fig. 8, 9B; P ⁇ 0.05, non-injected tumors in BatfT MVAAE3L-hFlt3L vs. BatfS 7" PBS; Fig. 8, 9A).
• the markedly reduced numbers of TAMs in tumors of Batf3-/- mice suggest the generation of TAMs might be linked to the CD8+ T cell infiltration within the tumors. TAMs have been shown to promote tumor progression and metastasis. Effective depletion of TAMs from both injected and non-injected tumors by intratumoral injection of MVAAE3L-hFlt3L might contribute to the effectiveness of this therapy.
• the inventors next analyzed dendritic cell (DC) populations in both injected and non-injected tumors.
• DC dendritic cell
• Tumor infiltrating DCs are characterized as CD45 + Ly6C-MHC-i CD24 hi F4/80'° cells (Broz et al., Cancer Cell, 2014).
• CD24 hl DCs there are two DC populations, CDl lb + DC and CD103 + DC.
• the percentage of CD24 hi DCs (CD24 + ) out of CD45 + cells in both injected and non-injected tumors was investigated.
• CD103 + DCs is a subset of peripheral DCs that are specialized in cross-presenting antigens.
• Batf3 is a transcription factor that is important for the differentiation of CD103 + DCs.
• CD103 + DCs play important roles in host anti-tumor immunity.
• the inventors of the present disclosure have previously shown that Batf3-dependent CD103 + DCs are required for inactivated MVA-mediated antitumor effects (WO2016/168862).
• CD103 + DCs undergo dynamic changes after intratumoral injection with viruses.
• Intratumoral injections of MVAAE3L or MVAAE3L-hFlt3L are also effective in a murine triple-negative breast cancer 4T1 bilateral implantation model
• TNBC triple-negative breast cancer
• mice 5 days post tumor implantation, the larger tumors on the right flank were injected with either MVAAE3L or MV ⁇ 3 L-hFlt3L (2 x 10 7 pfu) twice weekly. Mice were monitored daily and tumor sizes were measured twice weekly. The survival of mice was monitored. It was found that intratumoral injection of MVAAE3L or MVAAE3L-hFlt3L led to dramatic decrease of tumor volumes of the injected tumors compared with PBS-treated tumors ( Figure 15A). The tumor volumes of the injected and non-injected tumors at 18-day post treatment were shown ( Figure 15, B, C; P ⁇ 0.0001, MVAAE3L or MVAAE3L-hFlt3L vs. PBS).
• MVAAE3L or MVAAE3L-hFlt3L with immune checkpoint blockade such as anti-CTLA-4 or anti-PD-l/PD-Ll antibodies would also be more effective than virotherapy alone in this bilateral 4T1 implantation model.
• Intratumoral injections of MVAAE3L-hFlt3L is effective in a murine prostate cancer TRAMP -C2 unilateral tumor implantation model, which requires STING
• mice 17 days post tumor implantation, the tumors (around 3-4 mm in diameter) on the right flank were injected with either PBS or MVAAE3L-hFlt3L (2 x 10 7 pfu) twice weekly. Mice were monitored daily and tumor sizes were measured twice weekly. The survival of mice was monitored. It was found that intratumoral injection of MVAAE3L- hFlt3L led to dramatic decrease of tumor volumes of the injected tumors in the WT mice compared with PBS-treated tumors, but it was less effective in STING-deficient mice (Figure 16, A-D). The initial tumor volumes and 24-day post injection tumor volumes were shown in Figure 16 E and F, respectively.
• Intratumoral injection ofMVA-hFlt3L is also effective in a bilateral B16-F10 melanoma implantation model
• MVA-hFlt3L or MVAAE3L-hFlt3L or PBS was injected into the larger tumors twice a week and tumor sizes and survival were monitored.
• the inventors have previously shown that the combination of intratumoral injection of inactivated MVA and systemic delivery of immune checkpoint blockade results in enhanced efficacy compared to either agent alone in bilateral B 16-F10 and MC38 tumor implantation models.
• mice treated with intratumoral injection of MVAAE3L-hFlt3L in combination with systemic delivery of immune checkpoint developed immunity against challenge of a different tumor type
• the inventors examined whether the surviving mice developed antitumor memory T cell immunity against B 16-F10 and MC38 colon cancers after treatment with intratumoral injection of MVAAE3L-hFlt3L alone or in the presence of intraperitoneal delivery of anti- CTLA-4 antibody by using Enzyme-linked ImmunoSpot (ELISpot). Briefly, CD8 + T cells were isolated from splenocytes and 1 x 10 5 cells were cultured overnight at 37°C in anti-IFN- ⁇ -coated BD ELISPOT plate microwells.
• ELISpot Enzyme-linked ImmunoSpot
• CD8 + T Cells were stimulated with either B16-F10 or MC38 cells irradiated with an ⁇ -irradiator and cytokine secretion was detected with an anti-IFN- ⁇ antibody. Whereas CD8 + T cells from naive mice did not show any reactivity to either B16-F10 or MC38 cells, CD8 + T cells from MVAAE3L-hFlt3L-treated mice showed reactivity to both B 16-F10 and MC38 cells ( Figure 18, A and B).
• mice implanted with B16-F10 and subsequently treated with MVAAE3L-hFlt3L intratumorally but have never been exposed to MC38 cells similar reactivity to MC38 cells were also observed (data not shown).
• intratumoral injection of MVAAE3L-hFlt3L for the treatment of B16-F10 led to the development of antitumor immunity against not only B 16-F10 melanoma but also an irrelevant tumor type, in this case, MC38 colon adenocarcinoma.
• Example 15 Intratumoral injection with Heat-inactivated MVA induces type I IFN and inflammatory cytokines and chemokines in both injected and non-injected tumorsin a bilateral B16-F10 tumor implantation model
• Heat-inactivated MVA Heat-inactivated MVA
• MVAAE3L infection of tumor cells or dendritic cells leads to the induction of type I IFN and proinflammatory cytokine and chemokine production
• the inventors performed the following experiment. Briefly, 2.5 x 10 5 B 16-F10 melanoma cells were intradermally implanted to the left flank and 5 x 10 5 B16-F10 melanoma cells to the right flank of 6-8 weeks old C57B/6 mice.
• the release of tumor DNA can be sensed by the cGAS/STING cytosolic DNA-sensing pathway in the immune cells including dendritic cells, macrophages, and monocytes, which leads to the induction of Ifrib, 116, Ccl4, Ccl5, Cxcl9, and CxcllO gene expression.
• MVAAE3L induces higher levels of type I IFN and proinflammatory cytokines and chemokines than MVA in both immune cells and tumor cells, through the activation of both the cGAS/STING-dependent cytosolic DNA- sensing pathway and MDA5/MAVS-dependent cytosolic dsRNA-sensing pathway.
• the tumors were 4 mm in diameter, they were treated with intradermal injections of Heat-MVA twice 3 days apart. 7 days post the initial injection, TDLNs were incubated and cell suspensions were prepared and incubated for 30 mins at room temperature with anti-FcyR ⁇ (2.4G2) antibody and PE-H-2 Kb TRP2 (SVYDFFVWL) tetramer (MBL), followed by staining with anti-CD3 and anti-CD8 antibodies. Cells were analyzed by FACS.
• Intratumoral injection with MVAAE3L-hFlt3L is effective in the treatment of large established B16-OVA melanoma in a unilateral tumor implantation model
• the inventors compared the anti-tumor efficacy of intratumoral injection of MVAAE3L- hFlt3L with Heat-inactivated MVA (Heat-MVA) or poly (I:C) in a large established B16- OVA unilateral tumor implantation model.
• Heat-MVA Heat-inactivated MVA
• I:C poly
• Extracellular poly (I:C) can activate the endosomal localized Toll-like receptor 3 (TLR3), whereas intracellular poly (I:C) can activate the cytosolic dsRNA sensor Melanoma Differentiation-Associated protein 5 (MDA5). Because CD103 + DC in the tumor microenvironment and CD8D + DC in the tumor draining lymph nodes express higher levels of TLR3, poly (I:C) could be an effective immune modulator for the cross-presenting DCs (Salmon et al., Immunity 2016). Intratumoral injection of poly I:C (50 ⁇ g per mouse) twice weekly also led to tumor shrinkage in this large established unilateral B 16-F10 melanoma model ( Figure 21B).
• mice treated with poly (I:C) exhibited systemic illness including fatigue and wasting. It is possibly than intratumorally delivered poly (I:C) was leaked into the systemic circulation which caused immune-related side effects.
• intratumoral injection of MVAAE3L-hFlt31 or Heat-MVA is effective in treating large established highly aggressive B16-OVA in a unilateral implantation model.
• the inventors further tested whether the combination of intratumoral injection of Heat- inactivated MVA (Heat-MVA) and systemic delivery of immune checkpoint blockade such as anti-CTLA-4, anti-PD-1, or anti-PD-Ll antibodies have enhanced potency in eradicating large established B 16-F10 in an unilateral tumor implantation model.
• Heat-MVA Heat- inactivated MVA
• systemic delivery of immune checkpoint blockade such as anti-CTLA-4, anti-PD-1, or anti-PD-Ll antibodies have enhanced potency in eradicating large established B 16-F10 in an unilateral tumor implantation model.
• B16-F10 melanoma cells (5x 10 s cells) were implanted intradermally into the shaved skin on the right flank of WT C57BL/6J mice. After 9 days post implantation, tumors that are 5-6 mm in diameter were injected with Heat-MVA (equivalent of 2 x 10 7 pfu of MVA), or PBS.
• poxviruses for cancer the lead example of JX-594. Current pharmaceutical
• VMA modified vaccinia virus Ankara
• Cyclic GMP-AMP synthase is a cytosolic
• Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826-830 (2013).
• Double-stranded RNA is a trigger for apoptosis in vaccinia virus- infected cells. J Virol 71, 1992-2003 (1997).

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